WSRC-TR-96-0088

Final Report for TTP# SR-16-PL-42 (Formerly SR-141019J -Bioremediation of Aqueous Pollutants Using BiomassEmbedded in Hydrophilic Foam (U)

E. W. Wilde

Westinghouse Savannah River Company ; : •" • • { ' - ' "Savannah River Site • ( ; t .- ^ -Aiiceri, South Carolina 29808 t-r-i O J fJ. C. Radway * *"1 "k ""

J. Santo Domingo O

R. G. Zngmark

M. J. Whitaker

DOE Contract No. PE-AC09-89SR1S035

This paper was prepared in connection with work done under the above contract number with the U. S.Department of Energy. By acceptance of this paper, the publisher and/or recipient acknowledges the U. S.Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper,along with the right to reproduce and to authorize others to reproduce all or part of the copyrighted paper.

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WSRC-TR-96-0088

WSRC-TR-96-0088

FINAL REPORT FOR TTP# SR-16-PL-42 (FORMERLY SR-141019)

BIOREMEDIATION OF AQUEOUS POLLUTANTS USING BIOMASSEMBEDDED IN HYDROPHILIC FOAM

by

E. W. Wilde, J.C. Radway, J. Santo Domingo, R.G. Zingmark and M.J.Whitaker

Prepared for the Department of Energy Office of Technology Development,Plumes Focus Area

Approved by: D.B. Moore-ShedrowEnvironmental Sciences SectionSavannah River Technology Center

WSRC-TR-96-0088

Table of Contents

1.0«Abstract «*.'.\ 3

2.0 Introduction .: 4

3.0 Identification, Characterization and Selection of MetalContaminated Waste Sites and Waste Streams at SRS 7

4.0 Selection and Culturing of Algal Strains 17

5.0 Characteristics of the Foam Used for Embedding 25

6.0 Bioremoval Capabilities of Foam-Embedded Microbes 27

7.0 Polyurethane Foam Immobilization of TCE-Degrading Bacteria:Entrapment Effectiveness and Influence on Metabolic Activity 35

8.0 Degradation of Trichloroethylene and Benzene by Embedded Bacteria 61

9.0 Conclusions 80

10.0 Recommendations for future work 82

11.0 Acknowledgments 83

12.0 Literature Cited 84

Appendices A1

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1.0 ABSTRACT

The major objective of this project was to examine the potential of a novel hydrophilicpolyurethane foam as an immobilization medium for algal, bacteria, and other types ofbiomass, and to test the resulting foam/biomass aggregates for their use in cleaning upwaters contaminated with heavy metals, radionuclides and toxic organic compounds.Initial" investigations focused on the bioremoval of heavy metals from wastewaters atSRS using immobilized algal biomass. This effort met with limited success for reasonswhich included interference in the binding of biomass and target metals by variousnon-target constituents in the wastewater, lack of an appropriate wastewater at SRS fortesting, and the unavailability of bioreactor systems capable of optimizing contact oftarget pollutants with sufficient biomass binding sites. Subsequent studies comparingalgal, bacterial, fungal, and higher plant biomass demonstrated that other biomasssources were also ineffective for metal bioremoval under the test conditions.Radionuclide bioremoval using a Tc-99 source provided more promising results thanthe metal removal studies with the various types of biomass, and indicated that the algaCyanidium was the best of the tested sources of biomass for this application. However,all of the biomass/foam aggregates tested were substantially inferior to a TEVA resinfor removing Tc-99 in comparative testing.

We also explored the use of hydrophilic polyurethane foam to embed Burkholderiacepacia. B. cepacia is an efficient degrader of trichloroethylene (TCE), a contaminantof considerable concern at SRS and elsewhere. However, it does not adhere well tosurfaces and hence is ill-adapted to use in bioreactors. We first optimized theconditions of foam manufacture so as to achieve a high degree of bacterial retentionwithin the foam matrix. The type and concentration of surfactant and the biomassdensity used in the foaming process proved to be of crucial importance. Secondly, thegeneral physiological status of the embedded bacteria was examined. The embeddedpopulation proved to be incapable of growth on nutrient media, but retained respiratoryactivity. Lastly, the degradative capabilities of embedded G4 were examined. Phenol-or benzene-induced bacteria retained the ability to degrade TCE; they were alsocapable of benzene degradation. Degradation of both compounds was inhibited in thepresence of readily metabolizable carbon sources. Preliminary tests indicated that,once TCE-degrading enzyme activity was induced, it was of relatively short duration,potentially limiting shelf life of foam/bacterial aggregates. However, by appropriatemanipulation of induction conditions, we were successful in inducing enzyme activityafter the organisms had already been embedded. It may therefore be feasible togeographically and temporally separate the induction and use of the material (possiblyfor several consecutive cycles) from its growth and immobilization, greatly facilitatingpractical applications.

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2.0 INTRODUCTION

The principal objective of this TTP was to evaluate, with the aid of an industrial partner,Frisby Technologies Inc., a new bioremediation scheme involving filter media consistingof biomass embedded in a hydrophilic foam matrix. Figure 2.1 depicts a simplifieddiagram of the process. Novel features of the conceptual process include: (1) the useof unique biomass strains, (2) use of a unique polymer to produce a biofilter mediumconslsfing of unaltered biomass embedded within a hydrophilic matrix; and (3) use of aunique bioreactor system designed to optimize contact of bioagents with targetpollutants. The utility of the foam embedded with biomass was investigated with actualand simulated wastewater contaminated with toxic heavy metals, radionuclides andtoxic organic compounds, particularly TCE.

There is great potential for processes that utilize natural, biodegradable materials (e.g.microbial biomass) to remove toxicants (such as metals, radionuclides, and chlorinatedhydrocarbons) from wastewaters. A major stumbling block to the development of suchprocesses has been the lack of suitable containment or immobilization of the biomassto fully facilitate their bioremediation capabilities. This project was aimed at overcomingthis liability, demonstrating the capability of clean-up of a variety of waste sites andwaste streams at the SRS with biomass/foam combinations. The longer term goal is tobroaden applicability to clean-up efforts at government and commercial facilities world-wide.

This project began in FY 1994. The principal focus of the project during the first yearincluded the identification, characterization, prioritization and selection of SRS wastesites and waste streams potentially amenable to bioremediation; and the selection ofand growth of algal strains well suited for the bioremediation process underdevelopment. During FY 1995, work was primarily directed toward evaluating theconceptual process using a novel laboratory test rig developed by Frisby, along withother laboratory apparati, for removal of metals from a coal pile runoff basin by the useof foam embedded with algal biomass. A wider scope of potential applications for theprocess was developed for work which occurred from late FY 1995 through the end ofthe project in March 1996. The wider scope was designed to give the foam a betterevaluation, and enhance the possibility of developing a technology with commercialpotential. The project was expanded in two directions. First, additional types ofwastewater with additional contaminants were evaluated, and secondly, additionaltypes of biomass were evaluated for their ability to remediate pollutants whileembedded in foam. Thus, we expanded the technology evaluation from merelybioremoval of heavy metals by algae to both bioremoval and biodegradation of severaltypes of pollutants by a variety of bioagents.

Wastewaters considered for the expanded program included a variety of groundwatersand process streams that contain TCE, toluene, and benzene in addition to heavymetals. Non-algal biomass tested for bioremoval included metal removing

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mMICROBIALSTRAINSELECTION

BIOREMEDIATION USINGFOAM-ENCAPSULATED

BIOMASS

MASSPRODUCTIONOF BIOMASS ENCAPSULATION

OF BIOMASSIN FOAM

TREATMENTIN BIOREACTOR

PURIFIEDWATER

Figure 2.1 Simplified Diagram of Conceptual Process

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bacteria (based on the literature), including a strain currently used by ORNL for Ubiosorption, and two fungal strains. A radionuclide uptake experiment includedevaluation of the comparative uptake by algae, bacteria, fungi, higher plant biomassand an ion exchange resin. Biodegradation was evaluated by testing bacteria known tobe tiigh rate biodegraders containing oxygenases suitable for treating multiplehazardous organic wastes.

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3.0 IDENTIFICATION, CHARACTERIZATION AND SELECTION OF METALCONTAMINATED WASTE SITES AND WASTE STREAMS AT SRS

3.1 Introduction

An investigation was conducted to identify and prioritize heavy metal-containing wastewaters at the Savannah river Site (SRS) in terms of their suitability for testingpf andpotential clean-up by bioremediation processes (Wilde and Radway, 1994).

The investigation included a review of information on surface and/or groundwaterassociated with all known SRS waste sites, as well as waters associated with all knownSRS waste streams. Following the initial review, waste waters known or suspected tocontain potentially problematic concentrations of one or more of the toxic metals (listedin Appendix Table A3.1) were given further consideration.

3.2 Waste Sites

Information on SRS waste sites was obtained by numerous discussions withEnvironmental Restoration (ER) personnel (see Appendix 3) and by reviewingpublished and unpublished documents provided by ER and other SRS personnel (e.g.the Savannah River Site Environmental Report for 1992, WSRC-TR-93-075). Thesereports provided a starting point for the process of investigating and selecting the mostappropriate waste sites for future bioremediation.

The initial screening effort resulted in the identification of over 30 ER waste unitsthat were reported to contain surface and/or groundwaters with heavy metals aspollutants, often in combination with radionuclides and/or toxic organic compounds(Appendix Table A3.2). The current ER contact person for each of these sites wasidentified and interviewed to obtain the most recent information about each site.

In addition to holding discussions with ER personnel, recent (1993) groundwatermonitoring data were obtained and screened for measurements of toxic metal andradionuclides that exceeded regulatory limits. Sites with known or alleged metalcontamination were also compared with groundwater monitoring data (summarized inWSRC-TR-93-075) to determine if drinking water standards were exceeded at thesesites in 1992. This screening resulted in the selection of 25 RFI/RI waste sites thatclearly have heavy metal polluted water. They are listed in Appendix Table A3.3 alongwith information used for evaluating the sites in terms of their suitability for incorporationinto metal bioremoval studies.

A thorough review of WSRC-TR-93-075 also resulted in the identification of severalother SRS facilities at which groundwater monitoring well samples contained heavymetal contamination. Appendix Table A.3.4 lists all sites displaying heavy metal

WSRC-TR-96-0088

containing groundwaters along with the specific metal contaminant(s), the drinkingwater standard (DWS) for the metal, the highest concentration of the contaminantobserved in 1992, the number of wells sampled, and the number of wells wheredrinking water standards were exceeded. As shown in the table, much of thegroujidwater contaminated with toxic metals is also contaminated with radionuclidesand toxic organic compounds.

.Following the data gathering process, the authors prioritized the ER waste units inferms"6f their potential for remediation by and compatibility with the heavymetal/radionuclide bioremediation process being developed. Since the studies wereplanned to be conducted in a non-radiation control area, the initial selection processwas restricted to RFI/RI sites with non-radioactive, metal-containing waste waters thatappear to require remediation and can be readily sampled by the researchers. Theonly SRS wastewaters fulfilling these criteria were the coal pile runoff basins and theirassociated contaminated groundwaters.

Although only non-radioactive sites were selected for the initiation of laboratory studies,it should be emphasized that radioactive metal-containing wastewaters may be moreamenable to the bioremediation process being developed since it is expected that theprocess will be equally or more efficient at sequestering some radionuclides than non-radioactive heavy metals. Thus, the logistics of conducting the initial laboratory workwas the principal criterion used in selecting the sites listed above and described in moredetail below.

3.2.1 Coal Pile Runoff Basins (CPRBs)It was concluded that the coal pile run-off basins were the best available ER waste unitsfor the initial testing of a bioremoval process because they are non-radioactive,contaminated with a variety of heavy metals, readily available for sample collection(especially the surface waters) and believed to be in need of future remediation.

There are seven CPRBs at SRS located in A,C,D,F,H,K, and P-Areas. They providereceptacles for runoff from rainfall on coal piles located at these seven sites. The coalwas used to fuel facilities producing steam and electricity for SRS. The facilities at A-and D-Areas are currently active, while the facilities in the other five areas have beenshut down. Coal piles in C- and F-Areas were removed in 1985. Currently, rainwaterrunoff from the remaining coal piles (A,D,H,K, and P) flows into the CPRBs via gravityflow through ditches and sewers. The coal is generally of low sulfur content (1-2%).Chemical and biological oxidation results in water that has a very low pH (due to theformation of sulfuric acid) and high concentrations of dissolved heavy metals.Contaminants leaching into the coal pile runoff basins during rainfall eventuallycontaminate underlying soil and groundwater. Principal toxic metal contaminants ofconcern include Al, As, Be, Cd, Cr, Cu, Hg, Ni, Pb, and Se. All of these metals havebeen measured at levels above drinking water standards in samples collected from thebasins. Appendix Table A.3.5 shows levels of metal contaminants in the basins from

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three studies, including a recent one made by the authors (Wilde et. al., 1994,unpublished data). Maximum levels ranged from 107-11300% of the drinking waterstandards. Drinking water standards were exceeded by the largest margins for Al, Cd,Ni, Be, and Pb. The D-Area CPRB typically had the highest levels of metalcontaminants and was thus our first choice as a source of wastewater for initialexperimental work.

J3.2.2 TNX Burying GroundThe TNX Burying Ground is located within the fence that surrounds TNX near thewestern border. This waste site was created in 1953 when an experimental evaporatorcontaining 590 kg of uranyl nitrate exploded. Contaminated material included structuralsteel, tin, timber, drums, rags, and other items. The contaminated material was buriedin four trenches, 6-8 feet below land surface. The waste trenches were rediscovered in1980 during construction of buildings. Most of the contaminated material was removedin 1982 and 1983. However, an estimated 27 kg of uranyl nitrate along with othercontaminants remain under buildings or in locations where the use of excavationequipment was restricted. This site contains Pb and Hg above DWS (WSRC, 1993).Recent (1993) groundwater monitoring data also revealed high levels of Al. This sitealso has substantial contamination by toxic organic compounds. The TNX BuryingGrounds was considered a prime site for the metal bioremoval research programbecause of its proximity to and association with other bioremediation activities beingconducted by the ESS Biotechnology Group based within the TNX complex.

3.2.3 Road A Chemical BasinThe Road A Chemical Basin is located about 0.5 mile southwest of the intersection ofHighway 125 and SRS Road 6. This basin was 100f tx175f tx10f t deep. It reportedlyreceived miscellaneous radioactive and chemical aqueous waste for several years, butno records of the materials disposed of at the basin are available. The basin was closedand backfilled in 1973. It is currently part of the RFI/RI program. Recent data fromgroundwater monitoring wells below the basin reveal levels of Pb and Hg abovedrinking water standards. No other contaminants were observed above DWS during1992 (WSRC, 1993). Thus, the site was considered for obtaining water samples fromthe groundwater monitoring sampling program and for testing of metal removaltechniques in laboratories not set up for handling radionuclides or carcinogens.

3.2.4 Other SitesAdditional waste sites that appeared particularly well suited for bioremediation by theprocess being developed are listed below, followed by a brief description:

Burning Rubble PitsD-Area Ash basinsChemicals, Metals & Pesticide Pits (CMPs)Miscellaneous Chemicals Basin/Metals Burning PitRetention Basin in H-AreaSeepage Basins

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Silverton Road Waste SiteBurial Ground ComplexAcid/Caustic BasinsL-Area Oil & Chem. Basin

3.2.4.1 Burning Rubble Pits (BRPs)There are numerous BRPs with heavy metal contaminated underlying ground water atSRS. The BRPs are primarily unlined pits that have received combustible wastes whichwere allowed to accumulate and periodically burned . These pits have subsequentlybeen taken out of service, and backfilled with soil and sediments to grade level. Eightburning rubble pits were operated in A-, K-, P-, C-, L-, R-, and G-Areas for severalyears. Groundwater below these pits has been contaminated with heavy metals alongwith radionuclides and organics. Metals of concern include Pb, Cr, and Hg.Groundwater remediation is deemed necessary and remediation plans are beingdeveloped. Based on recent groundwater monitoring data (WSRC 1993), the BRPs inL-.N-.P-, and K-Areas have metal contaminated groundwater with no radioactivecontaminants. Thus, water from these sites should be suitable for experimentation in anon-radiation controlled laboratories.

3.2.4.2 D-Area Ash BasinThe 488-D basin is located in the southwestern part of D-Area. It began operation in1951 and was used to intercept, stabilize, and provide passive treatment of ash sluicewater prior to discharge to local surface streams. The basin ceased receiving sluicewater when two additional basins were constructed. The basin was subsequently usedfor placement of dry ash and coal crusher reject materials. Monitoring wells in theunderlying ground water consistently show heavy metals and toxic organics aboveregulatory limits in the groundwater below this basin.

3.2.4.3 Chemicals, Metals & Pesticide Pits (CMPs)The CMP Pits are located approximately one mile north of L-Area and one milenortheast of the 131-3L Rubble Disposal Area. This complex originally consisted ofseven unlined pits which were designed to receive non-radioactive wastes, such asspent solvents, pesticides and toxic metals. The pits were used from 1971 until 1979.In 1984, the pits were excavated and waste materials were removed. Then the areawas backfilled and capped with a geosynthetic material. Recent groundwatermonitoring has demonstrated significant contamination by heavy metals. Remediation isdeemed necessary and a formal remediation plan has not been developed.

3.2.4.4 Miscellaneous Chemicals Basin/Metals Burning PitThis waste unit actually comprised two separate facilities in close proximity. Both aresuspected to have polluted underlying ground waters. Contaminants of concern fromthe miscellaneous chemicals basin include Al (3483-7488 ppm), and Pb (2.65-10.5

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ppm). Contaminants of concern from the metals burning pit include Al (range 1430-95,570 ppm) (Wilde and Radway, 1994).

3.2.4.5 H-Area Retention BasinThe old H-Area Retention basin (281-3H) is located just south of Road E near theintersection of Road E and Road 4. This basin was used for temporary emergencystorage of cooling water containing radionuclides and trace quantities of otherchemicals from the chemical separations process. Groundwater monitoring data showAl, Pb, and Sb to be among the contaminants exceeding DWS.

3.2.4.6 Seepage BasinsSeveral seepage basins at SRS are considered waste sites and have potential forclean-up using bioremediation. These include the 716-A Motor Shop Seepage Basin,the D-Area Oil Seepage Basin, the new TNX Seepage Basin, the old F-and H-Areaseepage basins, the Ford Building seepage basin and seepage basins in all the reactorareas. Some of these basins still contain standing waters and all have underlyinggroundwater contaminated with metals and other pollutants, especially radionuclides.

3.2.4.7. Silverton Road Waste SiteThe Silverton Road Waste Site is located about 1.5 miles west-southwest of A/M Area.This unit consists of an approximately 700 ft. x 300 ft. x 7 ft deep area that existed asan open pit prior to construction of SRS. During and after construction of SRS, the pitand surrounding area was used for the disposal of construction debris such as metalshavings, drums, and storage tanks. Operations at this location ceased in 1974, andthe waste material is presently covered with soil and vegetation. Underlyinggroundwater contains several constituents exceeding DWS. These include Sb, Be, andPb.

3.2.4.8. Burial Ground Complex (BGC)The BGC occupies approximately 194 acres in the central part of SRS between the Fand H Separations Areas. It consists of several adjacent facilities which were formerly,or are currently disposal sites for hazardous and radioactive wastes and spent solventsgenerated from plant processes. Groundwater below the BGC is contaminated withnumerous toxic metals in addition to radionuclides and toxic organic compounds.

3.2.4.9 Acid/Caustic BasinsAcid/caustic basins are located in several areas (F,H,K,L,P, and R) of SRS. Thesebasins are unlined earthen pits, approximately 50 ft x 50 ft x 7 ft deep, that receiveddilute sulfuric acid and sodium hydroxide solutions used to regenerate ion-exchangeunits in water purification processes at the reactor and separations areas. Otherwastes discharged to the basins included water rinses from the ion-exchange units,

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steam condensate, and runoff from the spill containment enclosures in the storagetanks. The basins allowed mixing and neutralization of the dilute solutions before theirdischarge to nearby streams. All of the basins were constructed between 1952 and1954. They were taken out of service between 1964 and 1982. These basins are partof the RFI/RI program and closure, characterization and remediation plans-are invarious stages of development within the various areas. Basins in L- ands R- Areas arethe farthest along in this process. However, all of the basins are expected to requireremediation in the future.

3.2.4.10 L-Area Oil and Chemical BasinThe L-Area Oil and Chemical Basin is located in the southeastern portion of L-Area,just outside the L-Area perimeter fence. This 118 ft x 79 ft basin was put into operationin 1961 and continued to receive waste liquids until 1979. Contaminants of concerninclude Cd, Pb, Cr, and Hg, along with radionuclides and organics (Radway and Wilde,1994). Groundwater monitoring data revealed that concentrations of Cd and Pbexceeded DWS in groundwater below the basin.

3.3 WASTE STREAMS

In contrast to the case with waste sites at SRS, documents comprehensively describingwaste streams in various sectors of SRS could not be found. Thus, a slightly differentapproach was taken to identify and prioritize the waste streams in terms of theirsuitability for the bioremoval process. Key personnel throughout the site, such asenvironmental coordinators and site waste coordinators, were canvassed in an attemptto obtain information relevant to the selection process (Appendix Table A-3.8).

The SRS (Fig. 3.1) is subdivided into 18 principal areas. These are listed in AppendixTable A3.6, along with major activities previously and/or currently conducted at them.In compiling information about waste streams in these areas, we attempted todetermine:(1) the general nature of the waste-generating process, (2) presence ofradionuclides, (3) major metals present, (4) whether waste is currently generated, (5)volume stored and/or rate of generation, (6) availability of analytical data, (7) currentmethod of treatment or disposal, and (8) need for further treatment; problems withtreatment or disposal.

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TO WH.L5T0N6 mile s

Q j 10 «CLLISION' < miles

Figure 3.1. Map of SRS Showing Principal Site Areas

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Information gained during the investigation is catalogued (according to site area) inTable A.3.7. Only metal-containing aqueous wastes are included. Those areas listedin Table A3.6 but not in Table A.3. 7 proved not to contain wastes of interest within thescope of our study.

Three major criteria were used in selecting those waste streams most amenable tobioremediation process development. First, there must be a need (or an anticipatedneed^for present or future treatment of the waste. Second, streams withoutradioisotopes are best suited to process development, with those containing low levelcontamination being less suitable and those with high level contamination beingunsuited to experimental purposes. Third, the streams should be generated or storedin sufficient quantities to make the cleanup effort worthwhile.

In this manner, the streams catalogued in Table A.3.7 can rather quickly be reduced toa handful of candidates. Among these are the CIF (Consolidated Incineration Facility)blowdown water and a D Area lab waste containing Hg thiocyanate. These contain lowlevel radiation, requiring the use of an RCA or of simulated wastes. Likewise, it ispossible that algal biosorbents might provide a useful alternative or adjunct to thepresent ion exchange resin used to remove Hg from various lab wastes andWastewater Neutralization Facility wastes. Future candidates are sanitary wastewatertreatment facilities in which metals may eventually pose a sludge disposal problem.Bioremediation processes might also find application in the ongoing cleanup of reactordisassembly basins, but these wastes are not suitable for the initial development ofsuch processes due to their high radiation levels.

The waste streams fell into several major groups. These are briefly discussed below:

3.3.1. Photographic wastes.A number of on-site operations generate sizable amounts of spent fixer which cancontain up to 4500 ppm silver. These wastes are currently being treated by an ionexchange process at a silver recovery unit in A-Area and one in N-Area. The treatmentreduces Ag content to levels classified as non hazardous, allowing disposal via thesanitary sewer system.

3.3.2. Radioactive laboratory wastes (mixed wastes).These include wastes collected via low level (high and low activity) drains in SRTC labsand stored in tanks in A Area, along with lab waste water generated during testsassociated with the development of vitrification processes (S Area). The waste isperiodically treated with Duolite GT-73 resin to remove mercury introduced by acontaminated drain, and will eventually be sent to the tank farm when tanks are full.

Two wastes containing Hg and heavy water are generated in D Area. The WaterQuality Laboratory analyzes samples from reactor cores. Excess sample water (towhich standard mercury solution has sometimes been added) is currently processed bythe Heavy Water facility for recovery and recycling of its heavy water content. A second

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lab waste, containing Hg thiocyanate, heavy water, permanganate, and a variety oforganics, is no longer being generated. However, 100 drums (classified as hazardous)remain in storage at D Area, and 46 drums (classified as hazardous) are stored in NArea. These require cleanup before their heavy water can be recycled. The currenttreatment option consists of ion exchange using Duolite GT-73 resin, which reduces Hgcontent to acceptable levels. Resin performance is sometimes impaired due toclogging of the columns, and the system is currently undergoing modification^toOvercqme this problem.

3.3.3. Non radioactive laboratory waste.These include wastes generated by the M-Area Analytical and Metallurgy Laboratoriesand containing a variety of metals. They are currently treated at the Dilute EffluentTreatment Facility (DETF) prior to discharge. The treatment involves precipitation ofmetals followed by pressure filtration and is considered adequate. The laboratories areexpected to move to an undecided location in the near future. Acidic wastes containingCr and other metals are also generated by the A-Area Metallurgy Lab. They arecurrently being neutralized and stored pending the establishment of a Cr precipitationprocedure which will allow their discharge via the sanitary sewer system.

3.3.4. Reactor wastes.Although no reactors are operating at the present time, disassembly basins at C, K, L,P, and R Reactors contain water and sludge contaminated with radionuclides andmetals (e.g. Cs, Pu, Al, Fe). In K, L and P disassembly basins, a mixed bed ionexchange resin is used intermittently to reduce cation and anion levels in standingwater. This is not done at C and R basins because little or no fuel is present andhence contamination levels are much lower. Resin is regenerated by a RBOF, (reactorbasin for offsite fuel), facility in H Area and the eluted contaminants sent to a waste tankfor storage. Spent resin is also stored pending the selection of a disposal method. Anupgrade of the deionizing system is planned and may involve reverse osmosis carriedout by an outside vendor. Sludge is periodically vacuumed from disassembly basinsand is currently being stored in the absence of a disposal method.

3.3.5. Separations wastes.High level radioactive wastes, containing Hg, Al, and various fission products, aregenerated (albeit currently at about 20% of previous levels) as a result of separationsprocesses in F and H areas. During periods of ongoing separations activities, muchlarger amounts containing a variety of radionuclides and metals would be produced.Separations wastes undergo a series of treatments at the tank farms (H Area),ultimately leading to the discharge of purified water and shipment of contaminatedsolids and liquids to S and Z Areas respectively for immobilization and permanentstorage.

3.3.6. Waste processing facilitiesA large number of on-site facilities deal with the processing of wastes, and maythemselves generate effluents which must be treated or disposed of. During full

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operation, high level radioactive and mixed waste streams will be processed at the tankfarms (F/H Areas) before being sent to S-Area or Z-Area for further processing intoimmobilized forms, while some liquid waste streams generated by the latter processesreturn to H Area for additional processing.

A Consolidated Incineration Facility (CIF) is slated for completion in 1996. It isexpected to produce about 75,000 gal/yr of blowdown water, containing Pb, Hg, 9C)Sr,i?7Cs,.and possibly other metals. Evaluation of a treatment scheme, involving pHadjustment and coprecipitation of metals with iron followed by sulfide treatment toremove Hg, indicated that Cs and Sr were not affected by the treatment, and that theuse of sulfide presented a disposal problem (Wilde and Radway, 1994). Current plansare to solidify the blowdown waste in the same manner as solid CIF wastes in order tominimize permitting requirements and avoid delays in startup of the facility. However,a cost-effective means of reducing the volume of blowdown waste requiringsolidification and permanent storage would be of value.

Domestic waste water, which sometimes contains significant amounts of Pb, Zn, Cu,and/or Al, is processed by a system of sanitary waste water treatment plants located invarious areas. The treated liquid meets water quality standards, but it has been notedthat the sludge sometimes contains metals at levels approaching allowable limits forland application. It is conceivable that additional treatment might be needed if metallevels should rise or regulatory limits should change. Several waste water treatmentunits are expected to close soon because of the transition to a centralized facility, butthe treatment method (and hence the sludge composition) is not expected to change.

3.3.7. Miscellaneous WastesOther wastes, such as those from the F Area cooling maintenance shop and cleanupactivities in N Area, are generated as a result of miscellaneous activities. Most aregenerated infrequently, in small volumes, or one time only, and hence are notconsidered in detail here.

3.4 Selection Of Wastewaters for Study in the TTPThe purpose of this endeavor was to identify potential SRS waste waters that areamenable to clean-up by a novel bioremediation process being developed at SRS, andto select three sites for experimentation while the process is in the development stage.The three site selected for study in the short term were the CPRBs, the TNX BuryingGrounds and the Road A Chemical Basin. These sites all have significant heavy metalcontamination, they are not radioactive, and samples are readily obtainable.Furthermore, the CPRBs have standing water and all three waste water sources havemonitoring wells where contaminated ground water samples can be obtained. All ofthese sites are in need of remediation and no formal remediation plan is available.

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4.0 SELECTION AND CULTURING OF ALGAL STRAINS AT THE UNIVERSITY OFSOUTH CAROLINA

4.1 Introduction

Various microalgae have been shown to have potential for the bioremoval of pollutantssuch as toxic heavy metals from contaminated water sources (e.g. Wilde and."Benemann, 1993). However, only a few of the tens of thousands of existing algalspecies and varieties have been tested for their effectiveness and specificity. Based onearlier work by SRTC (unpublished data), several algal strains were shown to be veryeffective at removing selected metal ions from simulated wastewaters. These strainsincluded a strain of Mastigocladus laminosus, isolated from a reservoir receivingthermal effluent from a nuclear reactor, as well as strains of Cyanidium caldarium andChlorella pyrenoidosa. The main objective of the work described in this section of thereport was to obtain additional strains to evaluate in process development experiments.To this end, stock cultures of a variety of promising microalgal species representingseveral algal divisions were obtained to determine their growth characteristics in smalland large volumes of culture media. The selection of the strains was based on thefollowing criteria:

1. Absence of toxin production.

2. Ease of maintenance in a unialgal state.

3. Ability to grow in extreme environments (e.g. extremes of salinity,pH, or temperature).

4. Rapid growth in defined media.

5. Ability to achieve a high density in stationary phase.

6. Ease of harvesting (e.g. by means of filtration, phototropism, and/orflocculation).

Each strain was maintained at the University of South Carolina Algal Culture Collectionand inocula of species showing promise were made available to scientists at SRS forfurther testing and in developing innovative approaches for maximizing bioremediation.

4.2 Materials and Methods

Eighteen freshwater and marine unialgal cultures obtained from various sources weretransferred and maintained in 125 ml Erienmeyer flasks containing approximately 75 mlof appropriate autoclaved liquid media (Table 4.1). Cultures were kept in environmental

17

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Table 4.1 Algal species used, their sources and culture media.

Strain. No.Algal species

Chlorella capsulataC. fusccivar.-yacuoiataC. stigmatophoraDunaliella salinaD. tertiolectaScenedesmus acutiformisS. quadricaudaCricosphaera carteraeIsochrysis galbanaAmphiprora paludosa

Chaetoceros gracilis

Navicula pelliculosa

PhaeodactylumtricornutumSurirella ovata

Peridinium trochoideumRhodomonas sp.Cyanidium caldarumPorphyridium cruentum

Algal division

ChlorophytaI I

i i

I I

t i

t i

11

Chrysophytai t

Bacillariophyta

11

I I

n

n

PyrrophytaCryptophytaRhodophyta

i t

Source

UTEXUTEXUTEXUTEXUTEXUTEXUTEXUTEXUTEX

Groningen

UTEX

Bigelow

UTEX

Groningen

BigelowUTEXUTEXUTEX

20742519931644999416614

2167

2375

642

24192393161

:, Media

. F/2Bristol's + Proteose

F/2F/2F/2

Bristol'sBristols

F/2F/2

F/2 +SilicaF/2 +SilicaF/2 +SilicaF/2

F/2 +SilicaF/2F/2

CyanidiumF/2

18

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chambers maintained at 20 °C and a 12 hr light: 12 hr dark photoperiod with cool whitefluorescent lighting at a photon fluence rate of approximately 100 uE m~2 sec' ' .Culture media are shown in Tables 4.1 and 4.2. Enriched seawater media such as F/2(Guillard and Ryther, 1962) was prepared using 30 %o seawater collected from NorthInlet Estuary, SC and filtered through Gelman GF/F glass fiber filters. Mediapreparation instructions and general maintenance procedures were taken from Starrand Zeikus (1993). Growth experiments were performed by transferring 5 ml pi stock"cultures to 75 ml of fresh media in 125 Erlenmeyer flasks and placing them intoenvironmental chambers under the above temperature and light regime. Allexperiments were done in triplicate.

Additional experiments were run on some species to determine the effects of culturevolume, salinity, pH, and growth media on their growth rates and maximum yields.Large volume experiments were conducted by inoculating Chlorella capsulata andPhaeodactylum tricornutum into 8 L of autoclaved,, F/2 media (30 %o salinity) containedin 10 L polycarbonate carboys kept at 25° C with aeration and constant cool-whitefluorescent lighting at approximately 70 uE m~2 ^

To determine the effect of salinity on growth rate, cells of Navicula pelliculosa wereinoculated into triplicate sets of 125 ml flasks containing quartz-glass-distilled water to7.5, 15, 30, or 37 %o salinity and enriched with F/2 + silica stocks (Guillard and Ryther,1962). Cultures were maintained at 25 °C and constant cool white fluorescent lightingat approximately 100 uE m~2 sec~1.

The effect of pH and medium type on growth rate was tested simultaneously on N.pelliculosa, inoculated into either F/2 seawater media + silica, or F/2 + silica mediamade up using 'Instant Ocean©1 salts in distilled water, both at 30 %o and adjusted to apH of either 6.5 using HCL or to 8.2 with NaOH and buffered with Tris©'.

Population densities were determined by counting cells daily or other frequent intervalsusing a Model ZM Coulter Counter® until cultures had reached stationary phase(maximum yield). Maximum growth rates were calculated from cultures in log phase asdoublings per day (U2) using the formula:

in which NQ and N are cell concentrations at the initial stage and after the time period trespectively.

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Table 4.2 Culture media used and their origins

MEDIUMASP-2

ft..

Bristol'sBristol's plus proteose

CyanidiumF/2

F/2 with silica

TYPE OF WATERArtificialSeawaterFreshwaterFreshwater

FreshwaterMarine

Marine

SOURCEProvasoJiet al (1957)Bold (1949)Starr and Zeikus(1993)Schlosser(1982)Guillard and Ryther(1962)Guillard and Ryther(1962)

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4.3 Results

4.3.1 Initial Growth ExperimentAlthough each strain of microalgae was successfully transferred and grown in batchcultures, there was substantial variation in the growth rates and maximum'densitiesamong the species in the media and the growth conditions selected. Growth rates inthe initial growth rate experiments varied from a low of 0.28 doublings d-1 in Phlorella

"iuscayar, vacuolata to a high of 3.85 doublings d~1 in Dunaliella tertiolecta. Maximumdensities also varied greatly from 5.00 x 104 cells mM in Pehdinium trochoideum to3.33 x 10^ cells mM in Navicula pelliculosa. A summary of the results of the initialgrowth rate experiments can be seen in Table 4.3 and in Appendix Figures A.4.1-A.4.8.

4.3.2 Large Volume ExperimentBoth C. capsulata and Phaeodactylum tricornutum reached maximum densities earlierthan in the initial growth experiments, though their doubling rates and final densitieswere somewhat lower than those reached in the initial growth experiments (AppendixFigs A.4.9-A.4.10, Table 4.3). Maximum growth rates (U2) for the two species were3.24 and 1.60 doublings d~1. Cells remained suspended in the media during the growthcycle until about day 10 when they began to settle to the bottom. Aeration preventedsettling but did not result in increases in cell density of either species.

4.3.3 Salinity ExperimentCells of N. pelliculosa grew in all of the experimental salinities. Cells inoculated into thelower salinities (7.5 and 15 %o) grew at higher rates than those in the higher salinities(30 and 37.5 %o). Maximum densities were reached in 8 days at all salinities, with thehighest density obtained at 7.5 %o, approximately 3 time higher than that of cells grownat 30 %o in this and the initial growth experiments (Appendix Fig. A.4.11).

4.3.4 Growth Medium and pH ExperimentCells of N. pelliculosa grew in both growth media and pH levels. Cells grown at a pH of8.2 grew similarly in F/2 and 'Instant Ocean1 media, expressing virtually identical growthrates and maximum densities in five days. However, cells grown at a pH of 6.5 inNnstant Ocean1 grew at a lower rate and reached a maximum density of only about athird that of the other cultures (Appendix Figs. A.4.12-A.4.13).

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Table 4.3. Summary of initial growth rate experiments.

SPECIES

Chlorella capsulataC. fusca var.vacuolataC. stigmatophoraDunaliella salinaD. tertiolectaScenedesmusacutiformisS. quadricaudaCricosphaeracarteraeIsochrysis galbanaAmphiprnrapaludosaChaetocerosgracilisNavicula pelliculosaPhaeodactylumtricornutumSurirella ovataPeridiniumtrochoideumRhodomonas sp.CyanidiumcaldarumPorphyridiumcruentum

MAXIMUMGROWTHRATE(Doublingsd-1)1.970.28

0.413.113.852.87

3.132.89

3.170.91

0.38

3.131.23

1.020.72

2.771.11

2.67

MAXIMUMDENSITY(Cells ml-1)

6.33x1061.41 x106

3.53 x10 &

3.76x1067.61 x 1066.65x106

9.49x1064.56x106

5.53x1068.37 x104

8.41 x 10t>

3.33 x10/5.43x106

9.51 x 104

5.00x104

5.73x1067.65x100

5.33x106

GROWTHPROFILES

Fig.A.4.1Fig. A.4.2

Fig. A.4.3

Fig. A.4.4

Fig. A.4.5Fig. A.4.6

Fig. A.4.7Fig. A.4.8

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4.4 Discussion And Recommendations

Several algal culture collections have been developed for various objectives, such as arepository for teaching materials (e.g. Carolina Biological Supply Co.) or general basicresearch organisms (e.g. Starr and Zeikus, 1993), as well as for specific'purposes suchas those species selected and maintained for their ability to produce and store lipids forpossible biofuel production (Barklay et al, 1986). The present small collection (Table4,1) has been assembled to provide the basis of readily available cultures of speciesthat may have desirable properties for successful bioremoval programs and that couldbe propagated and used in future screening studies.

Algal media and growth conditions have been developed which allow reasonablyadequate growth of a large number of species. However, each species of algae is agenetic entity each with its unique optimum growth requirements. Thus, in the initialgrowth rate experiments, maximum rates and maximum yields expectedly varied greatlyamong the 18 species (Table 4.3). These results should not be taken as absolutevalues, as further experimentation might show other outcomes if adjustments weremade in media formulation, culture volume, and/or light and temperature regimes . Thiscan be seen when comparing the growth characteristics of Chlorella capsulata andPhaeodactylum tricornutum in the initial growth experiments with those in the largevolume experiments. In large volume cultures (constant light) both species reachedmaximum density in less than half the time it took in smaller volumes (12 hr L:12 hr Dphotoperiod), but the yields were substantially higher in the smaller volumes (CompareAppendix Figs. A.4.1 and A.4.6 with A.4.9 and A.4.10). Without further experimentationit is not possible to determine whether it was the volume, the photoperiod, or both thatresulted in the observed differences in growth characteristics.

Several of the species had high growth rates and high yields [e.g. C. capsulata,Dunaliella salina, D. tertiolecta, Scenedesmus acutiformis, S. quadricauda,Cricosphaera carterae, Isochrysis galbana, Navicula pelliculosa, P. tricornutum,Rhodomonas sp., and Porphyridium cruentum (Table 4.3)] and appear to be suitablecandidate species for further testing.

Two of the species, Chlorella fusca var. vacuolata and P. cruentum, producedobservable amounts of what looked like mucus. The former species is known toproduce mucilaginous phytochelation complexes that are effective in sequesteringheavy metals (Gekeler et al., 1988). These two species in particular should beexamined further to determine the optimum culture conditions for maximizing growthand the synthesis of chelators.

It is obvious that microalgae grown in large volumes would more efficiently producebiomass than when grown in smaller volumes. Numerous species are already routinelygrown in large volumes to produce food for humans and hogs (e.g. Spirulina), food forrearing larval aquatic and marine invertebrates and fish (e.g. Chlorella, Cyclotella,Isochrysis, Nitzschia, Prymnesium, and Thalassiosira), and Pharmaceuticals (e.g.

23

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Dunaliella). It would be of value to optimize large scale cultivation of algal species thatshow promise at removing unwanted chemicals from water sources

Salinity has been shown to influence (sometimes dramatically) algal growth (e.g.Prirrgsheim, 1964). Navicula pelliculosa grew at all salinities tested, but grew best atthe lower portion of the range (Appendix Fig. A.4.11). This species was originallydescribed from freshwater (Joyce Lewin, pers. commun.), although the strairtused in"our experiments was isolated from a brackish pond (Robert Guillard, pers. commun.).

The composition of the culture medium provides adequate and balanced amounts ofthe essential macro- and micronutrients required for growth, and pH controls in part theavailability of many nutrients (particularly CO2 and heavy metals) to the algal cells.Thus, both the composition of the culture medium and the pH ought to profoundly affectgrowth of microalgae. In the case of N. pelliculosa, the choice of media had a greatereffect on growth than did pH (Appendix Figs. A.4.12-A.4.13). There are also otherindications that some media allow better growth than others. For example, ASP-2media, an artificial seawater medium commonly used to grow a variety of marine algaewas not a good choice for growing Amphiprora paludosa, Chaetoceros gracilis, Surirellaovata or Peridinium trochoideum, as their growth rates and yields were among thelowest of all the species tested (Table 4.3). Time did not allow us to grow these speciesin other media.

24

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5.0 CHARACTERISTICS OF THE FOAM USED FOR EMBEDDING

5.1 Introduction

Studying the physical and chemical characteristics of the biomass-embedded foam wasof interest for two reasons. First, it was important to know how biomass/foamaggregates would hold up when exposed to harsh conditions such as might be

^experienced during wastewater treatment. Secondly, we wanted to know whatchemical treatment would be necessary should we want to dissolve the spent foamfollowing bioremediation treatment for purposes of metal reclamation and/or wastedisposal.

5.2 Methods

The chemical integrity of the foam filter media was investigated by exposing it toconcentrated acid, base, and oxidizing solutions. Three experiments were conductedon the plain foam and foam impregnated with three different algae types(Cyanidium,Phaeodactylum, and Chlorella). No heat was provided to enhance the reactions. Thefirst experiment involved exposing the foam to a variety of acids, bases and oxidizingagents. In the second experiment, plain foam (no biomass) was exposed to 1:1mixtures of acid or base with hydrogen peroxide. The third experiment involvedexposing the three algae impregnated foams to a 1:1 concentrated mixture of H2SO4and H2O2.

5.3 Results

Results of the experiments are shown in Tables 5.1a, 5.1b, and 5.1c. Table 5.1ashows the response of the foam and foam/biomass to the concentrated solutions.Concentrated sulfuric acid (94% H2SO4) dissolved the foam/biomass completely, butnot immediately Sodium hypochlorite (5.6% NaOCI) was also a formidable dissolutionagent for the foam. The mixture of 94% H2SO4 and 30% H2O2 (hydrogen peroxide)dissolved the foam in <5 minutes (Table 5.1b). The reaction generated heat, enhancingthe dissolution of the foam. All foams dissolved in <5 minutes when exposed to a 1:1concentrated mixture of H2SO4 and H2O2 (Table 5.1c). What seemed like sandparticles remained in the solution containing Phaeodactylum and Chlorella.

5.4 Discussion

It appears that the foam is very resistant to degradation when exposed to hydrogenperoxide or various basic solutions. The foam can be dissolved when subjected tostrong acids or hypochlorite solutions. The most effective chemical treatment fordissolving the foam was a combination of H2SO4 and H2O2 .

25

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Table 5.1a Foam and Algae Impregnated Foam Response

Chemical30% H2O250% NaOH

28% NH4OH. 69% HNO394%H2SO4

38% HCL85% H3PO45.6% NaOCI

Cyanidium+1--

+2---

+4

Phaeodactylum+1--

+2+2+2+2+4

Chlorella---

+2+3+2+2+4

Plain Foam- •

-

+ 2 •*.

+3+2+2+4

(+) indicating some response. (-) indicating no response.

1. Some initial fizzling and bubbling upon addition of H2O2. No apparentdegradation of Cyanidium algae-foam after two weeks of observation.

2. Initial degradation activity observed. After two weeks dissolution stillincomplete.

3. Initial degradation activity observed. Foam/biomass completely dissolvedafter two week.

4. Foam/biomass begins to dissolve immediately. Solution becomes clearovernight. A fine white precipitate remains at the bottom of the container aftertwo weeks.

Table 5.1b Response of Plain Foam to Mixture of HNO3, H2SO4, NaOH with H2O2

Plain FoamHNO3 + H2O2

+1H2SO4 + H2O2

+2NaOH + H2O2

-

1. Initial degradation activity observed. Most of foam dissolved overnight, only afew particles remain.

2. Complete dissolution in <5 minutes.

Table 5.1c Response of Algae Impregnated Foam to H2SO4 + H2O2

H2SO4 + H2O2Cyanidium

+1Phaeodactylum

+2Chlorella

+2

1. Dissolved in <5 minutes.2. Dissolved in <5 minutes. A few sand-like particles remain in the bottom of the

container.

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6.0 BIOREMOVAL CAPABILITIES OF FOAM-EMBEDDED MICROBES

6.1 Introduction

It has been suggested by numerous authors (e.g. Wilde and Benemann; 1993; Gadd,1990; Volesky, 1990; Macaskie, 1991) that there is considerable potential fordevelopment of a cost-effective and otherwise superior processes for sequestering toxicrnetal^from wastewaters by the use of microorganisms, a process known as"bioremoval". Therefore, numerous experiments were conducted in attempt to developsuch a process, at least on a bench scale. A process for metal and/or radionuclideremoval using a novel biomass immobilization scheme, where the microbes areembedded in a hydrophilic foam, was a principal focus of the work of this TTP in FY1994 and most of FY 1995. Despite an expansion of the scope of the project to includethe biodegradation of toxic organic compounds (see Sections 7 and 8), processdevelopment for the bioremoval of metals and radionuclides remained a principalobjective throughout the study until it was concluded at the end of March 1996.

6.2 Methods and Materials

This research involved an attempt to develop a novel process and thus involved a largenumber of unknowns. Initial attempts to develop a highly detailed experimental plan forthe entire project (TTP) in an a priori manner proved to be of limited value. We found itnecessary to make continuing modifications to the research plan and develop detailedstep-by-step procedures experiment by experiment. Principal factors that went into thedesign of a given experiment included the results of previous experiments, the numberof algae/foam samples to be prepared and the number of samples to be subjected tochemical analysis. These later two factors required consideration because they eachrepresented specific costs that were built into the budgeted costs of work scheduled(BCWS) for the TTP and could not be exceeded without additional funding. A total of14 bioremoval experiments were conducted during the study. These experiments andtheir results are described in detail in Appendix 6. An overview of the experimentalprocedures is provided here.

6.2.1 Biomass TypesTable 6.1 lists biomass types and sources as well as the experiments in which theywere used. The blue-green algal strains Mastigocladus and Nostoc were grown in NDMedia (Castenholz 1982). The green alga Chlorella was grown in Bold Basal Medium(Nichols and Bold 1965). The red alga Cyanidium was grown in Doemel's Cyanidiummedium (Carolina Biological Supply, 1978), and the diatom Phaeodactylum was grownin F/2 medium (Guillard and Ryther, 1962). The bacteria , Burkholderia andPseudomonas, as well as the two yeast strains, #14 and #42, were all grown inPseudomonas medium (Atlas 1993). Antifoam A was added as required to minimizefoam formation by bacteria and yeasts. All algal species were grown in aerated, 4-I

27

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Table 6.1 Microbes used in Metal Removal Studies

Microbial StrainAlgae

Mastigocladus laminosus #113Phaeodactylum tricornutumCyanidium caldarium

Chlorella pyrenoidosaNostoc sp.

BacteriaPseudomonas aeruginosaBurkholderia cepacia G-4

FungiYeast strain #R-14

Yeast strain #R-42

Higher PlantsAzolla Sp.Datura Sp.

Source

Isolated from SRSUniv. SC (see Sect. 4)Carolina BiologicalSupply Co.same as abovesame as above

ORNL (Dr. Faison)Univ. W. Florida (Dr.Shields)

Georgia State Univ.(Dr. Crow)same as above

Frisby Technologiessame as above

Experiments

2,4,5,9,10;11,12,13,143,5,8,97,8,9,10,11,12,13,14.

8,9,10,11,1213,14

13,1413,14

13,14

13,14

13,1413,14

28

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bottles with 1% CO2 enhancement, under continuous illumination of 300 jj.E/sec/m2.Bacteria and yeast were grown similarly but without the lighting and CO2. Thethermophiles, Mastigocladus and Cyanidium, were grown at 45°C. All other microbialstrains were grown near room temperature (20-25°C). All strains were harvested byceritrifugation after they had reached stationary phase growth.

6.2.2 imbedding ProceduresFollowing harvesting from the culturing systems described above, the organisms werewashed in deionized water (and/or other chemicals in the case of Experiment #11),and resuspended to a slurry concentration of approximately 10% dry weight ofbiomass per slurry. The slurry was analyzed with a moisture meter for solidsconcentration. Then, biomass slurry, surfactant, prepolymer and Dl water were mixedin amounts calculated to obtain 10% biomass, by dry weight, in the completed foamproduct. Foam production was done at room temperature and the foam was groundand sieved prior to being used in metal removal experiments.

6.2.3 Laboratory Testing ApparatiiMost of the experiments (Exps.# 1,2,3,4,9,10,11,12,&14) were conducted usingbioreactor systems developed by Frisby Technologies (see Appendix 6B). However, afew experiment were conducted using shake flasks and/or Bio-rad columns in anattempt to elucidate the cause of poor results in some of the bioreactor experiments(Exps. # 5,6,7,8) or to evaluate radionuclide uptake(Exp.# 13).

6.2.4 Chemical analyses proceduresWith the exception of Experiment 13, where Tc-99 concentrations were determinedusing a Packard-Tri-Carb 20-50A liquid scintillation spectrometer, concentrations of Fe,Al, Cr, Ni, and in some cases other toxic metals, were detected by inductively coupledplasma spectroscopy. Either ICP-ES or ICP-MS analyses were made Depending onthe concentration of metal in the wastewater and the desired level of detectionnecessary to determine if significant bioremoval was occurring . For example, Fe andAl were analyzed by ICP-ES, while Cr and Ni were analyzed by ICP-MS.

6.3 Results

Despite the inherent positive factors in the conceptual design (e.g. we had shown inearlier unpublished work that algal biomass can typically remove >90% of toxic metalfrom mock waste solutions spiked with metal standards), our success in providing abench scale demonstration was very limited. A brief summary of the results from all ofthe experiments appears in Table 6.2. Details of the 14 experiments, listed in

29

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Table 6.2 Summary of Metal Removal Experiments

EXP.No.

1b

2

3

4

5

6

7

TYPE OFEXPERIMENTUse of Frisbylaboratory test rigwith D-Area coalpile run-off basinwater

same as above

same as above

same as above

same as above

Incubation ofalgae/foam inshake flask withD-CPRB waterPretreatment of D-CPRB

Mini-columns

SUBJECT

startup

particle size (noalgae)

particle size w/algal species #1

particle size w/algal species #2

effect ofalgae/foamquantity

effect ofalgae/foambioremoval inshake flasksTesting effect ofpH adjustmentsand aeration onmetal content ofwaste water

Bioremoval ofpretreated wastewater and metalstandards: Test#1

OBJECTIVE

Evaluate new lab test rig withwastewater but no filter media toevaluate the integrity of the test systemin terms of: (1) leaching of metals fromtest system, (2) plating of metals on testsystem, and (3) uniformity of columnsEvaluate effect of foam particle size onmetal uptake and flow dynamics (noalgae)Evaluate the effect of particle size onmetal uptake and flow dynamics withan algal - foam aggregate containing6% MastigocladusEvaluate the effect of particle size onmetal uptake and flow dynamics withan algal - foam aggregate containing6% PhaeodactylumDetermine effect of the quantity ofalgae/foam (0,2,8,12 &16 g/column offoam embedded with 6%Mastigocladus by dry wt.) onbioremoval efficiencyDetermine bioremoval of foam plugsimbedded with Mastigocladus andPhaeodactylum in shake flask 1g foamper 50 ml D-CPRB waterEvaluate the change in metalconcentrations of D-CPRB waterfollowing pretreatments includingaeration and pH adjustments to 3,4,5,&6 from ambient pH of 2.5

Evaluate the bioremoval efficiency offoam containing algae (6% Cyanidium)or no algae with foam granulated (8mesh) or not granulated (plug) with D-CPRB water adjusted for pH 5 orambient (pH 2.5) and with metalstandard solutions containing 0.8 ppmNi and 1.5 ppm Zn adjusted to pH 2.5and 5.0

Results

No significantleaching or plating.Good uniformityamong columns

No significantdifferencesdetectedNo significantmetal removal withany particle size

No significantmetal removal withany particle size

No significantmetal removal byany amount tested

No significantmetal removal

Aeration wasinsignificant.Concentrations ofmetals greatlyreduced with pHincreases. Somemetal still aboveregulatory limits atpH6.No significantremoval at eitherpH from runoff.Enhanced Ni andZn removal fromtest solutions atpH 5. Solid foamless effective thangranular foam.

30

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Table 6.2. Cont.

EXP.No.8 n

9

10

TYPE OFEXPERIMENTMini-columns

Modified test rig

Modified test rig

SUBJECT

Bioremoval ofpretreated wastewater and metalstandards: Test#2

Bioremoval ofcontaminatedground water byvarious algalstrains

Bioremoval ofcontaminatedground water byvarious algalstrains

OBJECTIVE

Determine reasons for the poor -. -performance of foam-algal aggregateswith coal runoff compared to standardmetal solutions in previous experiment.Compare pretreated D-CPRB waterand a mixture of metal standardscontaining Al, Be, Cd, Co, Cr, Cu, Fe,Ni, and Zn at levels similar to that ofthe runoff. Also test single-metalsolutions of Ni and Zn and watercollected from a monitoring well belowaCMPpit(CMP-13). Use granulatedalgae/foam aggregates and modifymini-columns for reduced flow rate toincrease contact time. Algae werePhaeodactylum and Chlorella.Test metal removal efficiency bypassing metal contaminated groundwater through columns containingfoam-algae aggregates comprised offour different algal strains (Cyanidium,Mastigocladus, Phaeodactylum andChlorella). Measure Ni, Al, Cr, and Febefore and after treatment.

Metal Removal of water from Well DC-4A using Cyanidium, Mastigocladus,and Chlorella in the modified Frisbytest bed. (Repeat of Exp. #9, exceptPhaeodactylum deleted). Attempt toverify removal indicated in Exp. 9

Results

Phaeodactylumand Chlorellaremoved moretnetal from pH 5runoff and Ni, Znsolutions than didCyanidium, but Niand Zn remainedabove waterquality criteria.More removalfrom mixed metalsthan runoff, butless than fromsingle metalsolutionsSignificantremoval of Al andFe by all algae.Also significantremoval of Cr byChlorella andCyanidium. No Niremoval by anyalga.No significantremoval with anyof the algal/foamaggregates,relative tocontrols.

31

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Table 6.2 Cont.

EXPNO.11ft

12

13

14

TYPE OFEXPERIMENTModified packedbed bioreactor

Modified packedbed bioreactorand static mixerbioreactor

Mini-columns

Modified packedbed bioreactor

SUBJECT

Bioremoval of metal -contaminated groundwater by various algalstrains subjected tovarious pretreatments

Bioremoval ofcontaminated groundwater by the use oftwo bioreactorconfigurations

Bioremoval of aradionuclide in spikedDl and river watersamples

Bioremoval ofcontaminated groundwater by the usevarious types of algaland non-algal biomass

OBJECTIVE

Metal Removal of water from WetlDC-4A using three types of algae(Cyanidium, Mastigocladus, andChlorella) and six biomasspretreatment schemes with themodified Frisby test bed.

Compare packed bed vs. static mixerbioreactors for removal of Cr, Ni, Fe,and Al by two algal species

Compare Tc-99 removal by algae (2spp), fungi (2 spp), bacteria (2 spp),higher plants (2 spp) and ionexchange resin

Compare Ni, Cr, Al, and Fe removalby algae (2 spp), fungi (2 spp),bacteria (2 spp), and higher plants (2spp).

RESULTS

Limited removalwith any algaeand pretreatmentcombination. Bestresults with acidpretreatedCyanidiumLeaching ofmetals from pumpof static mixer. Nosignificantremoval withpacked bedBest biomassresults withCyanidium, butfar inferior to resin

Best bioremovalwith algae andbacteria but lessthan 25% removalof any metal withany type ofbiomass

32

WSRC-TR-96-0088

numerical and chronological order can be found in Appendix 6 along with descriptionsof the test rigs provided by our industrial partner Frisby Technologies.

Metal removal experiments with algae provided inconsistent results with,metal removalefficiency seldom exceeding 25% removal under any test conditions. Studies comparingalgal, bacterial, fungal, and higher plant biomass demonstrated that other biomasssources were also ineffective for metal bioremoval under the test conditions,.Radionuclide bioremoval using a Tc-99 source provided more promising results thanthe metal removal studies with the various types of biomass, and indicated that the algaCyanidium (~35% removal) was the best of the tested sources of biomass for thisapplication. However, all of the biomass/foam aggregates tested were substantiallyinferior to a TEVA resin (>95% removal) for removing Tc-99 in comparative testing.

6.4 Discussion

Three potential reasons for our lack of success in converting the conceptually designedprocess into an effective bench-scale process for metal removal included the following:

(1) interference in biomass-metal binding in "real" wastewater relative to"simulated" wastewaters,

(2) inappropriate wastewater for testing, and(3) inadequate bioreactor systems

Numerous authors have described interference by one or more metals in the binding ofothers (Ting et a l , 1989; Ting et aL, 1991a,b; Harris and Ramelow, 1990. Non-metallicconstituents can also interfere with metal binding and it was not surprising that we wereable to obtain higher removal efficiencies using mixed metal standard solutions thanwith real wastewater solutions containing similar metal concentrations. In addition, weobserved higher metal removal for a given metal using single metal solutions than withmixed metal solutions with the same amount of the given metal. Thus, as is usually ifnot always the case, simulated wastewater proved easier to clean up than realwastewater.

Despite an intensive search at SRS (see Section 3), we found a paucity of wastewatersources having: (1) toxic heavy metal concentrations in need of remediation, and (2) alack of other contaminants that necessitate the use of special precautions for study (i.e.additional laboratory training and procedural controls). The coal pile runoff basins andtheir underlying groundwaters were the only waters that fit these two criteria.Unfortunately, these waters were far from ideal for biosorption due to very low pH(typically ca. 2.5) and extremely high iron and/or aluminum concentrations. Ironconcentrations ranged from 79 to 1780 ppm in the D-CPRB basin water samples, butwere < 1 ppm in the samples from monitoring well DCB-4A.. Al concentrations in theraw wastewater ranged from 64-470 ppm in basin water samples and from 10-64 ppmin the well water (DCB-4A) samples. The only other wastewater included in the metalremoval experiments was a sample from Monitoring Well CMP13, representing ground

33

WSRC-TR-96-0088

water below a former chemical, metals, and pesticide (CMP) disposal site. This water,while relatively low in Fe and Al, was also only slightly above the very low drinkingwater limits (see Appendix Table A.3.10 for a few toxic metals including Be, Cd, and Cr.Thus, experiments designed to detect substantial metal removal, relative.-to controls,were virtually impossible with this source.

Another problem that cropped up during the course of the study was inadequacy of ourbioreaptor systems. These systems were developed on a fixed-cost subcontract basis,which for the most part, precluded modifications to the systems after experimentationhad commenced. The first bioreactor (B.E.S.T.) (Appendix 6.B) proved to be sub-optimal for adequate contact between biomass and wastewater because the flow ratescould not be maintained below 0.3 gpm, which appeared to be excessively high forgood metal binding with the biomass. The size of the chambers holding the biomassalso proved problematic in that our algal production capability for filling a chamber wastaxed to provide sufficient biomass in a timely manner. Results improved somewhatwhen the bioreactor system was modified to accommodate lower flows and smallercolumns for housing the foam/biomass aggregates. However, it is suspected that thismodification resulted in a suboptimal ratio of biomass per unit wastewater. Themaximum amount of biomass that could be packed in a column was ca. 0.4g dryweight biomass and the minimum amount of wastewater necessary to maintain systemflow was about 1-liter. Thus, with this bioreactor system, we were not able to use thebiomass to wastewater ratio of 1.6 mg/ml used in the earlier work where high(>90%)removal percentages were obtained with solutions spiked with metal standards(unpublished data). A static mixer bioreactor provided confounding results when itbecame apparent that metals such as Cr, Fe, and Ni. were being leached into thesystem by metallic components of the bioreactor pumping system .

In conclusion, future research in a quest for a bioremoval process using biomassembedded in foam should focus on the selection of appropriate wastewaters whichwould preferably be of near neutral pH and have higher concentrations of the targetmetals (those especially desired to be removed) than non-target (e.g. relativelyinnocuous metals of little value) constituents. It will also be necessary to developbioreactor systems, containing all non-metallic components, that can maximize contactbetween the embedded biomass and the metal ions targeted for removal.

34

WSRC-TR-96-0088

7.0 POLYURETHANE FOAM IMMOBILIZATION OF TCE-DEGRADING BACTERIA:ENTRAPMENT EFFECTIVENESS AND INFLUENCE ON METABOLIC ACTIVITY

7.1 Introduction

The immobilization of bacteria in artificial matrices as a means of exploiting theirmetabolic activities is receiving increasing attention by the biotechnology industry. Oneapplication of this technology relates to the production of chemicals in continuoussystems or bioreactors. For example, immobilized fungal cells have been used for theproduction of cellulases and xylanases (Haapala et al., 1995), while immobilized yeastcells have been used to produce ethanol (Tanaka et al., 1986). The advantages of thisapproach over the classical fermentor/chemostat approach are the higher microbialdensities and greater biomass retention attainable in immobilized systems.

Immobilized degradative bacteria are also potentially useful in the ex situ treatment ofhazardous chemicals (Levinson et al., 1994). The transformation of phenol, p-nitrophenol, pentachlorophenol, p-cresol, and other compounds by immobilizedmicrobial cells has been documented (e.g. Bettmann and Rehm, 1984; O'Reilly andCrawford, 1989a). Embedding media can protect immobilized cells against chemicaltoxicity by absorbing toxic compounds. For example, Flavobacterium sp. wascapable of transforming pentachlorophenol (PCP) when immobilized in agarose beadsbut not as free cells (O'Reilly and Crawford, 1989b).

Entrapment or encapsulation appears particularly attractive in cases where thedegradative bacteria are poorly adhesive and hence are not suited for use inbioreactors. One such organism is Burkholderia cepacia (formerly Pseudomonascepacia) G4, which degrades trichloroethylene (TCE) and several other compounds.Due to its poor attachment to surfaces, G4 is generally washed out from bioreactorsystems. In one case, it was replaced by native microbes within days after a tricklingfilter reactor was opened to water from a contaminated aquifer (Berry, unpublishedresults). Partly because of this problem, G4 has not achieved widespread use for theex situ treatment of TCE contaminated groundwaters.

Numerous techniques for immobilizing bacteria have been evaluated, most of whichcan be classified as either entrapment or absorption methods (Woodward, 1988).Immobilizing agents include polyacrilamide beads, agarose beads, alginate beads,carrageenan beads, clay, granular activated carbon, and polyurethane foams (Levinsonet al., 1994). Although many factors will affect the success of an immobilizationtechnique in a biotechnological application, the immobilization or entrapment efficiencyand the material's effect on microbial activity are crucial. The ability to retain cells in theimmobilization medium during exposure to flowing water will undoubtedly affect theeffectiveness and functional lifetime of the material. In addition, the immobilizationtechnique should not impair metabolic activities required for the desired application.Examination of these factors is therefore important when evaluating immobilizationtechniques.

35

WSRC-TR-96-0088

In this study, we evaluated the use of a novel hydrophilic polyurethane based foam forthe immobilization of B. cepacia. Previous workers have noted difficulties in achievingsatisfactory entrapment of bacteria in polyurethane. Therefore, we first examined theefferets of foam formulation (surfactant type and concentration, embedding "temperature,presence of binding agents, and biomass density) on entrapment efficiency. Secondly,we explored the viability and physiological status of embedded cells, using colony"counts, the most probable number (MPN) technique, respirometry, and 16S rRNAtargeting oligonucleotide probes. Our objective was the identification of embeddingconditions which allow effective entrapment of a metabolically active bacterialpopulation.

7.2 Materials and Methods

7.2.1 Bacterial strains and growth conditions.The strain Pseudomonas (Burkholderia) cepacia G4 and the constitutive mutantPR131 were kindly provided by M. Shields (University of West Florida). To assureculture purity, original stocks were kept at -70°C. Prior to each experiment, stockmaterial was spotted onto PTYG plates (Appendix 7.1) to provide inoculum for batchcultures. Axenic batch cultures were grown in Pseudomonas medium (Atlas, 1993) ora yeast-glucose medium (YGM) (Shields and Reagin, 1992), which consisted of basalsalts medium (BSM)(Shields et al., 1989) plus 1 g/l glucose and 0.5 g/l yeast extract(Appendices 7.2, 7.3). Small-scale (100 - 250 ml) batch cultures were grown in shakeflasks (200 rpm, 30°C). Larger amounts of axenic biomass were grown by inoculating20 ml of 1- 3 day old culture into 4000 ml polycarbonate bottles containing 3000 mlYGM or Pseudomonas medium. These cultures were maintained at 26 ± 2°C andaerated through a sterile 0.2 (xm filter to provide mixing and 02. Cultures were routinelyharvested for foam embedding after 3 days' growth, at which time biomass yield wasabout 0.5 g dry weight / I . For experiments requiring the induction of the toluenemonooxygenase gene (Shields et al., 1989), G4 cells were exposed to 2 mM phenol for2h prior to harvesting. Harvesting was by centrifugation (10,000 rpm, 10 min, 15 -25°C). Pellets were resuspended in BSM or Pseudomonas medium to a density of 2.2-17.7% dry weight. Aseptic technique was not used during the harvesting process.

7.2.2 Immobilization of bacterial cells.Bacteria were routinely embedded in hydrophilic polyurethane foam within 24 h afterslurry preparation. Colony counts indicated that storage of up to one week did notresult in viability loss (data not shown). The foam samples were prepared by FrisbyTechnologies (Freeport, NY) at their Aiken, SC facility. Ingredients of the foam were:slurry, 20 g; prepolymer, 13.33 g; surfactant, 0.54 g. Thus, a 5% (dry wt) slurry wouldyield approximately 3 g dry wt bacteria per 100 g wet wt foam. Three surfactants werecompared in the course of the study. These were HS-3 (lecithin-based), F-88 (apluronic surfactant consisting of a mixture of ethylene oxide and propylene oxide), and

36

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DC198 (a silicone-based surfactant). The prepolymer Bipol 6B (NCO=6) was routinelyused, with three other prepolymers of lower NCO values (Bipol 3, #350, #802) beingused for comparison purposes in viability studies. The addition of silane as a bindingagent was tested in certain foam formulations. Prepolymers, surfactants,.-and silanewere provided by Matrix, Inc.

Control (cell - free) foams were generated by substituting 20 g medium for bacterials|urry,v Temperature was not controlled during the reaction, but did not exceed 42°C.Foam samples were reduced to a particulate state by means of a Waring blender andstored at ca 4°C prior to use.

7.2.3 Washout experiments.Entrapment efficiency was measured using 10 ml Poly-Prep chromatography columns(Bio-Rad Laboratories) modified by the replacement of the stock fritted disk with 75-80mg glass wool. Duplicate columns were loosely packed with 2.0 ± 0.01 g (wet wt) ofeach foam type. Cell retention by various foam types was routinely compared bypassing 50 ml of autoclaved, 0.2 j^m filtered deionized water through each column(gravity feed) and collecting the effluent. Effluents were preserved with 0.2 ^m filteredformalin (3.7%) and their bacterial content was determined by direct counts. Certainfoam formulations were retested using larger volumes of deionized water. In oneexperiment, 1000 ml water was passed through duplicate columns in 50-ml aliquots,with 50-ml samples of effluent being collected when cumulative water addition hadreached 50, 150, 400, 550, 700, 850, and 1000 ml (intervening effluent aliquots werediscarded). A second experiment involved passage of 2000 ml through duplicatecolumns, with the accumulated effluent being sampled when cumulative water additiontotaled 50, 1000, 1500, and 2000 ml. Experimental parameters used in washoutexperiments are summarized in Appendix 7.4.

7.2.4 Viability, total cell numbers, and activity measurements.To evaluate the effect of foam polymerization on the viability of immobilized cells, serialdilutions of immobilized and slurry cells were inoculated onto PTYG plates andincubated at 30°C until stable colony numbers were obtained. Cells in the foam werereleased by vigorous vortexing (30 sees) prior to dilution and plating. In oneexperiment, we used the Most Probable Number (MPN) technique to measureculturability in liquid media. Percent viability was calculated from the ratio of CFU orMPN to total cell numbers, determined by a modification of the Acridine Orange directcount technique (Hobbie et al., 1977). Appropriate dilutions were spotted onto heavyteflon coated slides (Cel-Line Associates, Inc., Newfield, NJ), heat fixed, and stainedwith 0.01% of acridine orange for 2 min at room temperature. Excess stain wasremoved with 0.2 jxm filtered nanopure water. Slides were allowed to air dry andobserved under a Zeiss Axioskop epifluorescent microscope (filter set 09) using a 100Xobjective. Viable counts and direct microscopical counts were done in duplicates ortriplicates, with at least 20 microscope fields being counted per sample. Experimentalparameters used in viability tests are summarized in Appendix 7.4.

37

WSRC-TR-96-0088

To determine the effect of immobilization on physiological activity of embedded cells,rates of CO2 evolution and O2 uptake were measured using a Micro-Oxymax v5.12indirect closed circuit respirometer (Columbus Instruments, Columbus OH). Triplicatesamples consisted of 8 g of foam-embedded bacteria or the equivalent number of cellsfrom the bacterial slurry (5 ml). Cells were incubated at two different temperatures(20OC or 25OC) with or without agitation (130 rpm) to compare the effect of ,temperature and oxygen availability on the respiration rates of immobilized vs. slurrycells. Experimental parameters used in respirometry studies are listed in Appendix 7.5.

In one experiment, carbon sources (0.1% glucose, 0.05% yeast extract) were addedand ribosomal probes utilized to elucidate the effect of embedding on metabolicactivity. Immobilized and slurry cells were transferred to BSM, carbon sources wereadded, and the suspensions were incubated at room for 24h. Aliquots were taken after2, 4, and 18 h, fixed with 3.7% formaldehyde (final concentration) and stored at 4°Cfor 24 h. Cells were centrifuged to remove formalin, resuspended on 0.2 \im filterednanopure water, fixed onto slides and hybridized with a fluorescently labeled 16S rRNAtargeting probe in an Autoblot hybridization oven (Bellco Glass, Inc., Vineland, NJ)following the procedure of Braun-Howland et at. (1992). The probe binds to alleubacteria and is complementary to the 342-360 region (essEscherichia coli asp).Fluorescing cells were observed by epifluorescence microscopy using a Zeiss Axioskopmicroscope and a Zeiss 15 filter set.

7.3 Results

7.3.1 Washout experiments.A list of the different formulations evaluated in washout experiments is presented inTable 7.1. Foam 1 (made with 1% F-88) resembles the formulation used in previouschapters to immobilize algal cultures, while Foams 2 - 8 each vary from it in terms ofone characteristic (bacterial density, surfactant type, surfactant concentration,embedding temperature, or presence of silane). As shown in Fig. 7.1, decreasing theconcentration of F-88 from 1% to 0.5% or increasing it to 10% did not reduce bacterialwashout (in terms of the percentage of total embedded bacteria removed by passing50 ml deionized water through 2 g foam). In fact, increasing the concentration ofsurfactant considerably increased washout, possibly by increasing the number and sizeof interconnected, bacteria-containing pores in the matrix.

38

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Table 7.1 Foam Formulation Used to Test Washout of B. cepacia PR131

Foamno.

1

2

3

4

5

6

7

8

10"

11

12

13

% slurry

8.10

17.70

8.10

8.10

8.10

8.10

8.10

8.10

17.70

8.10

8.10

17.70

Type ofsurfactant

E88

E88

DC 198

HS-3

E88

E88

E88

E88

HS-3

HS-3

HS-3

HS-3

Surfactantconcentration

1%

1%

1%

1%

0.5%

10%

1%

1%

1%

1%

1%

1%

Temperatureof

embedding3

RT

RT

RT

RT

RT

RT

Cold

RT

RT

RT

RT

RT

Silaheaddition

No

No

No

No

No

No

No

Yes

No

No

Yes

Yes

a Temperature at which the embedding process was performed. RT = room temperature(22±2); Cold = iced water bath.b Foams 10, 11, 12, and 13 represent a separate experiment.

39

WSRC-TR-96-0088

80

70

60

I 503. 40

1 3020

10

0

Effect of Surfactant Concentration

0.5% 1% 10%

F88 Concentration

Fig. 7.1. Effect of surfactant concentration. Values represent mean percentages oftotal embedded PR131 cells removed from duplicate columns containing 2 gfoam during passage of 50 ml water.

40

WSRC-TR-96-0088

Bacterial washout declined somewhat (from 36% to 25%) when embedding wasperformed in the cold (Fig. 7.2). Adding silane to an F-88-containing foam also had abeneficial effect (Fig. 7.2). However, even greater retention of bacteria (i.e., less than8% washout) was obtained when the concentration of bacterial biomass \r\ the slurryused for embedding was increased from 8.1 % to 17.7% (Fig. 7.3). - *

Fig. 7.4 shows that surfactant type had a major effect on bacterial retention. jSurfactantHS-3,was more effective than either DC 198 or F-88, when equal levels of the threecompounds were used. Based on results obtained with Foams 1-8, we selected HS-3(1%) for use in all subsequent foam batches used in the study.

Using Foams 10-13 (Foam 9 was a cell-free control and is not included in this report),we retested the effects of biomass concentration and silane in foams made with HS-3,since both factors seemed to reduce washout from foams made with F88. Results aresummarized in Fig. 7.5. Increasing bacterial biomass to approximately 18% reducedwashout from nearly 6% (Foam 11) to less than 2% (Foam 10). This confirms our priorresults with F88. Silane addition gave inconsistent results at the two biomass densities(Fig. 7.5). For this reason, silane was not used in subsequent foam batches.

Foams 10-13 were also used to examine washout during passage of larger volumes ofwater (Fig. 7.6, 7.7). In the experiment shown in Fig. 7.6, the first 50 ml of waterliberated 0.2%, 4.5%, 5.5%, and 1.1% of the total embedded cells from Foams 10, 11,12, and 13. These initially low values declined even further with subsequent aliquots ofwater. Similar results are shown in Figure 7.7. Foams 10, 11, 12, and 13 released0.1%, 5.3%, 9.7%, and 0.6% of total embedded cells into the first 50 ml of water.Passage of 2000 ml water liberated 0.2%, 13.9%, 16.2%, and 6.0% of the totalembedded cells. Results of these tests thus serve to validate our use of 50 mlvolumes for routine comparisons between foams, since initial washout rates arehighest and provide a reasonable means of predicting the comparative performance offoams subjected to larger volume washout tests.

7.3.2 Viability experiments.Immobilization in polyurethane foam (made with 1% HS-3, no silane) caused B. cepaciaPR131 to undergo a drastic decline in culturability on solid media (Fig. 7.8). Thenumber of colony forming units dropped to 0.006 % of total cell numbers, a decrease ofnearly five orders of magnitude. Dramatic decreases in culturability were also observedfor other environmental isolates as well as for a mixed community previously enrichedon chlorobenzene minimal salts media (Fig. 7.8). Colony counts performed usingFoams 1-13 indicated that culturability was severely impaired by all the foamformulations shown in Table 7.1 (data not shown).

41

WSRC-TR-96-0088

inn

90

80

70

•g 60

1 50| 40

2? 30

20

10

0

Effect of Cold or Silane

---

;

- ^H • • •- ^H ^H • • •— • • HH flH—I

Control Cold Silane

Treatment

Fig. 7.2. Effect of cold or silane. All foams were made using an 8.1% slurry and 1%F-88 as a surfactant. Cold embedding was performed using an ice bath,while control and silane-containing foams were prepared withouttemperature control. Values represent mean percentages of total embeddedPR131 cells removed from duplicate columns containing 2 g foam duringpassage of 50 ml water.

42

WSRC-TR-96-0088

1 UU

9080

70

•g 60

"I 50

| 40£ 30

20

10n

Effect of Slurry Concentration

-----

8.1% 17.7%

Slurry Concentration

Fig. 7.3. Effect of slurry concentration. Foams were prepared using 1% F-88 as thesurfactant and either an 8.1% (dry wt) PR131 slurry or an equal volume of17.7% (dry wt) slurry. Values represent mean percentages of totalembedded PR131 cells removed from duplicate columns containing 2 gfoam during passage of 50 ml water.

43

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1001 \J\J

90

80

70

•5 60

I 50| 40# 30

20

10

0

Effect of Surfactant Type

---

;

- ^H ^ ^- ^H ^HI • • • • • • I

F88 DC 198 HS-3

Surfactant

Fig. 7.4. Effect of surfactant type. Foams were prepared using an 8.1% slurry andequal volumes of a 1% solution of each surfactant. Values represent meanpercentages of total embedded PR131 cells removed from duplicatecolumns containing 2 g foam during passage of 50 ml water.

44

WSRC-TR-96-0088

)OU

t)

Was

r

°

Effect of Slurry

inn1 \J\J

80

60

40

20

0

-

-

-

8.1%

Cone.

17.7%

And Silane

8.1%,silane

Treatment

with HS-3

—17.7%,silane

Fig. 7.5. Effect of slurry concentration and silane with HS-3. Foams were preparedusing either an 8.1% or a 17.7% slurry, with or without added silane (bindingagent). HS-3 (1%) was used as the surfactant. Values represent meanpercentages of total embedded PR131 cells removed from duplicatecolumns containing 2 g foam during passage of 50 ml water.

45

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Sequential Washout

E

8

o17.1%. silane

. 1 % . silane8.1%

17.7% F o a m

Ml effluent ( cumj

Fig. 7.6. Sequential washout (A). Foams were as described in Fig. 7.5. 1000 mlwater was passed through duplicate columns in 50-ml aliquots. Total cellnumbers were determined in 50-ml effluent samples collected at the pointsindicated. Cell content of other aliquots was not determined.

46

WSRC-TR-96-0088

Sequential Washout

oO

1.50E+08

1.00E+08

5.00E+07

0.00E+00

17.1%, silane8.1%, silane

8.1%17.7%

Foam

ooo

Ml Effluent (cum.)

Fig. 7.7. Sequential washout (B). Foams were as described in Figs. 7.5 and 7.6.2000 ml water was passed through duplicate columns. The first 50 mleffluent was collected, followed by samples containing 950 ml, 500 ml, and500 ml. Values represent mean cell density (cells/ml) in each sample.

47

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Viability of immobilized microorganisms

<u

cu 1 H

0

% viable cells

B. cepacia PR13I Yeast strain #14 Bacterial strain 01 -b Bacterial community

embedded microorganism

Fig. 7.8. Viability of immobilized microorganisms. Two bacterial strains, a yeast(Candida sp.), and a mixed bacterial community were embedded. Colonycounts and total cell counts were performed on samples of cells removedfrom foam by vortexing. Values represent means of duplicate or triplicatesamples.

48

WSRC-TR-96-0088

During the polymerization process, initial pH of the reaction mixture is approximately 7;the pH then briefly drops to ca. 5 before returning to ca. pH 7 (P. Hermann, personalcommunication). We also observed a brief temperature increase to <42°C during foamproduction. To test whether these factors caused viability loss by embedded cells, weexposed PR131 slurries to heat shock (using a 42°C water bath) and a pH*7-» 5-^-7shift (accomplished by adding dilute HCI, then dilute NaOH). A control slurry wasmaintained at constant (room) temperature and pH 7. Total and culturable cell numbersljyere>then determined for all three preparations . Fig. 7.9 shows that viability of theslurries underwent relatively little change as a result of temperature or pH shock. Theeffect of temperature on culturability was also tested by carrying out the polymerizationreaction in an ice bath to prevent temperature increase (Fig. 7.10). Biomassconcentration in slurries used for embedding was also varied to determine whether thisfactor affected viability. However, culturability of the embedded bacteria was extremelylow under all conditions tested (Fig. 7.10).

To test whether embedding simply caused PR 131 to become unable to grow on agar-solidified media, we compared colony counts with results obtained using the MostProbable Number (MPN) technique. Both methods yielded exceedingly low viabilityestimates (data not shown), indicating that previously embedded cells were unable togrow on either liquid or solid media.

Toluene 2,4-diisocyanate (TDI) is used in the manufacture of polyurethane foams andis suspected to be a carcinogen. Therefore, we tested three additional prepolymerscontaining reduced TDI levels (ranging from 50% of the TDI content of Bipol 6B down toundetectable levels) in order to determine whether TDI was responsible for viabilityloss. However, culturability was again very low in all foam formulations tested (data notshown).

7.3.3 Respiration studies.Since it is possible for bacteria to remain metaboiically active but unable to formcolonies on artificial media, we investigated the effect of embedding on respirationrates. CO2 evolution and O2 consumption rates of embedded B. cepacia G4 werecompared with those of unembedded bacterial slurries. Surprisingly, embedded cellsshowed higher respiration activity than unembedded cells (Fig. 7.11). Respiration ratesof unembedded cells increased when temperature was increased to 25°C (Fig. 7.12),and remained higher than those of embedded cells even after temperature was againreduced to ambient (20°C) (Fig. 7.13).

We also examined respiration rates of B. cepacia G4 which had been exposed tophenol (to induce the oxygenase enzyme responsible for TCE degradation) prior toimmobilization. Immobilized cells again respired at higher rates than slurry cells whenboth populations were kept at 20°C (Fig. 7.14). When cells were incubated at 25°C withor without aeration by 140 rpm shaking, slurries were more active than immobilizedcells (Fig. 7.15, Fig. 7.16). However, immobilized cells retained more than 50% of the

49

WSRC-TR-96-0088

120

100 -

XiCO

Control pH shift

Treatments

Heat shock

Fig. 7.9. Effect of temperature and pH shock on culturability. Samples of PR131slurry were subjected to conditions simulating temperature increases and pHchanges that occur during embedding. Colony counts and total cell countswere then performed on the slurries. Values represent means of duplicateor triplicate samples.

50

WSRC-TR-96-0088

Temperature experiment

CO

"3

3CO

0.012

0.010-

0.008 -

0.006 -

0.004 -

0.002 -

0.0006.8%, RT 6.8%, Cold 13.4%, RT 13.4%, Cold

Treatments

Fig. 7.10. Temperature experiment. Foams were prepared using either a 6.8% or a13.4% PR131 slurry . Polymerizations were carried out both in an ice bathand without temperature control. Colony counts and total cell counts wereperformed on samples of cells removed from foam by vortexing. Valuesrepresent means of duplicate or triplicate samples.

51

WSRC-TR-96-0088

o -

-1 -

-2

- • — • — •

•o-—o—n-

-Q CO2 foam

- • CO2 slurry

-n O2 foam

-o O2 slurry

20i

40 60 80

h o u r s

Fig. 7.11. CO2 evolution and O2 consumption rates of foams and bacterial slurriescontaining equal numbers of 6. cepacia. Incubations were performed atroom temperature (approx. 20°C). Measurements represent means oftriplicate samples.

52

WSRC-TR-96-0088

CO2 foam

CO2 slurry

O2 foam

O2 slurry

hours

Fig. 7.12. Effect of temperature increase on respiration of B. cepacia G4 slurries.Foam and slurries from Fig. 7.11 were used in this experiment. Foams werekept at room temperature (20°C) while slurries were incubated at 25°C.Values represent means of triplicate samples.

53

WSRC-TR-96-0088

•61)

e

0.35-

0.15-

-0.05 -

-0.25 -

-0.45 -

-0.65

-Q CO2 foam

-*• CO2 slurry

-o 02 foam

-° O2 slurry

10 20

h o u r s

30

Fig. 7.13. Effect of temperature decrease on respiration of B. cepacia G4 slurries.Slurries from the experiment in Fig. 7.12 were returned to 20°C, after an 8 hrexposure to 25°C. Foam samples remained at 20°C. Values representmeans of triplicate samples.

54

WSRC-TR-96-0088

s

0.25-

0.05-

-0.15 -

-0.35 -

-0.55 -

-0.75 -

-0.95

Q CO2 rates foam

• CO2 rates slurry

a O2 rates foam

o O2 rates slurry

h o u r s

Fig. 7.14. Effect of phenol induction on respiration of B. cepacia G4. Bacterial cultureswere exposed to 2 mM phenol for 2 hours prior to embedding. Foams andslurries were maintained at room temperature (20°C). Values representmeans of triplicate samples.

55

WSRC-TR-96-0088

co2 rates foamco2 rates slurryo2 rates foamo2 rates slurry

-2

h o u r s

Fig. 7.15. Effect of aeration on respiration of phenol-induced B. cepacia G4. Aftercollection of the data shown in Fig. 7.14, foams and slurries were incubatedat 25°C with 130 rpm shaking. Values represent means of triplicate samples.

56

WSRC-TR-96-0088

CO2 rates foam

0.4

0.2-

o.o-

-0.2 -

-0.4 -

-0.6

* CO2 rates slurry

B O2 rates foam

~o O2 rates slurry

5 10

h o u r s

1 5

Fig. 7.16. Effect of decreased aeration on respiration of phenol-induced 8. cepaciaG4. The indicated respiration rates were measured after agitation ofsamples shown in Fig. 7.15 was discontinued. Temperature remained at25°C. Values represent means of triplicate samples.

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respiration activity shown by unembedded cells. This strongly suggests thatphysiological activity of B. cepacia is not as severely affected by the embeddingprocess as the culturability results had indicated. Microscopic observations alsoindicated that immobilization did not cause any visible physical damage to the cells(data not shown).

Respiration rates of B. cepacia G4 and B. cepacia PR131 were compared to determinewhether the two strains responded similarly to the embedding process (Fig. 7.17). Nomeaningful differences were observed. The slightly higher respiration rates of B.cepacia PR131 can probably be attributed to a small difference in slurry densities usedfor embedding (4.6% for PR131, 4.2% for G4).

7.3.4 Nutrient Amendments.Both unembedded and previously embedded cells responded to the addition of carbonsources by significantly increasing in volume within 18 h (data not shown). A fraction ofthe previously embedded bacterial population responded by forming elongated cells. Insitu hybridization of slurry and embedded cells with a fluorescently labeled rRNA-targeting probe revealed a simultaneous increase in ribosomal content as determinedby an increase in fluorescent signal intensity.

7.4 Discussion

It is clear that immobilized degradative bacteria are potentially of great value forgroundwater and wastewater treatment (Levinson et al, 1994). Several studies haveshown that entrapment systems can deliver bacteria capable of transforming manypollutants (e.g. Weir et al., 1995; Xu et al., 1996). The use of immobilized bacteriamight increase initial degradation rates of a compound by avoiding the lag time requiredfor biofilm formation. Moreover, immobilized bacteria could be cost effective inbioremediation projects since they can potentially be used several times withoutsignificant lost activity (Rhee et al., 1996).

The aim of this study was to evaluate the use of polyurethane based formulations forthe entrapment of degradative bacteria. Our main criterion was the ability of eachformulation to prevent the release of cells. This criterion is of great importance indetermining the functional longevity of this type of carrier system in bioremediationapplications. Moreover, in applications involving genetically engineered bacteria, theability to retain cells would seem critical in view of public concern about the release ofsuch organisms into the environment. This aspect has received little attention byresearchers evaluating immobilization agents, polyurethane foams in particular.

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0.6

3O

OX)

s

0.5-

0.4-

0.3-

0 .2-

0.1 -

0.0

21.5 25.5 25.5

21

a co2 foam 118

•• co2/slurry g4

co2 foam 119

co2/slurry pr131

10 20

h o u r s

30 40

Fig. 7.17. Comparison of respiration by B. cepacia strains G4 and PR131. Increase inrespiration rate coincides with periods of temperature increase (from 20°C to25°C) while decreases in respiration rates can be attributed to decreases intemperature from 25°C to 20°C. Measurements represent averages oftriplicates.

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Our results show that biomass concentration and the type and concentration >fsurfactant are major determinants of cell retention. The effect of surfactants isundoubtedly related to the observation that large numbers of embedded cells areactually located in fluid-filled pores and hence are readily released after the foam is tornor ctrt (unpublished data). Increasing the surfactant concentration visibly increasedtotal pore volume of the resulting foam and led to higher washout rates. The chemicalcomposition of the surfactant also influences the size, total volume and . yfnjtercQpnectedness of pores due to differences in surface tension, and hence will affectcell retention. The reason for the decrease in washout observed at higher biomassconcentrations is unclear, but may be related to the lower water content of foams madewith denser slurries. Based on our results, foam made with 1% HS-3 and an 18% dryweight bacterial slurry was most effective in retaining embedded B. cepacia. It remainsto be seen whether this formulation is equally effective with other organisms.

The dramatic decrease in culturability as a result of immobilization suggested thatviability was destroyed by the embedding process. However, we found no evidence thatcells were killed by temperature or pH changes during embedding. Culturability wasseverely reduced in the presence of all three surfactants, and was not affected by thefree TDI content of prepolymers used in foam manufacture. These findings suggestedthat some unknown factor inherent in the polymerization process was lethal to .microorganisms, and led us to question the ability of polyurethane-based formulationsto deliver functional bacterial cells.

However, respirometry data demonstrated that, although incapable of forming colonies,embedded 8. cepacia remained metabolically active. Indeed, respiration by embeddedbacteria compared favorably to that of free cells. This is consistent with the increase involume and ribosomal content of embedded cells after nutrient addition. We concludethat embedding caused most cells to become viable but nonculturable (Roszak et al.,1984), and that culture techniques are not reliable indicators of the metabolic status ofpolyurethane-embedded cells. In light of these findings, it appears likely thatembedded B, cepacia will retain its degradative capabilities and its potential forbioremediation.

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8.0 DEGRADATION OF TRICHLOROETHYLENE AND BENZENE BY EMBEDDEDBACTERIA

8.1 Introduction -Du#to its widespread use as a degreaser and solvent, trichloroethylene'(TCE) is acommon contaminant of groundwater at hazardous waste sites, and represents a threatto many aquifers. At the Savannah River Site, TCE concentrations well aboveregulatory limits have been reported from numerous monitoring wells at waste sites(WSRC, 1995). Consequently, there is strong interest on-site and elsewhere intechnologies for reducing levels of this compound. Currently, the most promisingtechnologies involve bioremediation, either by anaerobic or aerobic microorganisms.However, anaerobic TCE degradative processes are relatively slow and frequentlyresult in the production of other hazardous compounds, including vinyl chloride.Aerobic processes are more rapid and result in complete mineralization, but usuallyrequire the presence of a primary substrate to induce oxygenase activity.

The aerobic bacterium Burkholderia cepacia (formerly Pseudomonas cepacia) strain G4has recently received much attention (e.g. Folsom et al., 1990; Folsom and Chapman,1991; Shields and Reagin, 1992; Luu et al., 1995) due to its oxidative TCE degradationcapabilities. Although G4 requires a primary substrate to induce enzyme activity(phenol being the most commonly used, but benzene or toluene also act as inducers),constitutive expression of toluene monooxygenase has been reported in a mutant strain(PR1) developed by Dr. Malcolm Shields of the University of West Florida (Shields andReagin, 1992; Luu et al., 1995).

The use of G4 and the constitutive mutant in bioreactors has hitherto been limited inpart by their poor adhesion capabilities. This hinders their use in nonsterilewastewaters. In the previous section (Section 7.0), we have described the optimizationof techniques for embedding the constitutive mutant PR131 in hydrophilic polyurethanefoam in such a manner that cells are effectively entrapped and retain metabolic activity,as evidenced by ribosomal probes and measurement of respiration rates. However,loss of culturability by the embedded bacteria indicated that some cellular activitieswere impaired during the entrapment process. In this section, we compare the ability ofimmobilized and free cells of G4 and PR131 to degrade TCE. Our objective is apreliminary assessment of the potential of polyurethane-embedded B. cepacia for theremediation of TCE-containing groundwaters.

8.2 Materials and Methods

8.2.1 Bacterial strains, culture media, and growth conditions.Burkholderia cepacia strains PR131 and G4 were kindly provided by Dr. MalcolmShields. Strain G4 requires induction of toluene monooxegenase activity for TCEdegradation, while the mutant strain PR131 has been reported (Shields and Reagin,1992) to produce the enzyme constitutively.

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To assure culture purity, original stocks were kept at - 70°C in 20% glycerol. Prior toeach experiment, stock material was spotted onto PTYG plates (Appendix 7.1) toprovide inoculum for batch cultures. Axenic batch cultures were grown in a yeast-gluSbse medium (YGM) (Shields and Reagin, 1992), which consisted of basal saltsmedium (BSM; Shields et al., 1989) plus 1 g/l glucose and 0.5 g/l yeast extract(Appendix 7.3). Pseudomonas medium (Atlas, 1993) was used for comparison"purposes in one experiment (Appendix 7.2). Small-scale (100 - 250 ml) batch cultureswere grown in shake flasks (200 rpm, 30°C). Larger amounts of axenic biomass weregrown by inoculating 20 ml of 1- 3 day old culture into 4000 ml polycarbonate bottlescontaining 3000 ml YGM. These cultures were maintained at 26 ± 2°C and aeratedthrough a sterile 0.2 \xm filter to provide mixing and 02- Cultures were routinelyharvested for foam embedding after 3 days' growth, at which time biomass yield wasabout 0.5 g dry weight / I . Harvesting was by centrifugation (10,000 rpm, 10 min, 15 -25°C). Pellets were resuspended in BSM medium to a density of 2.2 - 8.9% (typicallyca. 5%) dry weight. Aseptic technique was not used during the harvesting process.

8.2.1 Preparation of hydrophilic polyurethane foam.Bacteria were routinely embedded in hydrophilic polyurethane foam within 2 h afterslurry preparation. In two experiments involving the induction of enzyme activitysubsequent to slurry preparation, slurries were stored overnight at 4°C prior toinduction and embedding. Colony counts indicated that storage of up to one week didnot result in viability loss (data not shown). The foam samples were prepared by FrisbyTechnologies (Freeport, NY) at their Aiken, SC facility. Ingredients of the foam were:slurry, 20 g; prepolymer, 13.33 g; surfactant, 0.54 g. Thus, a 5% (dry wt) slurry wouldyield approximately 3 g dry wt bacteria per 100 g wet wt foam. A 1 % solution of thelecithin-based compound HS-3 (Amisol) was used as the surfactant, based on ourprevious results indicating that this material enhanced retention of cells within the foam.The prepolymer Bipol 6B (Matrix, Inc) was selected on the basis of availability and ourprevious respirometry data. Control (cell-free) foams were generated by substituting 20g BSM medium for bacterial slurry. Temperature was not controlled during the reaction,but did not exceed 42°C. Foam samples were reduced to a particulate state by meansof a Waring blender, stored at ca 4°C, and used for experiments within 2-4 h unlessotherwise stated.

8.2.2 Induction of enzyme activity.Toluene monooxygenase activity in G4 was induced in one of several ways. Unlessstated otherwise, 2 mM phenol was added to cultures 2 h before the commencement ofharvesting. Benzene (2 mM) was sometimes used as an inducer for comparisonpurposes. In one experiment measuring the kinetics of TCE disappearance, 2 mMphenol was added to the 5.6% (dry wt) bacterial slurry 2 h prior to centrifugation andresuspension of the cells in BSM, after which the slurry was embedded in foam. Inattempts to induce enzyme activity in pre-embedded G4, 0.1 g quantities of G4/foamaggregate were placed in quadruplicate 22 ml headspace vials (Hewlett-Packard) to

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which 10 ml BSM and 2 \i\ phenol or 2 ^l benzene were added. Quadruplicate"control(uninduced) vials received G4/foam and medium only. Vials were sealed and shaken(200 rpm) for 2, 4, or 21 h. After induction, excess liquid was pipetted from the vialsand replaced with 10 ml fresh BSM. For comparison purposes, 0.295 mfof 64 slurry(equivalent to the biomass content of 0.5 g G4/foam) was placed in each of triplicatebottles containing 50 ml BSM medium and 10 jal phenol, 10 jul benzene, or no inducingagent. Bottles were sealed and shaken in the same manner as foam samples, afterwhich-eells were pelleted (8000 rpm, 15 min, 15 - 25°C) and the supernatant decanted.Pellets were resuspended in 50 ml fresh BSM and 10 ml of each suspension wasplaced in quadruplicate headspace vials for use in TCE degradation assays.

8.2.3 Degradation assays.Assays for TCE biodegradative capability were typically conducted using quadruplicate22 ml headspace vials (Hewlett-Packard) containing 10 ml BSM and 0.059 ml bacterialslurry or 0.1 g foam/bacterial aggregate (these amounts of slurry and foam werecalculated to contain equivalent biomass). TCE (3 ppm) was added as a 1 pptmethanol solution, after which vials were immediately sealed with crimp caps andTeflon-coated septa, incubated 3 - 5 (typically 3) days at 30°C, and subjected toheadspace analysis by gas chromatography. Results were interpreted with the aid of astandard curve generated from TCE-inoculated BSM samples, which had beenincubated under the same conditions as experimental samples. Benzene consumptionwas measured similarly, using 2 ppm benzene added as a ca. 800 ppm methanolsolution.

In experiments with unembedded PR131, 10 ml aliquots of YGM-grown cultures wereplaced directly in headspace vials, and TCE or benzene was added as describedabove. Unembedded G4 cultures were tested after induction with phenol (2 mM, 2 h),centrifugation (15 min, 8000 rpm, 15 - 25°C), and resuspension in BSM equivalent tothe original culture volume. 10 ml aliquots of this suspension were then dispensed intoheadspace vials for testing.

8.2.4 Groundwater sampling.Several groundwater monitoring wells at SRS were sampled for use in the study. Well# 1TC-SZP-9D is located at the B-Area Sanitary Landfill, while Wells MSB 25A, MSB34A, and MSB 75B are located in M-Area. Chemical characteristics of the variousgroundwater sources are described in Appendix 8.1. All groundwater samples weretaken after purging ca. 3 well volumes of groundwater, and were collected inheadspace vials that were immediately sealed with Teflon-coated septa and crimpcaps. Only vials containing no air bubbles or bubbles of less than ca. 2 mm diameterwere used to provide water for experiments. Samples were used within 6h aftercollection. Samples containing bubbles of less than 6 mm diameter are consideredadequate for quantitative analysis of volatile organics (U.S. EPA, 1986).

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8.2.5 Analyses.TCE was measured on a Hewlett-Packard 5890A gas chromatograph equipped with aHewlett-Packard 19395A automated gas headspace analyzer, an electron capturedetector, and a 60-m Vocol (Supelco) column. Column temperature wasiield at 35°Cfor 7 min, increased 5°C/min to 55°C, and then increased 15°C/min to^O^C.Samples were equilibrated at 75°C prior to analysis. Detection limits for TCE andbenzene were 1.0 and 20.0 ppb respectively. . y

Dry weight of bacterial suspensions was measured in duplicate after centrifugation(10000 rpm, 10 min, 15 - 25°C), washing once with an equal volume of deionizedwater, recentrifugation, resuspension in deionized water, drying to constant weight at104°C, and cooling in a desiccator.

8.2.6 Data interpretation and statistical analyses.Data were stored magnetically and interpreted through linear regression analysis ofstandard curves generated during each experiment. The nonlinearity of the standardcurves required the fitting of 3 - 4 lines to various portions of the concentration range.Regression analysis, t-tests, and calculations of sample standard deviations wereperformed using Microsoft Excel. Error bars in all figures represent standard deviationsof triplicate or quadruplicate samples. Dates and major experimental parameters for allbiodegradation experiments are summarized in Appendix 7.4.

8.3 Results

8.3.1 TCE degradation.Initial experiments with PR131 cultures showed little or no significant TCE degradationunder our experimental conditions. Although a 36% removal efficiency can becalculated from data in Table 8.1, this value is dubious due to variability in the standardcurve used to interpret the results. A one-tailed t-test indicates no significant difference(P = 0.19) between raw TCE peak areas of PR131-containing samples and controlsreceiving the same TCE addition (1.043 ppm). A second PR131 stock culture wasobtained from the University of West Florida, but gave similar results. However, a G4culture removed substantial amounts of TCE from the solutions after 2 h induction withphenol (Table 8.1) The difference between TCE peak areas of G4-treated samplesexposed to ca. 1 ppm TCE and controls exposed to ca. 0.5 ppm TCE was highlysignificant (P = 0.0008), and calculated removal was 81%.

TCE degradation by PR131 has been demonstrated in other laboratories, and noobvious reason for its absence in our experiments immediately presents itself. In theinterests of expediting our investigation of the effects of polyurethane

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Table 8.1. Comparison of TCE Removal from 1.043 ppmSolutions by PR131 and G4 Cultures. -

Peak AreaSample or Standard (X107) TCE, ppma

Std 0.522 ppm 4.852Std 0.522 ppm 4.915Std 0.782 ppm 5.576Std 0.782 ppm 5.987Std 1.043 ppm 6.477Std 1.043 ppm 5.440Std 1.043 ppm 5.470Std 1.303 ppm 6.009Std 1.303 ppm 6.164Std 1.563 ppm 6.303Std 1.563 ppm 5.727

G4 2.097 0.224G4 1.458 0.156G4 1.480 0.158G4 2.577 0.275

PR131 5.243 0.626PR131 5.440 0.678PR131 5.635 0.729PR131 5.320 0.646

a Calculated from the following regression lines: ppm =1.069 X 108 A (for A < 4.9 X 107), PP™ = 2.61 X 108

A - 7.42 X 107 (for A + 4.9 X 107 - 5.8 X 107) whereA = TCE peak area.

WSRC-TR-96-0088

foam embedding on biodegradation, we therefore decided to direct our attention towardthe more cooperative G4 strain.

To select an appropriate TCE spike concentration for further experimentation, weadded 1, 2, or 3 ppm TCE to BSM suspensions of G4 (grown in YGM). A BSMsuspension of a culture grown in Pseudomonas medium was inoculated with 2 ppmTCE as a comparison. Fig 8.1 shows that TCE removal by YGM-grown cells^was linear"over the 1-3 ppm range, with 85 - 87% of the compound being degraded. Noconclusion can be drawn from the lower mean TCE removal by cells grown inPseudomonas medium, since no attempt was made to standardize biomass content ofthe two cultures. However, cells grown in Pseudomonas medium were much morevariable in their response to 2 ppm TCE, as shown by the very large standard deviationin Fig. 8.1. Therefore, all subsequent experiments were performed with YGM-growncells. A 3 ppm TCE addition was routinely used for subsequent work, since this valuegave adequate results and is similar to that of many groundwater monitoring wells inTCE-contaminated areas of SRS.

After TCE degradation had been demonstrated in G4 cultures, an experiment wasconducted to test whether it could occur after foam embedding (Fig. 8.2). Foam wasmade using slurries (2.2 - 2.4 % dry wt) suspended in BSM and YGM medium in orderto test the effects of primary carbon sources on the degradation process. Aliquots ofunembedded slurry were tested as a control. Embedded and unembedded G4removed similar amounts of TCE (Fig. 8.2). The BSM-suspended cells removed 95 -97.5% of the substrate. However, the presence of primary carbon sources (1 g/lglucose, 0.5 g/l yeast extract) dramatically decreased TCE removal.

Although 3-day incubations with TCE were routinely used in our experiments, the use ofembedded G4 in a bioremediation process would require information on the kinetics ofTCE degradation over various time periods. A preliminary examination of degradationover a 2-day period is shown in Fig. 8.3. A 4 - 8 h lag period occurred before TCElevels dropped below those of controls, and most degradation of the compoundoccurred in the first 24 h. Small amounts of TCE disappeared over a 48-h incubationfrom samples containing uninduced G4/foam aggregates, cell-free foam, anduninduced G4 slurry. This phenomenon might be due to sorptive processes.

TCE degradation efficiencies in Fig. 8.3 did not approach those seen in previousexperiments. Low degradation levels were seen in both foam and slurry samples,indicating that the embedding process was not responsible for the poor results. Oneexplanation is that phenol induction of toluene monooxygenase was carried out aftercells had been concentrated to a 5.5 - 5.6% (dry wt) slurry, rather than prior tocentrifugation as in other experiments. The total amount of phenol available per cellwas hence much smaller than previously, and induction may have been incomplete. Asecond possibility is that high bacterial densities in the foam could impair TCE removal.The foam used to generate data in Fig 8.2 contained about half as much biomass asthat used for Fig. 8.3. To test this idea, we conducted an experiment with foam of two

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TCE Removal by G4 Cultures

eexQ.

2.5 -

2 -

-oo 1.5

UJ

0.5

0.5 1.5 2

TCE added, ppm

2.5 3.5

Fig. 8.1. TCE removal by G4 cultures. TCE degradation is shown as a function of theconcentration originally present.

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TCE Removal by Embedded and Unembedded G4

120

100

• SlurryDFoam

BSM YGM

Medium

Fig. 8.2. TCE removal by embedded and unembedded G4. Quadruplicate foam andslurry samples containing 1.3 -1.4 mg dry wt bacteria were exposed to 3.1ppm TCE in 10 ml BSM or YGM. Induction was performed prior to slurrypreparation.

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Time Course of TCE Removal by Embedded G4

F/G4/P

o F/G4/N

F/ND S/PA S/N

10 20 30 40 50

Hours

Fig. 8.3 Time course of TCE removal by embedded G4. Quadruplicate foam and slurrysamples, each containing 3.2 - 3.3 mg dry wt bacteria, were exposed to 3.0 ppm TCE in10 ml BSM. Controls were incubated for 48 h only, while phenol-induced G4-foamaggregates were tested for various time periods. Induction was performed on slurrypreparations prior to foam manufacture. Legend: F/G4/P, foam containing phenol-induced G4; F/G4/N, foam with uninduced G4; F/N, uninduced cell-free foam; S/P,phenol-induced slurry; S/N, uninduced slurry.

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biomass densities (Fig. 8.4). Phenol induction was carried out immediately beforeharvesting and slurry preparation. A separate set of foam and slurry samples received6:1 g/l glucose and 0.05g/l yeast extract to validate our previous finding that thepresence of primary carbon sources impaired TCE degradation. Fig. 8.4-shows thatbotfra 5.1% slurry and the resulting foam degraded more TCE than a 2T5% slurry andassociated foam. The high degradation efficiency (compared with Fig. 8.3) of the 5.1%slurry and resulting foam suggests that phenol induction of growing cultures was

"superior to induction of concentrated slurries. However, foam made from 2.5% slurryshowed poor TCE removal, despite the cells having been grown and induced in thesame manner and concentrated to a similar density as those seen in Fig 8.2. Wespeculate that some factor involved in polymer handling, the polymerization reaction, orsubsequent foam processing may vary between individual foam samples and affectTCE removal.

Very little TCE degradation occurred in samples containing glucose and yeast extract(Fig. 8.4). This suggests that toluene monooxygenase activity is repressed in thepresence of readily metabolizabie primary substrates, and confirms the results shown inFig. 8.2.

Since G4 requires induction of toluene monooxygenase for TCE degradation to occur,its utility for groundwater treatment will in part depend on the duration of enzymeactivity, once induced. In a preliminary exploration of its useful lifetime, unused foamfrom the previous experiment was stored in a sealed container at 4°C and retestedafter 3 days (primary carbon sources were not used in the second test). Results areshown in Fig. 8.5, with data from Fig 8.4 (for samples without primary carbon sources)being repeated (as "3 h samples") for comparison purposes. TCE degradation droppedmarkedly in both foams and both slurries during storage. The useful lifetime of theproduct would therefore appear to be considerable less than 3 days.

For a G4-based process to be practical, it would therefore be desirable to induceenzyme activity on-site. This would be most conveniently done using cells alreadyembedded in foam. To test the feasibility of inducing pre-embedded G4, we exposedfoam/G4 aggregates to 2 mM phenol or 2 mM benzene for various time periods (Fig.8.6). A 2 h induction (such as was used in previous experiments) had some effect onslurry preparations, but had no effect on foam. TCE degradation by slurries wasmaximal after a 4 h induction, but activity of phenol-induced and possibly benzene-induced foam preparations was higher after a 21 h induction. It is not known whetherlonger induction periods would have further increased TCE removal. The experimentdemonstrates that, assuming retention of overall metabolic activity in embedded G4,the induction and use of the product could be geographically and to some extenttemporally separated from its manufacture. It also raises the possibility that enzymeactivity could be induced repeatedly, extending the useful lifetime of the product.Lastly, the data suggest that TCE removal efficiencies may be improved by longerinduction periods than those routinely used in our experiments.

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Effect of Biomass Density and Carbon on TCE Removal

100

90

Foam Foam, +C Slurry Slurry, +C

Fig. 8.4. Effect of biomass density and carbon on TCE removal. Quadruplicate foamand slurry samples containing 1.5 mg or 3.0 mg dry wt bacteria(corresponding to 2.5% or 5.1% slurry density) were exposed to 3 ppm TCE in10 ml BSM with or without added carbon (0.1 g/l glucose and 0.05 g/l yeastextract). Induction was performed prior to slurry preparation.

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Duration of Induction

Foam, 5.1% Foam, 2.5% Slurry, 5.1% Slurry, 2.5%

Fig. 8.5. Duration of induction. Foams and slurries from batches used in Fig. 4 wereexposed to 3 ppm TCE in 10 ml BSM after 3 days' storage at 4°C. 3 hourdata is repeated from Fig. 8.4 for comparison.

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Induction of Previously Embedded G4

110

100

90

80

70

S 60

1 50 h40

30

20

10

0

p-

10

Induction period

• • —

•

o

D

A

A

«

15

, h

F/G4/P

S/P

F/G4/BS/B

F/G4/NS/N

F/N

20 25

Fig. 8.6. Induction of previously embedded G4. Quadruplicate foam and slurrysamples containing 3.2 - 3.4 mg dry wt bacteria were exposed to 3 ppm TCE in 10 mlBSM. Induction was performed with phenol or benzene (2 mM) on foam or slurrysamples for 2, 4, or 21 h prior to TCE exposure. Uninduced controls were held for 2h or4 h prior to testing. Legend: F/G4/P, phenol-induced foam-G4 aggregate; F/G4/B,benzene-induced foam-G4 aggregate; S/P, phenol-induced slurry; S/B, benzene-induced slurry; F/G4/N, uninduced Foam-G4 aggregate; S/N, uninduced slurry; F/N,uninduced cell-free foam.

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It is expected that TCE removal from actual groundwaters may differ from thatin testsolutions, due to the presence of indigenous microorganisms, competing organics, andother factors. Two experiments were conducted using water samples collected fromSRS monitoring wells. The first batch of water was taken from well # ITOSZP-9D atthe*B Area Sanitary Landfill Site. This water contained TCE in quantities belowregulatory limits and was therefore spiked with 0.1, 1.0, or 3.0 (nominal) TCE tosimulate a more heavily contaminated source. Slurry samples removed 94 -400% ofThe compound at all TCE levels tested, while equivalent foam samples removed 86% atthe two lower levels and only 50% at the highest level tested (Fig. 8.7). Controlsamples of sterile BSM containing 3 ppm TCE and treated with foam or slurry gaveresults similar to 3 ppm-spiked groundwater. This suggests that TCE removal from thegroundwater was indeed accomplished by G4 and not by indigenous microorganisms.

In another experiment, groundwaters were sampled from the M-Area wells 25A (1.99ppm TCE), 34A (2.71 ppm), and 75B (1.09 ppm). These were exposed to G4 slurriesand foam without additional TCE spiking. Slurries removed 88 -100% of TCE from allgroundwater samples (Fig. 8.8). However, foam-embedded G4 yielded variable results,ranging from 12% removal in 34A samples to almost 80% in 25A samples. Thesedifferences are not correlated with the original TCE content of the samples. Since allsamples were treated with foam from a single batch, the data suggest that groundwatercomposition strongly influences TCE removal by embedded G4, but has much lessinfluence on unembedded bacteria.

8.3.2 Benzene degradation by G4.Embedded G4 (2.95 mg dry wt in 100 mg wet wt foam) degraded 90% of the benzenecontent of 2.07 ppm solutions (Fig. 8.9). This approached the degradation efficiency(97%) achieved by the equivalent amount of unembedded slurry. A 29% benzene losswas observed in controls containing cell-free foam. This suggests that the foammaterial itself binds some benzene. We do not at present know whether benzeneabsorbed in this manner is still available for bacterial degradation.

An experiment comparing removal of a 2 ppm benzene solution from BSM medium withthat from YGM medium demonstrated that benzene removal was more variable andwas (on average) impaired in the presence of glucose and/or yeast extract (Fig. 8.10).Thus, it appears that one or more of the organic compounds in YGM medium was usedpreferentially as a carbon source. Comparison of the Fig. 8.9 (experiment performedimmediately after embedding) with Fig. 8.10 (experiment performed 2 days afterembedding) indicates that benzene degradation activity was unimpaired by 48 hstorage at 4°C.

8.4 Discussion

In this study, we attempted to evaluate the practicality of using hydrophilic polyurethanefoam as a means of immobilizing G4 for use in bioremediation processes. Our resultsindicate that although foam-entrapped cells can potentially degrade TCE at levels

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TCE Removal from Spiked Groundwater

120 r

OE

LUO

100

80

60

40

20

-•— Foamo Slurry• Foam (BSM)a Slurry (BSM)

0.0 0.5 1.0 1.5 2.0

TCE spike, ppm

2.5 3.0 3.5

Fig. 8.7. TCE removal from spiked groundwater. Quadruplicate foam and slurrysamples containing 3.0 mg dry wt bacteria were exposed to 10 ml sanitarylandfill water amended with various TCE levels. Induction was performed priorto slurry preparation.

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TCE Removal from Groundwater

E

UJO

3.0

2.5 -

2.0 -

1.5 -

1.0 -

0.5 -

0.0

25A 34A

Source

75B

Fig 8.8. TCE removal from groundwater. Quadruplicate foam and slurry samplescontaining 3.1 mg dry wt bacteria were exposed to 10 ml of three M Areagroundwaters containing various TCE levels. Induction was performed prior toslurry preparation.

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Benzene Removal

Slurry Foam/G4 Foam

Fig. 8.9. Benzene removal. Quadruplicate foam and slurry samples containing 2.95mg dry wt bacteria were exposed to 2 ppm benzene in 10 ml BSM. Noinduction was performed. Cell-free foam was used as an additional control.

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Influence of Additional Carbon on Benzene Removal

BSM YGM

Medium

Fig. 8.10. Influence of additional carbon on benzene removal. Quadruplicate foam andslurry samples (identical to those shown in Fig. 8.2) containing 1.3 -1.4 mgdry wt phenol-induced bacteria were stored for 2 days at 4 C and thenexposed to 2 ppm benzene in 10 ml BSM or YGM.

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WSRC-TR-96-0088

similar to those of free cells, they apparently lose the ability to divide. Theirapplicability to use in groundwater treatment would therefore depend in part uponmicrobial densities already present in the groundwater and upon the susceptibility of thefoam material to colonization by indigenous microbes.

Our findings suggest several additional considerations that will be of importance indesigning a groundwater treatment process using G4. In particular, the fact that thepresence of readily metabolizable carbon sources inhibits the degradation of TCEwould affect process design. Also, the relatively short duration of toluenemonooxygenase activity, once induced, presents an obstacle. However, our success ininducing the enzyme in previously embedded cells raises the possibility thatfoam/bacterial aggregates could be prepared and stored for later use, with inductionbeing performed at the bioremediation site (perhaps during several consecutiveinduction/TCE degradation cycles). This would greatly increase the functional longevityof the material and its convenience for the end user.

Implementation of a G4/foam based process would require considerable work beyondthe scope of the current preliminary study. In particular, additional work is needed toachieve consistently high TCE degradation rates. We obtained varying results withidentically grown G4 cultures embedded at similar densities and tested under the sameconditions. The source of variability is therefore likely to be found in the embeddingprocess itself. The same proportions of prepolymer, surfactant, and slurry were usedthroughout the study and the polymerization reaction was carried out under consistentconditions. We surmise, however, that aging of the prepolymer and variability inprocedures used to bring it to a suitable temperature for foam manufacture may haveaffected the resulting G4/foam aggregates.

Although many questions remain to be answered before a G4/hydrophilic foam-basedprocess can be devised, the present study demonstrates that the potential exists for thedevelopment of such a process. We conclude that this possibility is deserving ofcontinued investigation.

79

WSRC-TR-96-0088

9.0 CONCLUSIONS

The SRS has numerous waste sites and waste streams contaminated with toxic heavymetals, radionuclides and toxic organic compounds. Yet, few wastewatereat SRS havesignificant heavy metal contamination with an absence of additional radiological and/orcarcinogenic pollutants. A notable exception is the coal pile runoff basins and theirassociated underlying groundwaters. However, these waters typically have extremelyTow pH and high levels of iron and aluminum that make them particularly difficult toremediate in terms of the adsorption of toxic metals.

Numerous algal strains with desirable characteristics for bioremediation can be culturedin defined media with rapid growth and the ability to achieve high densities when thecultures are in stationary phase. Furthermore, algal-foam aggregates can be preparedwith sufficient structural integrity when subjected to a range of chemicals typicallyencountered in wastewaters. In addition, the foam can be converted back to a liquidrather easily with the addition of chemicals if desired. This adds flexibility to aspects ofthe process relating to disposal and recovery of pollutants from the spent foam.

However, results from experiments designed to assess and optimize toxic metalremoval from actual SRS wastewaters in bioreactor systems packed withfoam/biomass aggregates were generally disappointing. An experiment withradionuclide uptake by the foam/biomass aggregates provided slightly moreencouraging results, with the alga Cyanidium caldarium proving to be the best ofseveral types of biomass tested. However, the performance of Cyanidium (~ 35%removal) was far inferior to that of a TEVA resin (>95%) in comparative tests. Thus,these results did not offer encouraging prospects for scale-up and commercialization.Lack of success in this area was partially attributable to the nature of the wastewateravailable for study and the lack of appropriate bioreactor systems for contactingoptimization.

More encouraging results were obtained evaluating the foam/biomass aggregateconcept for use in biodegrading toxic organic compounds. The TCE-degradingbacterium Burkholdia cepacia was used for this part of the study. This organism is notordinarily suitable for use in bioreactors due to its poor adhesion capabilities. However,we demonstrated that it is possible to embed B. cepacia so that passing 50 ml waterthrough 2 g particulate foam washes out only 0.1% of the embedded cells, and 2000 mlwater washes out only 0.2% of the cells. Surfactant type, surfactant concentration, andbiomass density were most important in determining bacterial retention.

Although incapable of growth (cell division) in culture media, foam-embedded B.cepacia G4 cells are metabolically active. The nonculturability of embedded cells is notdue to free TDI content of prepolymers or to temperature or pH changes during theembedding process.

80

WSRC-TR-96-0088

Foam containing phenol-induced B. cepacia G4 removed substantial amounts of TCEfrom test solutions. TCE removal by embedded cells sometimes equaled that by freecells. Substantial benzene degradation by embedded G4 was also seen. However,variable results obtained with actual groundwaters suggest that groundwatercomposition affects TCE removal by embedded G4.

TCE degradation activity by embedded G4 can be induced by benzene as well as"phenol;: Once TCE-degrading activity is induced, it is of limited duration. However, G4can be induced after it has been embedded. Growth and immobilization can thus beseparated from induction and use of the product. This may allow repeated inductionand reuse, extending the useful lifetime of the product.

81

WSRC-TR-96-0088

10.0 RECOMMENDATIONS FOR FUTURE WORK

Continued research to develop a metal or radionuclide removal/reclamation processutilizing selected microbes embedded in foam has some merit because of the followingfeatures:

(1) demonstrated ability to culture algal strains with severe environmental 'irequirements (for ease in maintaining a unialgal condition),

(2) an affinity of certain algae for certain metals and radionuclides, and(3) the successful demonstration of the foam's ability to maintain structural integrity

under harsh conditions, house viable organisms with minimal washout, andbe readily dissolved with defined chemical treatment.

Although foam-embedded biomass might prove to be an effective means of metalremoval in some applications, we do not feel that it holds enough potential for theremediation of SRS coal pile runoff water (basin or groundwater) to merit continuedinvestigation. Any further work along these lines would hinge on the identification of awastewater more amenable to treatment. Such a water source would preferablycontain a single toxic and/or valuable metal of concern. It should also be of appropriatepH, should not have excessive iron levels, and should not be contaminated with othertypes of pollutants (e.g. hazardous organics or radionuclides) requiring additionallimitations to laboratory studies. In view of our previous extensive survey of on-sitewastewaters, identification of such a wastewater at SRS is unlikely.

Continued work with TCE-degrading bacteria would appear potentially rewarding. Anyfurther research should be ultimately directed toward achieving control over the TCEdegradation process, maintaining it in a bioreactor system, and assessing the efficiencyand economic competitiveness of the technology.

82

WSRC-TR-96-0088

11.0 ACKNOWLEDGMENTS

Preparation of this report was part of a Department of Energy/Office of TechnologyDevelopment (DOE/OTD) funded project under the direction of OTD Program ManagerRashalee Levine. James Wright, Will Laveille, and Allison Blackmon served as theTechnical Program Officers representing DOE in overseeing the work at SRS. PaulHermann, a consultant to industrial partner Frisby Technologies Inc., was thedeveloper of the prepolymer, a unique ingredient in the hydrophilic foam upon whichmuch of the study was based. Other Frisby personnel who played major roles in thestudy included Tanya Youngblood and Tom Baranek, who provided sample preparationand test rig support activities from the Frisby branch lab in Aiken.

We would like to thank Christopher J. Berry and Ed Becker for assistance with GCanalyses, Greg Powers (RCS Corporation) and Denis Altman for the collection ofgroundwater samples, Dr. Robin Brigmon for assistance with respirometry, and Dr.Terry Hazen for advice and suggestions. B. cepacia strains and advice on culturetechniques were generously provided by Dr. Malcolm Shields (University of WestFlorida), P. aeruginosa was provided by Brenda Faison (Oak Ridge NationalLaboratory), and yeast strains were provided by Sid Crow (Georgia State University).We also wish to thank the following SRTC personnel who made major contributions tothe preparation of this report and/or the work described herein: Mary Anne Johnson,Marie Youmans, Mel Bryant, Laura Tovo, Bill Boyce, Donna Beales, and Bill Mackey.Some of the other SRS personnel who provided key information in support of theproject are listed in Appendix 3.

This research was supported in part by appointments of two of the authors (JCR andJWSD) to the Postgraduate Research Program at the Westinghouse Savannah RiverSite administered by the Oak Ridge Institute for Science and Education.

83

WSRC-TR-96-0088

12.0 LITERATURE CITED

Atlas, R.M. 1993. Handbook of Microbiological Media. CRC Press, Boca Raton. 1079

Barklay, W., J. Johansen, P. Chelf, N. Nagle, P. Roessler, and P. Lemke. 1986.Microalgae culture collection. Solar Energy Research Institute Publication SERI/SP-232-3079. pp. 1-147.

Bettman, H., and H.J. Rehm. 1984. Degradation of phenol by polymer entrappedmicroorganisms. Appl. Microbiol. Biotechnol. 20:285-290.

Bettman, H., and H.J. Rehm. 1985. Continuous degradation of phenol(s) byPseudomonas putida P8 entrapped in polyacrilamide-hydrazide. Appl. Microbiol.Biotechnol. 22:389-393.

Bold, H.C. 1949. The morphology of Chlamydomonas chlamydogama, sp. nov. Bull.Torrey Bot. Club 76:101 -108.

Braun-Howland, E.B., S.A. Danielsen, and S.A. Nierzwicki-Bauer. 1992. Developmentof a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.Biotechniques 13:928-932.

Carolina Biological Supply Company. 1978. Culturing Algae. #45-8192. CarolinaBiological Supply Co., Burlington, N.C.

Castenholz, R.W. 1982. In: Starr, M.P. (ed.) The Prokaryotes, Springer-Verlag, NewYork. 236-246.

Ehrhardt, H.M., and H.J. Rehm. 1985. Phenol degradation by microorganismsabsorbed on activated carbon. Appl. Microbiol. Biotechnol. 21:32-36.

Folsom, B.R. and P.J. Chapman. 1991. Performance characterization of a modelbioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4.

Folsom, B.R., P.J. Chapman, and P.H. Pritchard. 1990. Phenol and trichloroethylenedegradation by Pseudomonas cepacia G4: kinetics and interactions betweensubstrates. Appl. Environ. Microbiol. 56:1279-1285.

Gadd, G.M., 1990. Accumulation of Metals by Microorganisms and Algae, in K.J. Rehm(Ed.), Biotechnology Handbook 6B Special Microbial Processes VCHVerlagsgesselschaft: Weinheim.

Gekeler, W., E. Grill, E.L. Winnacker, and M.H. Zenk. 1988. Algae sequester heavymetals via synthesis of phytochelation complexes. Arch. Microbiol. 150:197-202.

84

WSRC-TR-96-0088

Guillard, R.R.L., and J.H. Ryther. 1962. Studies on marine planktonic diatoms I.Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol.8:229-239.

Harris, P.O., and G.J. Ramelow. 1990, Binding of metal Ions by participate biomassderived from Chlorella vulgaris and Scenedesmus. quadricaudaT Envrion. Sci. Tech. 24:220-227.

Hobbie, J.E., R.J. Daley, and S. Jasper. 1977. Use of Nuclepore filters for countingbacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:1225-1228.

Levinson, W.E., K.E. Stormo, H.-L Tao, and R.L. Crawford. 1994. Hazardous wastecleanup and treatment with encapsulated or entrapped microorganisms. In Biologicaldegradation and bioremediation of toxic chemicals, edited by R.S. Chaudhry.Dioscorides Press, Portland, OR.

Luu, P.P., C.W. Yung, A.K. Sun, and T.K. Wood. 1995. Appl. Microbiol. Biotechnol.44:259-264.

Macaskie, L.E. 1991. The application of biotechnology to the treatment of wastesproduced from the nuclear fuel cycle: biodegradation and bioaccumulation as a means oftreating radionuclide-containing streams. Critical Reviews in Biotechnology 11, 41-112.

Nichols, H.W. and H.C. Bold. 1965. Trichosarcina polymorpha Gen, et. sp. Nov. J.Phycol. 1:34-38

O'Reilly, K.T., and R.L. Crawford. 1989a. Kinetics of p-cresol degradation by animmobilized Pseudomonas sp. Appl. Environ. Microbiol. 55:866-870.

O'Reilly, K.T., and R.L. Crawford. 1989b. Degradation of pentachlorophenol bypolyurethane immobilized Flavobactehum cells. Appl. Environ. Microbiol. 55:2113-2118.

Pringsheim, E.G. 1964. Pure cultures of algae, their preparation and maintenance.New York, Hafner.

Provasoli, L., J.J.A. McLaughlin, and M.R. Droop. 1957. The development of artificialmedia for marine algae. Arch Mikrobiol. 25:169-173.

Rhee, S.-K., G.M. Lee, and S.-T. Lee. 1996. Influence of a supplementary carbonsource on biodegradation of pyridine by freely suspended and immobilizedPimelobactersp. Appl. Microbiol. Biotechnol. 44:816-822.

85

WSRC-TR-96-0088

Roszak, D.B., D.J. Grimes, and R.R. Colwell. 1984. Viable but nonrecoverable stageof Salmonella enteritidis in aquatic systems.

Schlosser, U.G. 1982. Sammlung von Algenkulturen. Ber. Deutsch. Bgt:J3es. 95:181-276!

JShields, M.S., S.O. Montgomery, P.J. Chapman, S.M. Cuskey, and P.H. Pritchard.1989:"'Novel pathway of toluene catabolism in the trichloroethylene-degradingbacterium G4. Appl. Environ. Microbiol. 55:1624-1629.

Shields, M.S., and M.J. Reagin. 1992. Selection of a Pseudomonas cepacia strainconstitutive for the degradation of trichloroethylene. Appl. Environ. Microbiol. 58:3911-3983.

Starr, R.C., and J.A. Zeikus. 1993. UTEX - The culture collection of algae at theUniversity of Texas at Austin. J. Phycol. 29 (Supplement): 1-106.

Tanaka, H., H. Kurosawa, and H. Murakami. 1986. Ethanol production from starch bya coimmobilized mixed culture of Aspergillus awamori and Zymomonas mobilis.Biotechnol. Bioeng. 28:1761-1768.

Ting, Y.P., F. Lawson and I.G. Prince. 1989. Uptake of cadmium and zinc by the algaChlorella vulgaris: Part 1. Individual ion species, Biotech. Bioeng., 34, 990-999

Ting, Y.P., F. Lawson, and I.G. Prince. 1991a. Uptake of cadmium and zinc by thealga Chlorella vulgaris II. multi-ion solution, Biotech. Bioeng., 37:445-455

Ting, Y.P., F. Lawson, and I.G. Prince. 1991b. The influence of cadmium and zinc onthe cell size distribution of the alga Chlorella vulgaris, Chem. Eng. J., 47:23-34

U.S. EPA. 1986. Test Methods for Evaluating Solid Waste, Third Edition. U.S.Environmental Protection Administration, SW-846.

Volesky, B., (Ed.), 1990. Biosorption of Heavy Metals, CRC Press Inc., Boca Raton,Florida.

Weir, S.C., S.P. Dupuis, M.A. Proventi, H. Lee. and J.T. Trevors. 1995. Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated andfree Pseudomonas sp. UG14r cells in creosote-contaminated soil slurries. Appl.Microbiol. Biotechnol. 43:946-951.

Wilde, E.W., and J.R. Benemann. 1993. Bioremoval of heavy metals by the use ofmicroalgae. Biotechnol. Adv. 11:781-812.

86

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Wilde, E.W. and J.C. Radway. 1994. Wastewaters At SRS Where Heavy Metals Are APotential Problem . Westinghouse Savannah River Company, Savannah RiverTechnology Center, Aiken, S.C. WSRC-TR-94-0387.

WS'RC, 1993. Savannah River Site Environmental Report for 1992. WSRC-TR-93-075.Westinghouse Savannah River Company. Savannah River Site. Aiken, SC 29808.

WSRG, 1995 Savannah River Site Groundwater Monitoring Program. 2nd Quarter1995. ESH-EMS-950394.

Woodward, J. 1988. Methods of immobilization of microbial cells. J. Microb. Methods.8:91-102.

Xu, P., X.M. Qian, Y.X. Wang, and Y.B. Xu. 1996. Modelling for waste water treatmentby Rhodopseudomonas palustris Y6 immobilized on fibre in a columnar bioreactor.Appl. Microbiol. Biotechnol. 44:676-682.

87

WSRC-TR-96-0088

APPENDICES

A - l

WSRC-TR-96-0088

APPENDIX 3

IDENTIFICATION, CHARACTERIZATION AND SELECTION OF METALCONTAMINATED WASTE SITES AND WASTE STREAMS AT SRS

A-2

Table A.3.1. Toxic Metals And Regulatory Limita1ioft*rTR-96-0088

Metal

Aluminum

Antimony

Arsenic

Barium

Beryllium

Cadmium

Chromium (111)

Copper

Lead

Mercury

Nickel

Selenium

SBver

Zinc

PriorityPollutant a

no

yes

yes

no

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

NationalDrinking

Water Std.b

mg/1

(.05)

0.005

0.05

2

0.004

0.005

0.1

1.3

0.015

0.002

0.1

0.05

(09)

Aquatic LifeFreshwaterStd. c ugl\

0.174

360

"

1.79

984

9.2

34

2.40

789

20

1.23

65

EPA QualityCriteria

FreshwaterLKj/1

1600

190

~

5.3

0:66°

120°

6.5°

1.3°

0.012°

56°

35°

1.2°

59°

WaterQualityCriteriaHuman

Health oq/I

4308

1.4

"

1.17

10

673077

50

0.153

4584

10

50

*

MaximumContaminant

Concentrationsmg/Ie

1

5

200

0.1

0.5

10

1.5

0.2

10

5

20

700

Waste Std.,RegulatoryCone. Units

ppm '

"

5

100

1

5

5

02

1

5

1 Classified .by the Clean Water Act. Section 307

5 Secondary OW standards in parentheses

' S.C. Aquatic life Standard, criterion maximum concentration

1 At hardness 50

e 40 CFR. 1. pg374. Section 261.24 (7/1 /86)

Federal Register 1992

A-3

Table A.3.2 ER Waste Units With Potential for Bioremediation ofToxic Metals and/or Radionuclides Showing Information Contacts

Section #*C-5C-9C-10C-11C-12C-13C-14C-15C-16C-17C-18C-19D-20-3CM

D-6D-6D-8D-9D-10D-12D-13D-14D-15

D-16D-17

E-1E-4E-6E-7E-10E-19E-20

Waste Unit NameC-Area Burning/Rubble PitCMP pits (7)D-Area Burning/Rubble Pits (2)F-Area Burning/Rubble Pits (3)K-Area Burning/Rubble PitK-Area Rubble PileL-Area Burning/Rubble PitL-Area Rubble Pit 131-1LL-Area Rubble Disposal Pile 131-3LMisc. Chem Basin/Metals Burn PitP-Area Burning/Rubble PitR-Area Burning/Rubble Pits (2)488-D Ash Basin716-A Motor Shop Seep. BasinCoal Pile Runoff Basins A,C,D,F,H,K,& P

D-Area Oil Seepage BasinF- and H-Area Retention BasinsK-Area Reactor Seepage BasinL-Area Oil/Chem BasinL- and R-Area Acid/Caustic BasinsNew TNX Seepage BasinOld F-Area Seepage BasinOld TNX Seepage BasinR-Reactor Seepage Basins

Road A Chemical BasinSRL Seepage Basins (4)

Tank 16Gunsite 218Burial Ground ComplexCentral Shops Sludge LagoonFord Building SeepageBasinSilverton Road Waste SiteTNX Burying Grounds

Building #131-C080-17g,-17.1g,-18g,18431-D,-1D231-F, 1F.-2F131 -K631-20G131-L131-1L131-3L731-4A.-5A131-P131-R.131-1R488-D904-101G189-C.-K.-P, 788-3A,489-D, 289-F.-H631-G281-3H.281-3F904-65G904-83G904-77G, 904-79G904-102G904-49G904-76G904-57G.-58G, 59G,60G,-103G,-104G904-111G904-53G1.-53G2,S4G.-55G241-H631-23G643-E, 643-7E080-24G904-91G731-3A643-5T

SRS ContactR. Plunkett (4-6797)R. Soucha (4-6908)J. Hammock (4-1801)J.Hammock(4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)J. Hammock (4-1801)K. Ward (4-6941)R. Plunkett (4-6796)K. Ward (4-6941)

R. Plunkett (4-6797)K. Kuelske (4-6659)G. Blount (4-6775)G. Blount (4-6775)G. Blount (4-6775)R. Soucha (4-6908)K. Kuelske (4-6659)K. Kuelske (4-6659)K. Wise (4-1819)

R. Soucha (4-6908)K. Jerome (4-6786)

T. Gaughan (4-6773)H. Hickey (4-1802)K. Lewis (4-6750)R. Plunkett (-4-6797)G. Blount (4-677S)K. Ward (4-6941)K. Kuelske (4-6659)

a - Section numbers referenced in RCRA Facility Investigation/CERCLA Remedial Investigation Workpian Summaries

A-4

Table A.3.3 ER Waste Units With Best Potential forBioremediation of Toxic Metals and/or Radionuclides Showing

Selection Criteria

Sect#"

C-9C-11C-12C-14C-15C-17C-18D-20-3D-4

D-6D-6D-8D-9D-10D-12D-13D-14D-1S

D-16E-1E-6E-10E-19E-20

Waste Unit Name

CMP Pits (7)F-Area Burning/Rubble Pits (3)K-Area Burning/Rubble PitL-Area Burning/Rubble PitL-Area Rubble Pit 131-1LMisc. Chem Basin/Metals Burn PitP-Area Burning/Rubble Pit488-D Ash Basin716-A Motor Shop Seep. BasinCoal Pile Runoff Basins A,CtD,F,H,K,

D-Area Oil Seepage BasinH-Area Retention BasinK-Area Reactor Seepage BasSinL-Area Oil/Chem BasinL- and R-Area Acid/Caustic bAsinsNew TNX Seepage BasinOld F-Area Seepage BasinOld TNX Seepage basinR-Reactor Seepage Basins

Road A Chemical BasinTank 16Burial Ground ComplexFord Building Seepage BasinSilverton Road Waste SiteTNX Burying Ground

Building

080-17g,-17.1g,-18g,231-F, 1F.-2F131-K1314.131-1L731-4A.-6A131-P488-D904-101G189-C.-K.-P, 788-3A,489-D, 289-F.-H631-G281-3H904-65G904-83G904-77G, 904-79G904-102G904-49G904-76G904-57G,-58G, 59G,60G,-103G,-104G904-111G241-H643-E, 643-7E904-91G731-3A643-6T

TypeWater"

GWGWGWGWGWGWGWSW&GWGWSW&GW

GWGW SWGW7GWGWGW&SWGW&SW7GWGW

GWGWGWGW&SWGWGW

Char.DataAvail.?YESYESYESYESYESYESYESYESYESYES

YESYESYESYESYESYESYESYESYES

YESNOYESYESYESYES

MetalContain.*

PbAl.SrAl.PbPbPbPbPb,AICd.Cr, As, AI,CSbCd,Cr.As,AI,C

AI,PbAI,Pb,SbAI,PbCd.PbAIAI,PbAlAl.CdPb, Hg,AIAi,Cd,Pb,Hg

Hg,PbAl.Cd.PbCd,Pb,Hg,Sb,NiPb,AIAL,Pb,Be,SbHg,Pb,AI

Rad.Contain.?

NOYESNONONOYESNONONEYESGWONLY

YESYESYESYESNONOYESNOYES

NOYESYESYESNOYES?

Remed.Req'd?

YESYESYESYESYESYESYESYES |YESYES

YESYESYESYESYESYESYESYESYES

YESYESYES?YESYES

a - Section numbers referenced in RCRA Facility Investigation/CERCLA Remedial Investigation Workplan Summaries

b - G W - Ground WaterSW-Surface Water

- Based on exceedances reported in 1992/1993 SRS Monitoring Reports

A-5

WSRC-TR-96-0088

Table A.3.4 SRS Waste Sites where heavy metal concentrations inunderlying ground waters exceed drinking water standards

(based on data from the SRS Environmental Report for 1992,WSRC-TR-93-075).

SiteA-Met Burn PITM-Area HWMF

Misc. Chem Basi

Motor Shop Oil B

Plume def. wells

Silverton Rd. Wa

C-Ois. Basin

C-Seep. Basin-K^Acid/Caustic BK- BR PitK-Dis. Basin

K-Ret Bas.

contaminantPbSbPbHgUtoxic organicsradionuclidesPbradionuclidestoxic organicsSbradionuclidestoxic organicsCdPbHgradionuclidesToxic organicsSbBePbRadionuclidesToxic organicsPbradionuclidesPbtritiumPbPbPbradionuclidesPbradionuclides

unitmg/lmg/lmg/lmg/lmg/l

mg/l

mg/l

mg/lmg/img/l

mg/Img/lmg/l

mg/l

mg/l

mg/lmg/Img/l

mg/l

standard0.0150.0050.0150.00200.0020

0.015

0.005

0/0050.0150.002

0.00500.00100.015

0.015

0.015

0.0150.0150.015

0.015

maximum0.0290.0150.0730.00240.029

0.036

0.0085

0.00810.140.0034

0.00970.00430.040

0.16"

0.044

0.0220.0330.090

0.017

#wells sa641424241424277777720320420920821029292929292244943344

Swells12512408216216212120111315152214223314

A-6

WSRC-TR-96-0088

Table A.3.4. (Cont'd)

SiteL-acid/caustic &

L-BR pitL-Dis. Bas.L-RX Seep. bas.

P-BR Pit

P-CPRBP-Dis. Bas.

P-Seep Bas.

R-Acid/Caustic b

R-Dis. bas.R-Seep Bas.

E-Area Haz. wast

Old Buna! grnd

contaminantCdPbPbCdTe-99radionuclidestoxic organicsPbPbPbtritiumPbtoxic organicsPbPbtritiumPbtritiumtoxic organicsPbradionuclides.Pb

Cd

PbHgSr-90radionuclidesSb

Pb

tritiumCd

PbHgradionuclidestoxic organics

unitmg/lmg/lmg/lmg/l

mg/lmg/lmg/l

mg/l

mg/lmg/l

mg/l

mg/l

•mg/Imgflmg/lmg/l

mg/lmg/l

mg/lmg/lmg/I

standard0.0050.0150.0150.005

0.0150.0150.015

0.015

0.0150.015

0.015

0.015

0.0150.0050/0150.0020

0.0050.015

0.00500.0150.002

maximum0.0680.0470.0330.0059

0.0690.0740.046

0.049

0.0340.093

0.047

0.022

0.0230.0960.0200.0080

0.00690.042

0.0280.230.004

Swells sa44444444244

4

4

42

27774

43

21

2121

21

ye - -44

4

37

3737

48 •30

tfwells11111223213

2

1

1

1

2571

1

12

16

16

4

50

131

8

13

5

356

A-7

WSRC-TR-96-0088

Table A.3.4. (Cont'd)

SiteRadioactive wast

F-Acid/Caustic B

F-8urma Rd. Rub

F-BRPits &RPF-canyon etc.

F-CPRB

F-Process Sewer(inactive)

F-Seep basin

•

F-sludge land Ap

contaminantPb

HgNiradionuclidestoxic organicsmore Pb &SbPbradionuclidesPbradionuclidesno metalsPbSr-90Cs-137Toxic organicsRadionuclidesPb

Radionuclidestoxic organicsPb

radionuclidestoxic organicsSb

Cd

PbHg

NiUradionuclidestoxic organicsNO2+NO3-NAs

Pb

Hgradionuclides

unitmg/lmg/lmg/l

mg/l

mg/l

mg/l

mg/l

mg/l

mg/lmg/lmg/lmg/Img/lmg/l

mg/Img/lmg/lmg/l

standard0.0150.0020.10

0.015

0.015

0.015

0.015

0.015

0.0050.0050.0150.0020.100.02

100.0500.0150.002

maximum0.0330.00260.11

0.097

0.025

0.86

0.15

0.020

0.0120.0370.130.0f20.385.4

8850.0960.150.0058

#wells sa45

45

45

45

45

6

6

5

5

10

9

310

10

55

53

3

3

67

67

6767

676773

27

6718

4

4

4

Swells21

1

33

17

1

2

4

3

5

2

1

6

5

31

1

1

2

23

19

196

32758

245

1

3

12

Table A.3.4. (Confd)

SiteOld F seep bas.

contaminantSr-90U

radionuclidestoxic organics

unit

mg/l

standard

0.02

maximum

0.077

Swells sa4

4

4

4

Swells21

3

2

A-8

WSRC-TR-96-0088

H-Canyon Bldg.

H-CPRB

H-inactive Proces

H-Ret. basins

H-Seep bas.

H-area tank farm

Z-Area SaltstoneN-BR PitsN-Diese! Spill

Ford Bldg Seep

Hydrofluoric AciD-CPRB & Ash b

Pb

radionuclidestoxic organicsPb

radionuclidesPb

tritiumSb

Pb

radionuclidesSb

AsCdCo-60Pb

HgNO2+NO3-Nradionuclidestoxic organicsCd

Pb

HgradionuclidesSb

PbPb

toxic organicsPbradionuclidesPbCd

Cr

radionuclidestoxic organics

mg/l

mg/l

mg/l

mg/lmg/l

mg/lmg/lmg/l

mg/lmg/lmg/l

mg/lmg/lmg/l

mg/lmg/lmg/l

mg/l

mg/lmg/lmg/l

0.015

0.015

0.015

0.0050.015

0.0050.0500.005

0.0150.00210

0.0050.0150.0020

0.0050.0150.015

0.015

0.0150.0050.10

•

0.063

0.036

0.020

0.0120.024

0.0130.100.0098

0.0710.007990

0.341.00.0039

0.0080.0540.021

0.022

0.0400.0300.82

4

4

4

4

4

7755

5108

108108

40

108

108

108

113

26

25

323232

3

49

5

5

54

14

1414

14

2

42

24

1

72

24

13

21

10

13

15

49

88

5

9

198181

11

31

112

3

4

4

Table A.3.4. (Confd)

SiteTNX Burying Gro

G-Area CMP Pits

IWT sites

contaminantPb

Hgradionuclidestoxic organicsPbtoxic organicsradionuclidesPb

unitmg/l

mg/l

mg/l

mg/l

standard0.015

0.002

0.015

0.015

maximum0.016

0.0029

L0.073

0.026

#wells sa5

55

5

19

19

19

2

#wells1

12

25

3

1

1

A-9

WSRC-TR-96-0088

Par sludge land aNPR site

Road A Chem. Ba

Sanitary Landfill

Pb

Cd

Pb

radionuclidesPbHgSbCdPb

Hgradionuclidestoxic organics

mg/lmg/lmg/l

mg/lmg/lmg/lmg/lmg/lmg/l

0.0150.0050.015

0.0150.0020.0050.0050.0150.002

0.0150.0080.044

0.0540.00270.0120.0310.0210.0029

4

14

14

14

55

304242

425G

5G

1

1

2

1

31

2121

1

4

15

A-10

WSRC-TR-96-0088

Table A.3.5. Maximum concentrations (mg/l) of selected heavymetals based on samples collected from Coal Pile Run Off Basins

(CPRBs) at SRS during three studies. Area of CPRB shown inparenthesis.

MetalAlAsBeCdCr(!ll)Cu

HgNiPbSe

DW Std.a

0.0500.0500.0040.0050.1001.30.0020.1000.0150.05

Study#1b

264 (D)0.077 (D)0.0274 (D)0.047 (D)0.222 (D)1.395 (D)0.00036 (K)4.7 (D)0.0149 (C)0.018 (H)

Study#2c

NSe

0.10 (D)NS

0.056 (D)0.42 (D)

NS0.005 (D)

NS0.09 (H)0.05 (D)

Study#3d

56.8 (D)0.086 (D)0.014 (D)0.024 (D)0.035 (D)0.296 (A)

NS0.657 (D)0.049 (D)0.200 (D)

% DW Std.528000

200685

11200420107250

4700600400

a National drinking water standard. AH are primary except Al which is secondary.

bO'Brien and Gere 1987.

cCorbley,A. L. 1992.

dWilde, et. al., 1994 (unpublished data)

eNot Sampled

A - l l

WSRC-TR-96-0088

Table A.3.6. Past and Present Activities Within Principal Areas ofSRS

AreaABCDEF

G*HKLMNPRs

TNXYZ

R--X-----'XX

•-XX

- •

-

• -

s-- •

-

-

- •

X-X

•

•

•

- •

. - • •

- •

•

• -

FF

-----

XT--

XR-

• -

-

. -

-

RWX--XXXXx--XX-

' • - •

X• .

-

X

swXXXX' -X .XXXXXXX-XX-

LXX-X-XXX--X---XX--

ActivitiesRSFFRWSWL

Code:ReactorSeparationsFuel Fabrication (R-reactor fuels; T=tritium)Radioactive or mixed waste managementSanitary waste treatmentLaboratory activities

^Facilities not confined to any of the other specific areas arecollectively considered to exist in G-Area.

A-12

Table A.3.7 Metal-Containing Wastes at SRS

Area

A

A

A

A

A

A, B,C, D,F, G,H, K,L, P,

s,TNXC

Description

Photo lab waste

Medical Dept.photo waste

SRTC lab wastes

WastewaterNeutralizationFacilityMet Lab wastes

Sanitarywastewatertreatment plants

Disassemblybasin

Radioactive?

No

No

Yes (low level)

No

No

No

Yes

Metals

Ag

Ag

Hg, Cr, U, Pu

Hg

Ni, Cr, Fe, AI,Cu, others?

Pb, Zn, Cu, AI

Cs, Pu, AI, Fe,others

Volume

1200-1600gal/yr350gal/yr

52,000gal onhand

5000 galon hand

4 gal/yrnow, upto 20 gal/mofutureUp to4,000,000 gal/day(H Area)

3,550,00Ogalpresent

CurrentlyGenerated?

Yes

Yes

Yes

No

Yes

Yes, butsome willshut down inMarch '95

No

AnalysisAvailable?

Sampledpost-treatmentSampledpost-treatmentYes

Yes

In progress

Yes

Partial

CurrentTreatment

Ion exchange(A-Area AgRecovery Unit)Ion exchange(N-Area AgRecovery Unit)Ion exchange,storage

Ion exchangeupon demand

Neutralization,storage

Conventionalwastewatertreatment, landapplication

None

AdditionalTreatmentNeeded?No

No

Eventual disposalviaLLW(haz.)and landfill(nonhaz.)No

Procedure beingestab. to ppt. Cr,allowingdischarge tosanitary sewerNot at present.Metal levelsoccas. near landapplication limits

Yes (

M

od

•3d

©o00co

Table A.3.7 (Conf d).

Area

D, N

D %

F

F

F

F

H

Description

Hg thiocyanatelab waste

Water QualityLab wasteSeparationswastes

Separations-FB-Line Waste(from chillers)CoolingmaintenanceshopSeparations-Evaporatoroverhead

CIF blowdownwater

Radioactive?

Yes

Yes

Yes

Yes

No

Yes

Yes

Metals

Hg

Hg

Similar to H-Areaseparationswaste

Pu, Cr

Pb

Similar tootherseparationswastes, butlower cone.Pb, Sr, Cs,Hg, poss.others

Volume

146drumson hand

?

Current:5000gal/mo.Future:20,000gal/mo.27 gal.on hand

<1drum/yr

formerly160,000gal/d,very littlecurrently75,000gal/yrprojected

CurrentlyGenerated?

No

yes

On smallscale

Intermittently

Yes

Yes

Projected for1996

AnalysisAvailable?

Yes

yes

Yes

In prep.-NMPSB0930009In prep.

Yes

Predicted,WSRC TR93623

CurrentTreatment

Ion exchange(Duolite GT-73)

Heavy waterrecoveryITP, ETF,immobilization

Recycled or sentto Mixed WasteStorage FacilityRecycling &reuse

ETF

Solidification @Saltstone

AdditionalTreatmentNeeded?Columns tend toplug

No

No

No

No

No

Volume reductiondesirable

COO5

i

ooCOCO

Table A.3.7 (Cont'd).

Area

H

H

K

L

M

Description

Separationswastes

Separations-Filter backwashwater, resinregenerationwaterDisassemblybasin

Disassemblybasin

Analytical andMetallurgy Labeffluents

Radioactive?

Yes

Yes

Yes

Yes

No

Metals

Current: Pu,Fe. Duringnormaloperation: Pu,Sr, Cs, Hg, Cr,U, Fe, Al, Ag,Ba, Tc, Pm,Ni, ThAl, Zn, Cs, Se,Co

Cs, Pu, Al, Fe,others

U, Th(sludge), Cs,Zn (water)

As, Ba, Cu,Ag, Pb

Volume

Current:5000gal/mo.Previous: 30,000gal/mo.

29,000-45,000gal/mo.

3,400,00Ogalpresent

3,400,00Ogalpresent

260gal/wk.

CurrentlyGenerated?

On smallscale

Yes

Yes (contam.continues dueto presenceof fuel)Yes (contamcontinues dueto presenceof fuel)Yes, but willmove soon

AnalysisAvailable?

Partial

Yes

Yes

YesSRTC-ADS-93-0411

Yes

CurrentTreatment

ITP, ETF, DWPF(vitrification),Saltstone (concrete)

pH adjustment,sent to tank farm(hi level) or GPevaporator(lower level)ion exchangesludgevacuumed

ion exchange,sludgevacuumed

DETF (metalppt. thenpressure flit.

AdditionalTreatmentNeeded?No

No

Disposal ofelutedcontaminants,resin, sludgeDisposal ofelutedcontaminants,resin, sludgeNo

ICO

©©00CO

Table A.3.7 (Confd).

X

Area

N

N

P

R

S

TNX

Description

Photographicwastes:EBASCO Svcs.,Medical Dept.,Paint Shop,Document Ctrl.Wash water-bubble tower andgas tank cleanupDisassemblybasin

Disassemblybasin

Lab & processwastewater (fromwasteimmobilizationactivities)Lab waste fromIDMS sampleanalyses

Radioactive?

No

No

Yes

Yes

Yes

No

Metals

Ag

Hg, Cr

Cs, Pu, Al, Fe,others

Cs, Pu, Al, Fe,others

Hg (current),variousradionuclides(future)

Hg

Volume

350 galon hand

4 drumson hand

4,800,000galpresent

4,500,000gal.present200,000galpresent

2320gal/yr

CurrentlyGenerated?

Yes

No

Yes (contam.continues dueto presenceof fuel)No

Cold runsonly

Yes

AnalysisAvailable?

Sampledpost-treatment

Yes

Yes

Yes

Yes- DHECpermitproposal,CPESdocumentYes

CurrentTreatment

Ion exchange (NArea AgRecovery Unit)

offsite vendor

Ion exchange,sludgevacuumed

None

Vitrification/storage. Steamstripping/reduct-ion of Hg

Offsite vendor

AdditionalTreatmentNeeded?No

No

Disposal ofelutedcontaminants,resin, sludgeYes

No

No

i

we

i?

o

oaiooooCO

Table A.3.7 (Conf d)

Area

Z

Description

Saltstone facilitywastes (fromwasteimmobilizationactivities)

Radioactive?

Yes (lowleve/mixedwaste)

Metals

Cs1 v i / , misc.beta/gamma,Cr(VI)

Volume

Current:15,000gal/mo,future: 3-6 X 1 0 6

gal/yr

CurrentlyGenerated?

Yes

AnalysisAvailable?

WSRCTR94-0364 (in prep)

CurrentTreatment

Immobilization/storage

AdditionalTreatmentNeeded?No

o

O5

ooCO00

WSRC-TR-96-0088TABLE A.3.8 ASSISTING PERSONNEL FOR WASTE SITE AND WASTESTREAM IDENTIFICATION AND CHARACTERIZATION

Numerous SRS personnel contributed information during our investigation toidentify metal contaminated wastewaters on the site. Many of them are listedbelow:

Name

R. AylwardJ. BakerD. K. BeasleyR. BeckC. BennettN.N. BhattJ. P. BiblerG. BlountD. BoringB. BoulineauD. BowmanE. BrassP. BrooksD.B. BurnsB. BushJ. ChenD. ClarkA.L CorblyD. CostnerB. CulliganW. DaughertyR.W.-DeibleE.L. DunbarG. FroidlS. FullerT. GaughanG.K. GeorgetonA. GibbsJ. GladdenM.D.D. GoodmanN. HalversonJ. HammockW. F. HarlowL. HaselowC.R. Hayes, Jr.H. Hickey

Organization

ERReactor Engineering RBOFSolid WasteHLW/WWPFEPDWater Quality LaboratoryTIWTERHPPhotographic ServicesRODSTHeavy WaterCIFHB-LineSaltstoneERESSSeparationsTLSMTSREHLW F Tank FarmCentral ShopsCSERHLW EngineeringRTSRTC/ESSHLWE H-ETFTLSERSeparationsEREPDER

A-18

WSRC-TR-96-0088

J. HowellR. HuffinesM.S. Jackson, Jr.R.W. JacksonK. JeromeW. JohnsonW.H. Jones, Jr.C. KnappKKuelskeC. LangtonK. LewisB.B. LooneyH.L. MartinJ. MayerH. MooreB. MyersM. NewmanR. NicholsT.O. OliverW.L PayneC. PickettJ. PickettR. PlunkettL.K. PressleyJR. PriceO.D. RosierG. RuckerH. SchultzB.D. SilasD. SimmonsW.R. SimsD.K. SingerDP. SkiffS.E. SmithR. SouchaS.M. SpearmanW. SpechtPJ. SpitzerSO. StallingsC.A. StanfordK. SteegC. StroganG. SwisstackD. ThompsonJ. Travis

SRTCPEHLW/DWPFSeparations H-CanyonERERBechtel ConstructionERERSRTCERSRTC/ESSRMEEPDHLW/DWPFReactor EngineeringSRELSRTC/ESSSep RBOFEPDF/H Tech SupportRME&TERSeparations MaintenanceDWPFReactorsERRMEnviron. & Chem. SystemsTLSERSite ServicesReactorsEnvironment & WaterERSRTCTESSeparationsReactor MaterialsDWPFFB-LineSRELSTZ/SaltstoneSSE

A-19

WSRC-TR-96-0088

L. Turner Separations F-Canyon

K. Ward ERF.A. Washburn Environ. RestorationR W. Weigel EPDM. Whitaker SRTC/ADSK. Wise ERD. M. Wittry RME

A-20

WSRC-TR-96-0088

APPENDIX 4

SELECTION AND CULTURING OF ALGAL STRAINS

A-21

WSRC-TR-96-0088

<o

*

a.

CE

LLS

ruu •

600-

500-

400-

300-

200-

100-

o"

/

. ; : • /

• • " • . y ^ - : -

• t . i i • . i i i - i

0 6 8 10 12 14 16DAYS

Figure /\.4.1 Initial growth curve for Chlorella capsulata

A-22

WSRC-TR-96-0088

120

100-

o

UJ

a.CO

QJ

o

10 15 20 25 30

40-

20-

0

Figure A.4.2 Initial growth curve for Chlorella fusca var. vacuolata

WSRC-TR-96-0088

90

80-

70-

60

DCLU0_(f)

i

LLJO

CO

cGJW

O. szt

50

40

30-

20-

10

Figure A.4.3 Initial growth curve for Amphiprora paludosa

A-24

WSRC-TR-96-0088

0 1 2 3 4 5 6 7 8 9 1050

Figure A.4.4 Initial growth curve for ChaetocerosgraoMs.

A-25

WSRC-TR-96-0088

0 2 4 6 8 10 12 14 16

1.00E+07 =

ccUJCL' 1.00E+06=

UJ

O

DAYS

Figure A.4.5 Initial growth curve for Navicala pelliculosa.

A-26

WSRC-TR-96-0088

600

00 2 4 6 8 10 12 14 16 18 20

DAYS

Figure A.4.6 Initial growth curve for Phaeodactyium Lricornutum.

A-27

WSRC-TR-96-0088

100

DCLUQ.C0

111O

WXScain

O

Figure A .4.7 Initial growth curve for SurireUa ovata.

A-28

WSRC-TR-96-0088

5000

10001

Figure A.4.8 . Initial growth curve for Peridiruum trochoideum.

A-29

WSRC-TR-96-0088

1.0E + 07

111

en_ i_ iLUO

1.0E + 06

1.0E + 05, , ,

0 1 2 3 4 5 6 7 8 9DAYS

Figure A.4.9 Large volume growth curve for Chlorella capsulata.

A-30

WSRC-TR-96-0088

1.0E + 07

1.OE + 05

FigureA.4.10 Large volume growth curve for Phaeodactylum tncornutum.

A-31

WSRC-TR-96-0088

1.0E + 07

1.0E+06;

LU

C/)

LU

o 1.0E + 05:

1.0E + 048

7.5 PPT

15PPT

30 PPT

37.5 PPT

Figure A.4.11Effect of salinity on the growth curve of Navicula pelliculosa.

A-32

WSRC-TR-96-0088

1.0E+07

DCoi

ULJ

o

1.0E+061

1.0E + 05

F/2 AT pH 6.5 j

F/2 AT pH 8.2 j

1 2 3 4 5 6 7 8 9

DAYS

Figure A. A. 12 Effect of pH on the growth curve of Navicula pelliculosagrown in F/2 media.

A-33

WSRC-TR-96-0088

1.0E+07—i r

1.0E + 06

DCLU

a.en

LU

O1.0E + 05

1.0E+04

I.O. AT pH 6.5 i

I.O. AT pH 8.2

Figure A. 4.13 Effect of pH on the growth curve of Navicula pelliculosa grownin F/2 enrichment salts added to "Instant Ocean solut.on

A-34

WSRC-TR-96-0088APPENDIX 6

BIOREMOVAL CAPABILITIES OF FOAM- EMBEDDED MICROBES

A-35

WSRC-TR-96-0088

APPENDIX 6A METHODS AND RESULTS FROM 14 EXPERIMENTS

Table A.6.1 Experimental Procedures and results for Experiment 1

Test Plan for Experiment 1 11/8/94

TESTING WITH EIGHT CHAMBERED TEST ASSEMBLY CONSTRUCTED BYFRISBY USING WASTEWATER FROM THE D- AREA COAL PILE RUNOFF BASIN

Experiment 1A

General Experimental ProtocolThe first experiments (1a and 1b) with the Frisby Test rig will be conducted usingwaste water from the D-Area coal pile runoff basin (D-CPRB). There are 11 metals ofconcern in D-CPRB water: Al, Be, Co, Ni, Zn Cd, Cr, As, Cu, Pb, and Se. All of thesewill be measured in triplicate by SRTC/ADS following the passage of selectedwastewater volumes through the test assembly using various formulations of filtermedia to assess metal removal efficacy. Flow rates, pressure drop and totalaccumulated flow volumes through each chamber will be monitored by pressure metersand flow/totalizer meters attached to each chamber. Collections of treated wastewaterfor metal analyses will be extracted from each chamber effluent line at four timeintervals (nominally 10, 30, 60, & 120 minutes). Untreated water samples will be usedas controls.PartObjective: Test system with wastewater but no filter media to evaluate the integrity ofthe test system in terms of:

leaching of metals from test system,plating of metals on test system, and

comparability between cartridges (chambers)

Experimental Procedure:

1. Select sampling location at the D-CPRB.

2. Collect about 34 gallons of D-CPRB water by filling eight 6-gallon carboys to the 4-gallon mark and one carboy to about the 2-gallon mark.

3. Measure pH, and other water quality parameters at the time of collection using aHoriba Water Quality Checker.

4. Record collection data in lab notebook and return to lab.

A-36

WSRC-TR-96-0088

5. Place two 50 ml aliquots of the raw waste water into labeled bottles (to be used ascontrols)

6. Calibrate and flush test assembly with Dl water and adjust flow to 0.03 gpm througheach chamber. Discard Dl water used to flush system.

7. Add 4 gallons of wastewater to each of eight feed tanks. Start pumps and feedwastewater at a rate of 0.03 gpm through the eight test chambers in a recycling mode.Collect 50 ml samples from the downstream side of each of the eight test chambersafter 10, 30, 60, and 120 min time intervals (representing approximately 0.3, 0.9, 1.8,and 3.6 gallons) and place in labeled bottles. Maintain flow at 0.03 gpm throughout run(by adjusting needle valves if necessary) and record pressure gage readings at 10minute intervals. Also record accumulative flow at each sample collection time as statedabove.

8. Preserve the 34 samples with acid HNO3 (0.1ml concentrated acid per samplebottle) for future ADS for analyses of the 11 metals described above.

9. Shut down test rig and re-zero flow/totalizer meters making sure all data arerecorded in lab notebook.

Experiment 1bObjective: Select an initial particle size configuration for subsequent experiments usingfoam and determine metal uptake by plain foam w/ no algae.

Experimental Procedure:

1. Have Frisby prepare two disks 1.27 cm (0.5 in) thick and 5.08 cm (2.0 in) in diameterusing a "standard" density. Prepare another batch of foam of the same density. Grindand sieve this batch of material to get three additional pairs of samples having threedifferent "particle size configurations and weighing the same as the two disks. Thus, theend result should be four pairs of samples all weighing the same, all made fromchemically identical foam prepared to the same density, and representing four differentparticle size configurations.

2. Pack the foam samples from step #1 into the eight test cartridges (one of each pairof duplicates samples on opposing sides of the test assembly).

3. Use same D-Area CPRB water as in Experiment #1. Restart system and maintainflow at 0.03 gpm while monitoring pressure drop and flow accumulations (volumes)through each test chamber for two hours while collecting chamber effluent samples atintervals of 10, 30, 60, and 120 minutes.

A-37

WSRC-TR-96-0088

4. Preserve the 32 50-ml samples along with two more controls from collection jug withoriginal controls and take all samples to ADS for analyses.

A-38

EXP1 .XLS

WSRC-TR-96-0088

(Table A..6.1 (Cont.)

Exp. 1- 1st tun with test riq, 11-8-94

PARAMETERS

EffluentDate coll.PHCond.

D coal runoff11/6794

2.43.18 mS/cm

TurbDOTempSal

Run 1 (To determine uniformity of columns

BlosorbentDurationModeFlow rate

Column

Rawwater

1

2

3

4

6

6

7

8

Sample

1-OA1-OBMean

1-1AM BMean

1-2A1-2BMean

1 3A1-3BMean

1-4A1^BMean

1-5A1-5BMean

1-6A1-68Mean

1-7A1-7BMean

1-6A1-8BMean

None30minRecirc0.30 gal/min.

Al

82.1783.0982.63

78.3977.9978.19

80.9380.84

80.88S

80.1381.1180.62

80.6480.0480.34

80.6280.7680.69

80.8680.65

80.7SS

79.8780.28

80.07S

80.2379.84

As

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

O.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.l<0.1

Be

0.02610.02610.0261

0.02490.02470.0248

0.02570.03090.0283

0.02630.0258

0.02605

0.02570.02570.0257

0.02260.021

0.0218

0.02060.02040.0205

0.02020.0201

0.02015

0.02030

0.01015

1

Cd

0.01720.0157

0.01645

0.01610.01510.0156

0.01510.0156

0.01535

0.0160.0155

0.01576

0.01580.0151

0.01545

0.01770.0154

0.01655

0.01540.0153

0.01535

0.01590.01590.0159

0.01610.0154

0.01575

-108.3

17.70.15%

Co

0.33270.32010.3264

0.57870.5666

0.57265

0.39060.37820.3844

0.34080.351

0.3459

0.35120.3583

0.35475

0.34820.3399

0.34405

0.34640.349

0.3477

0.35170.3484

0.35005

0.35170.361

0.35635

Cr

0.04490.04730.0461

0.04520.0471

0.04615

0.04770.05810.0529

0.0480.05780.0529

0.04810.05170.0499

0.04070.0492

0.04495

0.03920.0501

0.04465

0.03840.0421

0.04025

0.04260.001

0.0218

Cu

0.22580.22780.2268

0.22550.22490.2252

0.23350.2304

0.23195

0.22390.2256

0.22475

0.22310.2254

0.22425

0.19310.19090.192

0.19640.1976

0.197

0.18850.1886

0.18855

0.19170.19170.1917

Fe

471.3475.7473.5

456.8453.4455.1

474.4477.4476.9

472.8473.7

473.25

473.1472.7472.9

475471.8473.4

472.2471.1

471.65

470.5470

470.25

469.7469.8

469.75

Ni

0.93010.92350.9268

0.88450.8854

D.88495

0.91140.91880.9151

0.91980.918

0.9189

0.91790.92550.9217

0.90510.9146

0.90985

0.91050.9118

0.91115

0.90050.91210.9063

0.90640.9059

0.90615

Pb

<0.05<0.05<0.05

<0.05<0.05<0.05

<0.05<0.05<0.05^

<0.05<0.05<0.05

<0.05<0.05<0.05

<0.05"<0.05<0.05

<0.05<0.05<0.05

<0.05<0.05<0.05

<0.05<0.05<0.05

Se

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1Vr -

v<o:i<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

Zn

1.9681.917

1.9425

2.6032.562

2.5825

1.9141.9

1.907

3.8273.8633.845

1.9221.897

1.9095

1.8421.8621.852

1.8361.8121.824

1.814Y 1794

1.804

1.8031.7791.791

Flowgal.

9.38

9.91

10.22

10.16

8.42

8.63

9.53

9.49

A-39

EXP1.XLS

Table A..6.1 (Cont.)

Run 2 (To determine effect of foam particle s/ze^

BiosorbentDurationModeFlow rate

Column

1-cyl

S-cyt

Sample

1-1AM BMean

1-5A1-5BMean

Mean cylinder

2 12 mes

6-12 mes

1-2A1-2BMean

1-6A1-68Mean

Mean 12 mesh

3-10 mes

7-10 mes

1-3A1-3BMean

1-7A1-7B

Mean

Mean 10 mesh

4-tmesh

S-t mesh

1-4A J1-4BMean

1-8A1-8B

Mean

Mean 8 mesh

Foam only, no algae. Mesh sizes 8,10,12, unground cylinder. 4.44 g/cdumn30minRecirc0.30 gal/min.

At

79.9380.06

79.995

78.9779.1579.06

79.53

80.0279.63

79.825

77.7777.7377.75

78.79

79.2279.09

79.155

80.0280.53

80.275

79.72

79.2778.48

78.875

80.4480.65

80.545

79.71

As

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1

<0.1<0.1<0.1

<0.1

^0 ^

<0.1

<0.1<0.1

<0.1<0.1

<0.1

O.1<0.1<0.1

<0.1<0.1<0.1

<0.1

Be

0.01880.0189j

0.01885

0.02540.0255

0.02545

0.0222

0.02970.0262

0.02795

0.02470.02490.0248

0.0264

0.02570.02550.0256

0.0240.0213

0.02265

0.0241

0.0253.0.0253

0.0253

0.02060.0203

0.02045

0.0229

Cd

0.0160.0157

0.01585

0.01680.01660.0167

0.0163

0.0150.01680.0159

0.0160.01520.0156

0.0158

0.01540.0183

0.01685

0.01510.01610.0156

0.0162

0.01730.01650.0169

0.0160.0163

0.01615

0.0165

Co

0.43530.91790.6766

0.52580.515

0.5204

0.5985

0.45251.084

0.76825

1.2311.2

1.2155

0.9919

0.4010.8021

0.60155

0.97730.9392

0.95825

0.7799

0.9350.91920.9271

1.4311.4111.421

1.1741

Cr

0.04060.04220.0414

0.04930.0446

0.04695

0.0442

0.05250.0434

0.04795

0.04720.04420.0457

0.0468

0.04760.04560.0466

0.04090.04810.0445

0.0456

0.04480.0457

: 0.0452S

0.04180:0435

0.04265

0.044

Cu

0.21650.19890.2077

0.19650.19730.1969

0.2023

0.20850.2036

0.20605

0.20150.20210.2018

0.2039

0.21780.19860.2082

0.22160.2146

0.2181

0.2132

0.20370.2028

0.20325

0.21270.2104

0.21155

0.2074

Fe

467.7472

469.85

477.4476.5

476.95

473.4

473.6479.6476.6

472.547Z2

472.35

474.5

474.9477.3476.1

473.2467.8470.6

473.3

477473.2475.1

470.1470.3470.2

472.7

Hi

0.90150.91030.9059

0.9040.9119

0.90795

0.9069

0.91420.917

0.9156

0.89650.9032

0.89985

0.9077

0.91120.9108

0.911

0.91130.9034

0.90735

0.9092.

0.91230.90250.9074

0.90840.91020.9093

0.9084

Pb

O.05<0.05<0.05

<0.05<0.05<0.05

<0.05

<0.05<0.05<0.05

<0.05<0.05<0.05

<0.05

<0.05O.05<0.05

<0.05<0.05

<0.05

<0.05

O.05<0.05<0.05

O.05O.05<0.05

<0.05

/— 1 1

Se

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1

<0.1<0.1<0.1

<0.1<0.1

<0.1

<0.1

<0.1<0.1<0.1

<0.1<0.1<0.1

<0.1

• f -Qf i -O

In

1.821.7961.808

1.8171.8331.825

1.817

1.8521.87

1.861

1.8261.7821.804

1.833

3.8033.7693.786

1.8961.873

1.8845

2.835

1.8411.818

1.8295

1.8691.8651.867

1.848

088

Flow.g

8.46

7.17

7.815

8.36

7.82

8.09

8.12

7.78

7.95

9.14

7

8.07

A-40

WSRC-TR-96-0088Table A.6.2 Experimental procedures and results for Experiments #2

Experiments #2: 11/29/94

I. PURPOSEDetermine Effect of Particle Size on Bioremoval by Mastigocladus

II. Alga11-day-old Mastigocladus laminosus culture

III. FOAM:6% algae by dry weight air dried and seivedSamples obtained for 8,10, and 12 mesh

IV. EFFLUENTS: D-Area coal pile runoff basin - near outfall

V. TEST APPARATUS:Frisby Test rig

VI. PROCEDURE:Fill reservoirs w/ 4 liters of wastewaterFill columns with 4.4g. of foamRun at 0.3gpm for 30 minutesCollect samples and acidify (0.5ml cone. HNO3) prior to taking samples to ADSfor chemical analyses

VII. SAMPLE LABELS

The following labels will be used on samples sent to ADS: ;

.abel

2-1 a&b2.2a&b2.3a&b2.4a&b2,5a&b2.6a&b2.7a&b2.8a&b2.0a&b

Description

8mesh Mast.t i

10mesh Mast.i t

12mesh Mast.i t

#8mesh blank foami t

controls

A-41

EXP2F.XLS

Table A.6.2 (Cont.) WSRC-TR-96-0088

Exp. 2- Effect of particle size on bioternoval by 6% Mastigocladus -11 -29-94

PARAMETERS

EffluentDate coll.pHCond.

BlosorbentDurationModeFlow rate

Column Mesh

Raw NonewaterMean raw water

1 Control

2 Control

Mean control (8

3 8 mesh

4 8 mesh

Mean 8 mesh

6 10 mesh

6 10 mesh

Mean 10 mesh

7 12 mesh

8 12 mesh

Mean 12 mesh

D coal runoff11/28/94

2.45.88 mS/cm

248.419

0.30% •

Mastigocladus. 31, 6 % by dry wt, embedded 11-28. 4.44 g/column30minRecirc0.30 gal/min.

Sample Flow, ga

2-OA2-OB

2-1A2-18Mean

2-2A2-2BMean

mesh)

2-3A2-3BMean

2-4A

2-4BMean

2-5A2-58Mean

2-6A2-6BMean

2-7A2-7BMean

2-8A2-8B

Mean

9.28

9.639.455

8.34

10.379.355

8.68

10.239.455

8.56

8.66

8.S1

Al

250

246.2248.1

203.6258

230.8

246.4263.4254.9

242.85

222262.4242.2

220.48.045?

220.4231.3

242.3260.6

251.45

196.6237.3

216.95234.2

206.7243.2

224.95

189.1242.1

215.6

220.28

Be

0.05410.05380.054

0.03750.04870.0431

0:0470.0491

0.048050.0456

0.03950.0494

0.04445

0.03830.04890.04360.044

0.04250.04650.0445

0.06050.1256

0.093050.0688

0.07770.06690.0723

0.04350.053

0.04825

0.0603

Cd i

0.0450.045

0.045

0.03260.0433

0.03795

0.04270.04450.0436

0.0408

0.03710.04450.0408

0.03510.0442

0.039650.0402

0.03780.0411

0.03945

0.05340.1267

0.090050.0648

0.08120.0631

0.07215

0.04070.0449

0.0428

0.0575

Co

0.93870.9447

0.9417

0.86791.055

0.96145

1

1.171.085

1.0232

0.88251.274

1.07825

0.86031.415

1.137651.108

0.94931.208

1.07865

0.80040.9757

0.888050.9834

0.86061.002

0.9313

0.76630.9681

0.8672

0.8993

Cr

0.29310.291

0.2921

0.22760.27860.2531

0.27240.2787

0.276550.2643

0.24040.28060.2605

0.2336

0.2840.2588

0.2597

0.25770.27470.2662

0.22840.2736

0.2510.2586

0.23760.2767

0.25715

0.22630.2793

0.2528

0.255

Cu

0.61270.61470.614

0.48430.59930.5418

0.57860.6055

0.592050.567

0.51090.60610.5585

0.504, 0.60730.555650.557

0.55840.5981

0.57825

0.49990.5892

0.544550.561

0.50080.59160.5462

0.46690.5914

0.52915

0.538

Fe

17961767

1781.5

153619241730

18641973

1918.51824.3

16771968

1822.5

1654683.6

- 1168.81495.7

182019361878

144317331588

1733

151017701640

137717S2

1564.5

1602.3

Ni

2.9122.923

2.9175

2.2972.8152.556

2.7292.82

2.7745

2.6653

2.4032.818

2.6105

2.3662.82S

2.5955

2.603

2.5992.7432.671

2.32.6942.497

2.584»

2.378' 2.788

2.583

2.2392.8252.532

2.5575

In

5.8175.848

5.8325

4.5375.596

5.0665

5.4025.61

5.6065.2863

4.7395.674

6.2065

4.6585.557

5.10755.157

5.0475.416

5.2315

4.5735.2674.92

5.0758

4.6295.44

5.0345

4.4025.512

4.957

4.9958

A-42

EXP2FJCLS

WSRC-TR-96-0088Table A.6.2 (Cont.)

SUMMARY

Column Mesh Al Be Cd Co Cr Cu Fe Ni Zn

Mean raw water 248.1 0.05395 0.045 0.9417 0.29205 0.6137 1781.5 2.9175 5.8325

Mean control (8 me 242.85 0.045575 0.040775 1.023225 0.264325 0.566925 1824.25 2.66525 5.28625

Mean 8 mesh 231.3 0.044025 0.040225 1.10795 0.25965 0.557075 1495.65 2.603 5.157

Mean 10 mesh 234.2 0.068775 0.06475 0.98335 0.2586 0.5614 1733 2.584 5.07575

Mean 12 mesh 220.275 0.060275 0.057475 0.89925 0.254975 0.537675 1602.25 2.5575 4.99575

Percent Removal (% 8 mesh control)

Column Mesh Al Be Cd Co Cr Cu Fe Ni Zn

Mean 8 mesh 4.756022 3.400987 1.348866 -8.28019 1.768656 1.737443 18.01288 2.33562 2.445022

Mean 10 mesh 3.561869 -50.9051 -58.7983 3.896992 2.165894 0.974556 5.002056 3.04849 3.982029

Mean 12 mesh 9.295862 -32.2545 -40.9565 12.1161 3.537312 5.159413 12.16938 4.04277 5.495389

Percent Removal (% raw water)

Column Mesh Al Be Cd Co Cr Cu Fe Ni Zn

Mean control (8 me 2.116082 15.52363 9.388889 -8.65722 9.493237 7.621802 -2.39966 8.6461 9.365624

Mean 8 mesh 6.771463 18.39666 10.61111 -17.6542 11.09399 9.226821 16.04547 10.7798 11.58165

Mean 10 mesh 5.60258 -27.4791 -13.8889 -1.42285 11.45352 8.522079 2.722425 11.431 12.97471

Mean 12 mesh 11.21524 -11.7238 -27.7222 4.507805 1Z69474 12.38797 10.06175 12.3393 14.34634

A-43

_ . , A „ n ^ WSRC-TR-96-0088Table A.6.3 Experimental procedures and results for Experiments #3

Experiments #3: 11/29/94

I. PURPOSEDetermine Effect of Particle Size on Bioremoval by Phaeodactylum

II. Alga11 -day-old Phaeodactylum thcornutum culture

III. FOAM:6% algae by dry weight air dried and seivedSamples obtained for 8,10, and 12 mesh

IV. EFFLUENTS: D-Area coal pile runoff basin - near outfall

V. TEST APPARATUS:Frisby Test rig

VI. PROCEpURE:Fill reservoirs w/ 4 liters of wastewaterFill columns with 4.4g of foamRun at 0.3gpm for 30 minutesCollect samples and acidify (0.5ml cone. HN03) prior to taking samples to ADSfor chemical analyses.

VII. SAMPLE LABELS

The following labels will be used on samples sent to ADS:

abel

3-1 a&b3.2a&b3.3a&b34a&b3,5a&b3.6a&b3.7a&b38a&b

Description

8mesh Phaeo.i t

10mesh phaeo.i t

12mesh Phaeo.i t

#8mesh blank foamt i

A-44

EXP3F.XLS

Table A.6.3 (Cont.)Exp.3 - Effect of particle size on bioremoval by 6% Phaeodactyium -11 -29-94

PARAMETERS

EffluentDate coilpHCond.

BlosorbentDurationModeFlow rate

Column

Rawwater

Mesh

None

Mean raw water

1

2

Control

Control

D coal runoff11/28/94

2.45.88 mS/cm

248.419

0.30%

Phaeodactyium, GR10 31. 6 % by dry wt, embedded 11 -28, 4.44 g30minRecirc0.30 gal/min.

Sample

2-0A2-OB

3-1A3-1BMean

3-2A3-2BMean

Mean control (8 mesh)

3

4

8 mesh

8 mesh

Mean 8 mesh

5

6

10 mesh

10 mesh

Mean 10 mesh

7

8

12 mesh

12 mesh

Mean 12 mesh

3-3A3-3BMean

3-4A3-48Mean

3-5A3-5BMean

3-6A3-6BMean

3-7A3-7BMean

3-8A3-fiBMean

Flow, ga

8.72

9.559.135

9.8

8.81

9.305

9.28

7.828.55

10.18

8.829.5

Al

250246.2

248.1

173.5226.1199.8

188231.3

209.65204.73

177.4237.2207.3

180237.6208.8

208.05

207.6237.8222.7

179.4

235.1207.25

214.98

164.1227.6

195.85

184.6224.7

204.65

200.25

Be

0.05410.05380.054

0.03430.04850.0414

0.03840.04880.0436

0.0425

0.03420.04940.0418

0.03450.04890.0417

0.0418

0.04230.05010.0462

0.03450.04950.042

0.0441

0.03180.048

0.0399

0.0360.0469

0.041450.0407

Cd

0.0450.045

0.045

0.03060.0413

0.03595

0.03550.04190.0387

0.0373

0.03050.04150.036

0.0297

0.04210.03590.036

0.03560.0437

0.03965

0.03130.0444

0.037850.0388

0.02880.0423

0.03555

0.03290.0418

0.03735

0.0365

Co

0.93870.9447

0.9417

0.71440.97380.8441

0.73991.166

0.952950.8985

0.69951.198

0.94875

0.7146

1.3281.02130.985

0.80911.277

1.04305

0.69781.009

0.85340.9482

0.66111.023

0.84205

0.71381.083

0.8984

0.8702

/column

Cr

0.29310.291

0.2921

0.21320.2708

0.242

0.21620.2639

0.240050.241

0.20730.26990.2386

0.20780.26840,2381

0.2384

0.2370.2719

0.25445

0.2060.2685

0.237250.2459

0.19190.2568

0.22435

0.21020.24960.2299

0.2271

WSRC-TR-96-0088

Cu

0.61270.6147

0.614

0.44660.5799

0.51325

0.46040.57040.51540.514

0.44390.581

0.61245

0.44240.5837

0.51305

0.513

0.51480.589

0.5519

0.44420.58820.51620.534

0.39530.5406

0.46795

0.4399

0.53260.48625

0.477

17961767

1781.5

12521611

1431.5*•

14041729

1566.51499

132517491537

1320

« 4 31531:5

1534.3

153417381636

13121715

1513.51574.8

122217101466

136416501507

1486.5

Ni

2.9122.923

2.9175

2.0952.705

2.4

2.172Z651

2.4115

2.4058

2.0582.675

2.3665

2.0672.6772.372

2.3693

2.3512.704

2.6276

2.071Z687

• 2.3792.4533

1.9292.576

2.2525

2.0952.508

2.3015

2.277

Zn

5.8175.848

5.8325

4.2185.4524.835

4.3735.402

4.88754.8613

4.1435.464

4.8035

4.1315.468

4.7995

4.8015

4.8345.5285.181

4.1695.494

4.8315

5.0063

3.8755.2974.586

4.2845.213

4.7485

4.6673

A-45

EXP3F.XLS

Table A.6.3 (Coni

SUMMARY

Column Mesh

Mean raw water

Mean control (8 me

Mean 8 mesh

Mean 10 mesh

Mean 12 mesh

Al

248.1

204.725

208.05

214.975

200.25

• \

"' 1

Be

0.05395

0.0425

0.04175

0.0441

0.040675

Cd

0.045

0.037325

0.03595

0.03875

0.03645

Percent Removal (% 8 mesh control)

Column Mesh

Mean 8 mesh

Mean 10 mesh

Mean 12 mesh

Al

-1.62413

-5.00672

2.185859

Be

1.764706

-3.76471

4.294118

Percent Removal (% raw water)

Column Mesh

Mean control (8 me

Mean 8 mesh

Mean 10 mesh

Mean 12 mesh

Al

17.48287

16.14268

13.35147

19.28658

Be

21.22335

22.61353

18.25765

24.60612

Cd

3.683858

-3.81782

2.344273

Cd

17.05556

20.11111

13.88889

19

Co

0.9417

0.898525

0.985025

0.948225

0.870225

Co

-9.62689

-5.53129

3.149606

Co

4.584793

-4.60072

-0.6929

7.589997

Cr

0.29205

0.241025

0.23835

0.24585

0.227125

Cr

1.109843

-2.00187

5.767037

Cr

17.47132

18.38726

15.81921

22.23078

Cu

0.6137

0.514325

0.51275

0.53405

0.4771

Cu

0.306227

-3.83512

7.237642

Cu

16.19277

16.44941

12.97865

22.25843

Fe

1781.5

1499

1534.25

1574.75

1486.5

Fe

-2.35157

-5.05337

0.833889

Fe

15.85742

13.87875

11.60539

16.55908

WSRC-TR-96-0088

Ni

2.9175

2.40575

2.36925

2.45325

2.277

Ni

1.5172

-1.9744

5.35176

Nt

17.5407

18.7918

15.9126

21.9537

Zn

5.8325

4.86125

4.8015

5.00625

4.66725

Zn

1.229108

-2.98277

3.990743

Zn

16.65238

17.67681

14.16631

19.97857

A-46

Table A.6.4 Experimental procedures and results for ExpenmentW1"96"0088

Experiments #4: 11/29/94

I. PURPOSEDetermine effect of various biomass (Mastigocladus) amounts on bioremoval

II. Alga11-day-old Mastigocladus laminosus culture

III. FOAM:6% algae by dry weight air dried and 8 mesh

IV. EFFLUENTS: D-Area coal pile runoff basin - near outfall

V. TEST APPARATUS:Frisby Test rig

VI. PROCEDURE:Fill reservoirs w/ 4 liters of wastewaterFill columns with the following amounts of foam:

Cols. 1&2-2gCols. 3&4 - 8gCols. 5&6-12gCols. 7&8-16g

Run at 0.3gpm for 30 minutesCollect duplicate ca 50ml samples from each columnCenttifuge 10 min @ 10.OOOrpmAcidify w/ 50 ul HNO3Take to ADS for chemical analyses

VII. SAMPLE LABELSThe following labels will be used on samples sent to ADS

Label Description

4-1 a&b4-2a&b4-3a&b4-4a&b4-5a&b4-6a&b4-7a&b4-8a&b

2g foamI I

8g foam

12g foam

16g foamI I

A-47

EXP4F.XLS

WSRC-TR-96-0088

Table A.6 4 (Cont. It

Exp. 4 - Effect of foam amount on bioremoval by 6% Mastigocladus -11 -29-94

PARAMETERS

EffluentDate coll.pHCond.

BlosorbentDurationModeFlow rate

D coal runoff '11/28/94

2.4

5.88 mS/cm

248.419

0.30%

Mastigoeladus 6% by dry wt, embedded 11-2830minRecirc0.30 gal/mtn.

NOTE- USED LEFTOVER WATER FROM EXP. 3- SEE DATA FOR ZERO TIME VALUES FOR EACH COLUMN

Column

1

2

3

4

5

6

7

8

gfoam

2.0006

2.0061

2g

8.0013

8.0058

sg

12.0096

12.007812g

16.0089

16.006416g

Sample

4-1A4-1BMean

4-2A4-2BMean

Mean

4-3A4-3BMean

4-4A4-48Mean

Mean

4-5A4-5BMean

4-6A4-«BMean

Mean

4-7A4-7BMean

4-8A4-8BMean

Mean

Flowgal.

9.43

9.299.36

8.81

9.S39.17

8.99

9.229.105

9.47

9.029.245

Al

239.1239

239.05

237.9239.2

238.55238.8

242.7241

241.85

235.9

236.2236.05

238.95

239.3239.4

239.35

238.2234

236.1237.73

226.2223.8

225

222

219.8220.9

222.95

Be

0.04970.0492

0.04945

0.04930.04910.0492

0.0493

0.04930.0498

0.04955

0.05160.04960.0506

0.0501

0.04930.049

0.04915

0.04810.0482

0.048150.0487

0.04540.0453

0.04535

0.04450.0444

0.044450.0449

Cd

0.04470.0432

0.04395

0.0450.0453

0.045150.0446

0.04550.0442

0.04455

0.05250.05110.0518

0.0483

0.05050.04990.0502

0.04910.04890.049

0.0496

0.04770.04670.0472

0.04590.04690.0464

0.0468

Co

0.94681.049

0.9979

0.98640.96740.9769

0.9874

0.98410.9876

0.98585

0.98831.612

1.300151.143

0.95691.437

1.19695

0.91491.082

0.998451.0977

0.91621.188

1.0521

0.87341.16

1.01671.0344

Cr

0.26550.264

0.26475

0.26170.2616

0.261650.2632

0.27330.2687

0.271

0.26750.2708

0.269150.2701

0.27170.27070.2712

0.2670.26960.2683

0.2698

0.25710.25430.2557

0.25530.25510.2552

0.2555

Co

0.55730.563

0.56015

0.5580.5567

0.557350.559

0.57850.58050.5795

0.61330.6102

0.611750.596

0.620.6221

0.62105

0.61160.6119

0.611750.616

0.56470.57130.568

0.56660.5681

0.567350.568

Fe

179217921792

17881779

1783.51787.8

18181795

1806.5

174917411745

1775.8

176717551761

174417201732

1746.5

164516251635

158815661577

1606

HI -

2.6472.65

2.6485

2.6312.6352.633

2.6408

2.6882.675

2.6815

2.739

2.7382.73852.71

2.732.741

2.7355

2.7142.727

2.72052.728

• 2.5792.616

2.5976

2.5532.572

2.5625

2.58

Zn

5.4915.5015.496

5.475.46

5.4655.4805

5.4945.51

5.502

5.43

5.3815.4055

5.4538

5.4045.396

5.4

5.3455.368

5.35655.3783

5.1315.084

5.1075

5.0585.033

5.0455

5.0765

A-48

EXP4F.XLS

SUMMARY

Column

2 g, meanPrevious

% Removal

8 g, meanPrevious

% Removal

12g, meanPrevious

% Removal

16 g, meanPrevious

% Removal

Al

238.8204.725

-16.6443

238.95208.05

-14.8522

237.725214.975

-10.5826

222.95200.25

-11.3358

Table

Be

0.0493250.0425

-16.0588

0.0500750.04175

-19.9401

0.048650.0441

-10.3175

0.04490.040675-10.3872

A.6.4 (

Co*

0.044550.037325

-19.357

0.0483250.03595

-34.4228

0.04960.03875

-28

0.04680.03645

-28.3951

Cont.)

Co

0.98740.898525-9.89121

1.1430.985025-16.0377

1.09770.948225-15.7637

1.03440.870225-18.8658

Cr

0.26320.241025-9.20029

0.2700750.23835

-13.3103

0.269750.24585

-9.72137

0.255450.227125-12.4711

Cu

0.558750.514325-6.63753

0.5956250.51275

-16.1628

0.61640.53405

-15.4199

0.5676750.4771

-18.9845

Fe

1787.751499

-19.2628

1775.751534.25

-15.7406

1746.51574.75

-10.9065

16061486.5

-8.03902

Ni

2.640752.40575

-9.76826

2.712.36925

-14.3822

2.7282.45325

-11.1994

2.58Z277

-13.307

WSRfV

Zn

5.48054.86125-12.738

5.453754.8015

-13.584

5.378255.00625-7.4307

5.07654.66725-8.7685

0088

A-49

. „ „ _ . .. .* * r- WSRCJIR-96-0088

Table A.6.5 Experimental procedures and results for Experiment #5

Experiments #5: 11/29/94

I. PURPOSE

Determine bioremoval by foam embedded with Mastigocladus and Phaeodactylumafter mixing in shake fflasks with waste water for 30 minutes.II. Alga

11-day-old Mastigocladus laminosus, and Phaeodactylum tricornutum cultures

III. FOAM:

6% algae by dry weight air dried and 8 mesh

IV. EFFLUENTS: D-Area coal pile runoff basin - near outfall

V. TEST APPARATUS:Shake flasks in Pschrotherm incubator 20°C 150rpm 30min.VI. PROCEDURE:

Add 50 ml wastewater to 6 125ml Erlenmeyer flasksAdd 1 g foam to each flask as described below:

Flasks 1&2 = plain foam, 8 meshFlasks 3&4 = Mastigocladus, 8 meshFlask 5&6 = Phaeodactylum, 8 mesh

Incubate for 30 minutes at 20°C with shaker set for 120rpmRemove flasks from PschrothermCentrifuge liquid for 10 minutes at 10,000rpmAcidify w/ 50 ul HNO3Take to ADS for chemical analyses

VII. SAMPLE LABELSThe following labels will be used on samples sent to ADS

Label Description

5-1 a&b Plain foam5-2a&b Mastigocladus, 8 mesh5-3a&b Phaeodactylum, 8 mesh

A-50

EXP5F.XLS

Talh»lp A 6 -*» fCont.)WSET TR 96

Exp. 5 - 11-29-94. Incubation of Kastigocladus £ Phaeodactylun + foam in shake f lasks of runoff

PARAMETERS

EffluentDate coll.pHCond.

BiosorbentDurationMode

Flask

Rawwater

Alga

None

Mean raw water

1

2

3

Foam

Mast

Phaeo

0 coal runof11/28/94

2.45.88 tnS/cm

248.4

190.30X

Hastigocladus and Phaeodactylum, 6 X by dry wt, embedded 11-28, 1 g/50 ml runoff .30 minShake f lasks, 150 rpra, 20 C

Sample

2-OA2-OB

5-1A5-1BMean

5-2A

5-2BMean

5-3A5-38Mean

Al

250246.2

248.1

245.7256.7251.2

256.8

258.8257.8

259.9259.9259.9

Be

0.05410.0538

0.054

0.05430.052

0.05315

0.0506

0.05020.0504

0.04920.0495

0.04935

Cd

0.0450.045

0.045

0.05370.04850.0511

0.0491

0.04790.0485

0.04640.0487

0.04755

Co

0.93870.9447

0.9417

0.9810.9777

0.97935

0.9653

0.9430.95415

0.92510.9249

0.925

Or

0.29310.291

0.2921

0.30710.3014

0.30425

0.2997

0.30050.3001

0.30230.29630.2993

Cu

0.61270.6147

0.614

0.71480.68180.6983

0.6643

0.65850.6614

0.81460.8409

0.82775

Fe

17961767

1781.5

182718971862

1909

19241916.5

19311914

1922.5

Nl

2.9122.923

2.9175

3.0132.9732.993

2.951

^ 2.932.9405

2.8972.8792.888

Zn

5.8175.848

5.8325

5.725.7285.724

5.622

5.65.611

5.6395.632

5.6355

A-51

Table A.6.6 Experimental procedures and results forExperirift&£$sTR-96-0088

Experiments #6 1/9/95

I. PURPOSEDetermine effect of metal reduction in coal pile runoff following aeration and pH

adjustment.

II. Alganone

III. FOAM:

none

IV. EFFLUENTS: D-Area coal pile runoff basin - near outfall

V. TEST APPARATUS:

Bench top experiment w/ standard laboratory glassware

VI. PROCEDURE:Collect sample from D-CPRB and split into 6 250m! fractionsTreat samples as follows:

1. no treatment2. aertate overnight3. adjust pH to pH 3 with NaOH and aerate overnight4. adjust pH to 4 with NaOH and aerate overnight5. adjust to pH 5 with NaOH and aerate overnight6. adjust pH to 6 with NaOh and aerate overnight

Centrifuge samples (10,000rpm for 10 min.)Collect two 30 ml samples of the supernatant from each treatment and acidify

with 0.6ml cone. HNO3.

VII. SAMPLE LABELSThe following labels will be used on samples sent to ADS

Labef Description

6-1 a&b6-2a&b6-3a&b6-4a&b6-5a&b6-6a7b

Untreated control, pH 2.5Aerated, pH2.5

Aerated and pH adjustedAerated and pH adjustedAerated and pH adjustedAerated and pH adjusted

totototo

3.04.05.06.0

A-52

EXP6F.XLS

Table A.6.6 (Cont

Aer

noyesyesyesyesyes

J.)

Exp. 6- Effect of pH increase and aeration on metal content of runoff - 1 -9-95

EffiuontDate coil

Method

D coal runoff (pH 5)1/9/95

250 ml samples of fresh runoff were subjected to the following treatments:

1) no treatment2} overnight aeration3)adjustment to pH 3 with 18 drops NaOh, overnight aeration4)adjustment to pH 4 with 11 drops NaOh, overnight aeration5)adjustment to pH 5 with 33 drops NaOh, overnight aeration6}adjustment to pH 6 with 19 drops NaOh, overnight aeration

Following 16 h aeration, samples were centrifuged (10 min, 10000 rpm).'

pH

2.52.5

Z 52.5

33

44

55

66

WSRC-TR-96-0088

Two 30 ml samples of each supernatant were acidified with 0.6 ml cone. HNO3.

Aerated?

NoNo

YesYes

YesYes

YesYes

YesYes

Yes

Yes

SUMMARY

pH

ZS2.5

•3

456

Al

66.1966.54563.4949.744.7550.094

Sample it

6-1A6-1BMean

6-2A6-2BMean

63A-6-3BMean

&4A&48-Mean

6-SA6-56Mean

6-6A6-6BMean

Be

0.017650.016550.016450.016050.00250.0094

AS

65.9266.4666.19

66.6866.41

66.645

63.1663.8263.49

49.6149,8749.74

4.7554.7264.765

0.08570.1023

0.094

Cd

0.01530.014950.0106

0.006750.005

0.00345

Be

0.01790.0174

0.01765

0.01650.0166

0.01655

0:01640.0165

0.01645

0.01630.0158

0.01605

0.00250.00250.0025

-0.0014

0.02020.0094

Co

0.25520.256650.255750.2429

0.231650.1968

Cd

0.01520.01540.0153

0.01510.0148

0.01495

0.01070.01050.0106

0.0074• 0.00610.00675

0.00550.00450.005

0.00350:0034

0.00345

Cr

0.03190.029050.029250.0029-O/X3O7-0.0005

Co

0.25670253702552

025730.256

025665

0255402561

02557S

024420241602429

023190.2314

023165

0.1983

0.19530.1968

Cu

0.19210.19205

0.18430.171950.0867

0.01835

Cr

0.03190.03190.0319

0.02820.0299

0.02905

0.02850.03

0.02925

0.00360.00220.0029

-0.0004-0.001

-0.0007

-0.0020.001

-0.0005

Fe

397.8396.3

196.4523.42

9.94550.32095

Cu

0.19260.19160.1921

0.19110.193

0.19205

0.18470.18390.1843

0.17250.1714

0.17195

0.08680:08660.0867

0.0133

0.02340.01835

Ni

0.804850.805350.7589

0.691150.6368

0.49845

Fe

396.8398.8397.8

396.1396.5396.3

197.1195.8

196.45

23.6823.1623.42

9549.951

9.9455

02673

0.37460.32095

Zn

1.5511.554

1.53751.511

1.43650.71025

Ni

0.80180.8079

0.80485

0.80210.8086

' 0.80535

0.76090.75690.7589

0.69020.6921

0.69115

0.63640.63720.6368

0.4921

0.50480.49845

In

1.5S11.5511.551

1.5541.5541.654

1.541.535

1.6375

1.5171.5051.511

1.4391.434

1.4366

0.7102

0.71030.71025

A-53

Table A.6.6 (Cont.) EXP6F.XLSWSRC-TR-96-0088

1000

100

10

I 1a.

0.1

0.01

0

Effect of NaOH

4

pH

AI

Be

Cd

Co

Cr

Cu

Fe

Ni

Zn

A-54

WSRC-TR-96-0088Table A.6.7 Experimental procedures and results forExperiment #7

Experiment #7: Methods and Materials

Metal Removal using Cyanidium'm Mini-Columns

I. PURPOSE:To attempt to maximize metal removal, we will 1) use 10 ml Bio-Rad mini-columns packed with foam to allow slower flow and a greateralgae/wastewater ratio than is achievable using the test bed. 2) test coalbasin runoff at both the natural pH and after adjustment to pH 5 to removemost iron and aluminum, which may be competing with the other metals.3) compare metal removal from actual runoff with Zn and Ni standards atsimilar concentrations and pH values to further elucidate effects of pHand competing ions. Zn and Ni were chosen because they exceed waterquality standards and remain in solution at pH 5. 4) compare foamparticles with solid plugs to determine which gives the best exposure ofmetals to the algae.

II. ALGA:13-day old thermophilic Cyanidium caldarium (GR 1_6) Harvested bycentrifugation, washed once with DI, resuspended at 3% by dry wt in Dl.Embedded 1-23-95 at 6% in foam.

III. FOAM:8 mesh versus solid plugs (made directly in columns) About 2 g/column.

IV. EFFLUENTS:D area coal basin runoff, collected from basin0.8 ppm Ni standard1.5 ppmZn standard

V. TEST APPARATUS (GENERAL DESCRIPTION):

10 ml BioRad columns, 50 ml funnels

VI. PROCEDURE:

A. Preparation of effluent1. Collect surface water from D area coal pile runoff basin (1

carboy). Measure pH.

A-55

Table A.6.7 (Cont.)

WSRC-TR-96-0088

2. Upon returning to lab, prepare 2 flasks containing 1.5 literseach of runoff.

3. Adjust pH of one flask to 5 using NaOH. Record initial and finalpH values and volume of NaOH added (in drops; 1 drop = 0.042ml).

4. Centrifuge both batches of runoff to remove precipitates (10min, 10000 rpm). Decant or pipet off supernatant so as not todisturb pellet.

5. Recheck and record pH values of each batch of supernatant.

6. Take two 30 ml samples of each, preserve (0.6 ml cone. HNO3)for analysis.

B. Preparation of standards

1. Nickel, 100 ppm standard. Place 10 ml of a 1000 ug/ml NIST Nistandard (in 2% HNO3) in a 100 ml volumetric flask. Fill tomark with deionized water.

2. Nickel working standard, 0.8 ppm. Place 8 ml of 100 ppmstandard in a 1000 ml volumetric, dilute to 1 liter. Do twice.

3. Check the pH of each batch of standard. Adjust one to the pHof the non-adjusted effluent used in the experiment (probablyabout2:5), the othento pH^5-0. Use NaOH and/or HNO3 forthese adjustments. Record initial and final pH as well as dropsof acid or base added.

4. Centrifuge both batches of standard and decant supernatant inthe same manner as for effluent.

5. Take two 30-ml samples from each batch of effluent, preserveand send for analysis.

6. Zinc, 100 ppm standard. Place 100 ml of 1000 ug/ml NISTstandard (in 2% HNO3) into a 100 ml volumetric. Fill to markwith deionized water.

7. Zinc working standard, 1.5 ppm. Place 15 ml of 100 ppmstandard into a 1000 ml volumetric, dilute to 1 liter. Do twice.

A-56

Table A.6.7 (Cont.)

WSRC-TR-96-0088

8. Check pH, adjust to effluent pH and to pH 5, centrifuge, decant,and sample in the same manner as for Nt standards (Steps 3T5).

C. Preparation of columns

1. Columns containing solid plugs of foam or foam+algae areprepared by Frisby Technologies.

2. Pack 12 columns with 2 g of particulate foam + algae (8 mesh)each.

3. Pack 12 columns with 2 g plain particulate foam (8 mesh) each

D. Contacting biosorbent with effluent

1. Set up 12 columns containing ground foam + Cyanidium.Place funnels on top and acid-washed 125 ml flasksunderneath. Number columns GC1-12.

2. Place 50 ml standard or effluent in each funnel as follows:

Columns GC 1,2- Coal runoff (unmodified pH)Columns GC 3, 4 - Coal runoff, pH 5Columns GC 5, 6 - Ni standard, low pHColumns GC 7, 8 - Ni standard, pH 7Columns GC 9, 10 - Zn standard, low pH -Columns GC 11, 12 -Zn standard, pH 5

3. Wait 30 min or until liquid stops coming out of columns(whichever is longer). Remove 35 ml from each flask to alabeled centrifuge tube!

4. Centrifuge 10 min, 10000 rpm

5. Pipet off 30 ml from each tube to a labeled sample bottle.Preserve for analysis;

6. Set up 12 more columns containing plain ground foam (noalgae). Label them G 1-12. Repeat Steps 2-5.

7. Set up 12 columns with plugs of algae + foam. Label them PC1-12. Repeat Steps 2-5.

A-57

Table A.6.7 (Cont.)

WSRC-TR-96-0088

8. Set up 12 columns with plain foam plugs, labeled P 1-12.Repeat Steps 2-5.

9. Weigh leftover plugs and record weight of foam.

VII. APPARATUS

Coai basin runoff, at least 3 liters1000 ppm Ni standard1000 ppm Zn standardHNO3, 1 NHNO3, 10 NNaOH, 1 NNaOH, 10 NHNO3, ca. 7 N for acid washingDeionized water

CyanidiumFoam as described in methodsBio-Rad columns and funnels, about 30Carboy for effluent

The following glass and plasticware should be acid-washed.

50 125 ml flasks50 centrifuge tubesat least 60 sample bottles16 250 ml centrifuge bottles (may have to rewash halfway through)2 100 ml volumetrics:4 1000 ml volumetrics2 2 liter flasks8 1 liter flasks5 ml pipet tips, lots

VII. SAMPLE LABELS

The following labels will be used on samples sent to Analytical.

Label Description

7-1 Control - Effluent, unmodified pH7-27-3 Control - Effluent, pH 57-4 "7-5 Control - Ni standard, low pH

A-58

Table A.6.7 (Cont.)

WSRC-TR-96-0088

7-67-7 Control - Ni standard, pH 57-87-9 Control - Zn standard, low pH7-107-11 Control - Zn standard, pH 57-127-13 Sample from GC 17-14 Sample from GC 27-15 Sample from GC 37-16 " " GC47-17 " " GC57-18 " " GC67-19 " GC77-20 " " GC87-21 " " GC97-22 " " GC107-23 "•. "• G C 117-24 " " GC 127-25 " " G 17-26 " G27-27 " " G 37-28 " G47-29 " " -657-30 " " G67-31 " " G77-32 " " G87-33 " " G97-34 " " G 10

- 7-35 " " G117-36 " " G127-37 " " PC17-38 " " PC 27-39 " " PC 37-40 " " PC 47-41 " " PC 57-42 " " PC 67-43 " PC 77-44 " " PC 87-45 " " PC 97^6 " " PC 107-47 " " PC 117-4,8 " " PC 127-49 " " P 17-50 " " P2

A-59

Table A.6.7 (Cont.)

WSRC-TR-96-0088

7-51 .7-527-537-547-557-567-577-587-597-60

i i

i i

u

I I

i i

t i

t i

I I

I I

I I

i t

«

i i

t i

i t

i t

n

n

M

I I

P3PAP5P6P7P8P9P10P11P12

A-60

EXP7F.XLS

WSRC-TR-96-0088

Table A.6.1J (Coni

Exp. 7- Mini-columns - 1-26-95

Effluent

Date coll.

Foam

Method

Notes

Results

Sample

7 17_2Mean

7 37 4

Mean

7 57_6Mean

7 77 8Mean

7 97 10Mean

7 11

7 12Mean

7 137 14Mean

7 157 16

Eff.

RunoffRunoff

RunoffRunoff

Ni

Ni

NiNi

ZnZn

Zn

Zn

RunoffRunoff

RunoffRunoff

D coal runoff (pH 2.5 and adjusted to 5.0)cond = 2.9, turb = - 1 . DO = 11.2, temp 11.2, sal 0.1

0.8 ppm Ni standards, pH 2.5 and 5.01.5 ppm Zn standards, pH 2.5 and 5.0

1/26/95

Four foam types (made 1 -24)were used:8 mesh with Cyanidium caldarium (high temp; GR1 6.XLS) 2.00 +-0.01 g/column8 mesh, no algae. 2.00 +- 0.01 g/columnSolid plug with Cyanidium (avg wt of 3 was 2.35 g)Solid plug, no algae (avg wt of 2 was 1.49 g)

EXP7PRO.DOC

Adjusted runoff was actiually 4.76 after centrifugation, unmodified was 2.48Ni 2.5 req/d 3.8 ml/l for pH adjustmentNi 5.0 took 0.2 mlZn 2.5 took 3.3 mlZn 5.0 took 0.2 ml jRunoff 5.0 took5.0 ml

pH

2^5j2.5

55

2.52.5

55

2.5

55

2.52.5

55

Foam

ControlControl

ControlControl

ControlControl

ControlControl

ControlControl

ControlControl

8 mesh8 mesh

8 mesh8 mesh

Algae?

Yes

Yes

YesYes

AI

63.7664.3864.07

5.1845.1585.171

0.01230.0334

0.02285

ndndnd

ndnd

nd

0.42720.4277

0.42745

68.6269.4269.02

3.8683.797

Be

0.01910.01920.0192

0.00630.00490.0056

0.00030

0.0002

0.00010

0.0001

ndnd

0

nd

nd

nd

0.0180.0179

0.018

0.00380.0036

Cd

0.02050.02110.0208

0.00870.0066

0,00765

0.00O60.0007

0.00065

.. nd0.00020.0002

0.0010.0001

0.00055

0.00030.0004

0.00035

0.01950.01910.0193

0:00550.006

Co

0.2610.2613

0.26115

0.23250.23170.2321

ndndnd

0.001nd

0.001

0.0010.0025

0.00175

0.00150.00090.0012

0.2550.2557

0.25535

0.2282. 0.2242

Cr

0.0320,03470.0334

0.00010.00420.0022

0.0005L 0.00030.0004

0.0004nd

0.0004

nd

0.00170.0017

0.00140.00130.0014

0.03450.03340.034

0.0010.002

Cu

0.2

0.20.2

0.068GT0660.067

0.0080.0080.008

0.0090.0090.009

nd6E-046E-04

0.0020.0010.002

0.330.3270.328

0.060.065

Fe

380.4381.2380.8

9.9649.6949.829

0.01680.04640.0316

0.02920.02960.0294

*nd0.01080.0108

ndndnd

388.2387.6387.9

10.6210.3

Ni

0.83450.82850.8315

0.6260.623

0.6245

0.77070.77040.7706

0.77050.76910.7698-

0.010.00390.007

0.00890.00870.0088

0.79750.79990.7987

0.58570.5751

Zn

1.5711.566

1.5685

1.4141.3881.401

0.01040.0089

0.00965

0.01040.0117

0.01105

1.2931.2871.29

1.3821.3821.382

1.5831.562

1.5725

1.364

A-61

EXP7F.XLSWSRC-TR-96-0088

Mean

7 177 18Mean

7 197 20Mean

7 217 22Mean

7 237 24Mean

7 257 26Mean

7 27 .7 28Mean

7 297 30Mean

7 317 32Mean

7 337 34Mean

7 357 36Mean

7 377 38Mean

7 397 40Mean

7 417 42Mean

7 437 44Mean

7 457 46Mean

Ni

Ni

Ni

Ni

Zn

Zn

Zn

Zn

RunoffRunoff

RunoffRunoff

NiNi

NiNi

Zn

Zn

Zn

Zn

RunoffRunoff

RunoffRunoff

NiNi

NiNi

ZnZn

2.52.5

55

2.52.5

55

2.52.5

55

2.52.5

55

2.52.5

55

2.52.5

u 55

2.52.5

55

2.52.5

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

8 mesh8 mesh

PlugPlug

PlugPlug

PlugPlug

PlugPlug

PlugPlug

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

NoNo

NoNo

NoNo

NoNo

YesYes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

3.8325

0.35450.3752

0.36485

0.43950.1016

0.27055

0.35670.248

0.30235

0.41880.27740.3481

72.4472.15

72.295

5.7795.682

5.7305

0.3560.39860.3773

0.25590.2334

0.24465

0.2750.2571

0.26605

0.22080.1733

0.19705

66.9967.36

67.175

5.4235.5355.479

0.31580.30520.3105

0.18380.1027

0.14325

0.20050.1618

0.18115

0.0037

nd

ndnd

nd

ndnd

0.00050.00010.O003

0

0.00015E-O5

0.01810.0179

0.018

0.00410.00420.0042

ndnd

nd

0.00030.00060.0005

0nd

0

nd

0.00160.0016

.0.01780.01770.0178

0.00430.00430.0043

0.02070.008

0.0144

0.00210.00070.0014

0.00010

5E-O5

0.00575

nd

0.00010.0001

nd

nd

nd

0.0002nd

0.0002

nd

nd

nd

0.01840.01880.0186

0.00560.00680.0062

0.00010.0002

0.00015

0.0006nd

0.0006

0.08410.0004

0.04225

nd

0.00110.0011

0.0190.01920.0191

0.00620.0063

0.00625

0.01930.00830.0138

0.00140.0003

0.00085

nd

ndnd

0.2262

0.00260.00340.003

0.00390.00270.0033

0.00530.00390.0046

0.00110.0026

0.00185

0.26130.26550.2634

0.23980.2373

0.23855

0.00110.00050.0008

ndnd

nd

ndndnd

nd

0.00070.0007

0.25720.2503

0.25375

0.2350.23120.2331

0.02070.01410.0174

0.00210.0028

0.00245

0.0019nd

0.0019

0.0015

0.0001nd _,

0.0001

nd

00

0.00320.00010.0017

t i d

nd

nd

0.02980.03130.0306

0.00050.00110.0008

0.00030

0.0002

nd

0.00090.0009

0.00030.00090.0006

0.00180.00190.0019

0.02790.033

0.0305

0.00070.00090.0008

0.01970.00980.0148

0.00160.001

0.0013

0.0012nd

0.0012

0.063

0.0740.08

0.077

0.0310.0360.034

0.0690.0720.07

0.030.0290.03

1.0781.0891.084

0.2170.1950.206

0.1660.2130.19

0.1020.1010.101

0.20.1770:189

0.0910.0920.091

0.1790.1820.181

0.0530.0570.055

0.0220.0160.019

0.010.0060.008

0.0030.0030.003

10.46

0.01580.00940.0126

0.0031nd

0.0031

0.0024nd

0.0024

nd

nd

nd

435.7435

435.35

11.0710.8

10.935

0.00410.00090.0025

0.00420.00870.0065

0.0053nd

0.0053

nd

ndnd

402

402.5402.25

10.4510.46

10.455

ndndnd

0.0167nd

0.0167

nd

ndnd

0.5804

0.56580.53970.5528

0.21140.21120.2113

0.01850.01670.0176

0.01340.015

0.0142

0.80690.80460.8058

0.5990.59090.595

0.73180.72670.7293

0.72540.71910.7223

0.02420.01890.0216

0.01560.019

0.0173

0.78470.79160.7882

0.58410.59350.5888

0.72830.69320,7108

0.68890.70590.6974

0.01610.01690.0165

1.361

0.06680.0665

0.06665

0.03130.03230.0318

1.0281.059

1.0435

0.28210.3974

0.33975

1.5751.552

1.5635

1.3751.366

.1.3705

0.04140.0441

0.04275

0.03560.03140.0335

1.4061.363

1.3845

1.2941.3181.306

1.4931.4711.482

1.3321.3561.344

0.02090.02190.0214

0.02350.0116

0.01755

1.2741.275

1.2745• • i *

A-62

EXP7F.XLSWSRC-TR-96-0088

7 47

7 48Mean

7 49

7 50Mean

7 517 52Mean

7 537 54

Mean

7 557 56Mean

7 577 58Mean

Zn

Zn

Runoff

Runoff

Runoff

Runoff

Ni

Ni

Ni

Ni

ZnZn

55

2.52.5

55

2.52.5

55

2.52.5

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Yes

Yes

No

No

No

No

No

No

No

No

No

No

0.1717

0.13930.1555

69.95

69.23

69.59

5.428

5.496

5.462

0.11930.1338

0.12655

0.12910.07830.1037

0.12790.0194

0.07365

0

nd0

0.0184

.0.0181

0.0183

0.0480.0275

0.0378

0.0062

0.00080.0035

0.0004

0.0001

0.0003

0.0002

00.0001

nd

ndnd

0.01690.0198

0.01835

0.05810.03550.0468

0.00720.002

0.0046

0

nd

0

nd

nd

nd

0.00150.00030.0009

0.27660.27160.2741

0.30950.28450.297

0.0152nd

0.0152

nd

ndnd

nd

ndnd

nd

nd

nd

0.03090.02970.0303

0.07520.03640.0558

0.00940.00060.005

0.0004nd

0.0004

0.00110.001

0.0011

0.0020.0030.003

0.1890.1830.186

0.1040:0830.093

0.0150.0080.011

0.0080.0050.006

0.0040.0020.003

nd

nd

nd

419.4414.5

416.95

10.3910.6

10.495

0.0009nd

0.0009

nd

ndnd

nd

ndnd

0.01720.01650.0169

0.8

0.79840.7992

0.66170.63420.648j

0.74310.72540.7343

0.72560.737

0.7313

0.0310.02020.0256

1.2411.252

1.2465

1.561.539

K5495

1.4651.4471.456

0.03610.023

0.02955

0.01090.0094

0.01015

1.2971.3291.313

A-63

EXP7F.XLSWSRC-TR-96-0088

7 597 60Mean

Zn

Zn

55

Summary - Means

Effluent

Runoff

Runoff

Ni

Ni

Zn

Zn

Runoff

Runoff

Ni

Ni

Zn

Zn

Runoff

Runoff

Ni

Ni

Zn

Zn

Runoff

Runoff

Ni

Ni

Zn

Zn

Runoff

Runoff

Ni

pH

2.5

5j

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

5

2.5

Foam

Control

Control

C.ontrol

Control

Control

Control

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

-

8 mesh

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Algae?

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

No

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

Al

64.07

5.171

6.023

nd

nd

L 0.427

69.02

3.833

0.365

0.271

0.302

0.348

72.3

5.731

0.377

0.245

0.266

0.197

67.18

5.479

0.311

0.143

0.181

0.156

69.59

5.462

0.127

0.10670.01970.0632

Be

0.01915

0.0056

0.00015

0.0001

0

nd

0.01795

0.0037

nd

nd

0.0003

0.00005

0.018

0.00415

nd

0.00045

0

0.0016

0.01775

0.0043

0.01435

0.0014

0.00005

0

0.01825

0.03775

0.0035

0.0001nd

0.0001

Cd

0.0208

0.0077

0.0007

0.0002

0.0006

0.0004

0.0193

0.0058

0.0001

nd

0.0002

nd

0.0186

0.0062

0.0002

0.0006

0.0423

0.0011

0.0191

0.0063

0.0138

0.0009

nd

nd

0.0184

0.0468

0.0046

nd

nd

nd

Co

0.26115

0.2321

nd

0.001

0.00175

0.0012

0.25535

0.2262

- 0.003

0.0033

0.0046

0.00185

0.2634

0.23855

0.0008

nd

nd

0.0007

0.25375

0.2331

0.0174

0.00245

0.0019

0.0009

0.2741

0.297

0.0152

0.00130.0006

0.00095

Cr

0.03335

0.00215

0.0004

0.0004

0.0017

0.00135

0,03395

0.0015

0.0001

0

0.00165

nd

0.03055

0.0008

0.00015

0.0009

0.0006

0.00185

0.03045

0.0008

0.01475

0.0013

0.0012

nd

0.0303

0.0558

0.005

0.00160.003

0.0023

Cu

0.1997

0.0667

0.0082

0.0088

0.0006

0.0016

0.3281

0.0628

0.0769

0.0335

0.0705

0.0296

1.0835

0.206

0.1897

0.1013

0.1887

0.0911

0.1805

0.055

0.0191

0.0082

0.0029

0.0027

0.1857

0.0933

0.0115

0.0010.0020.002

Fe

380.8

9.829

0.032

0.029

0.011

nd

387.9

10.46

0.013

0.003

0.002

nd

435.4

10.94

0.003

0.006

0.005

nd

402.3

10.46

nd

0.017

nd

nd

4 1 7

10.5

9E-04

nd

ndnd

Ni

0.8315

0.6245

0.7706

0.7698

0.007

0.0088

0.7987

0.5804

0.5528

0.2113

,0.0176

0.0142

0.8058

0.595

0.7293

0.7223

0.0216

0.0173

0.7882

0.5888

0.7108

0.6974

0.0165

0.0169

0.7992

0.648

0.7343

0.01860.01870.0187

Zn

1.5685

1.401

0.0097

0.0111

1.29

1,382

1.5725

1.361

0.0667

0.0318

1.0435

0.3398

1.5635

1.3705

0.0428

0.0335

1.3845

1.306

1.482

1.344*

0.0214

0.0176

1.2745

1.2465

1.5495

1.456

0.0296

1.3421.343

1.3425

— i m.\.\ - ^

A-64

EXP7F.XLS

WSRC-TR-96-0088Ni

Zn

Zn

5

2.5

5

Plug

Plug

Plug

No

No

No

0.104

0.074

0.063

0.00025

0.0001

0.0001

0

nd

nd

nd

nd

0.00095

Summary - Means minus original metal levels in effluents and standards

Effluent

Runoff

pH

2.5

ppm Removed

% removed

Runoff 5ppm Removed

% removed

Ni 2.5ppm Removed

% removed

Ni 5ppm Removed

% removed

Zn 2.5ppm Removed% removed

Zn 5ppm Removed% removed

Runoff 2.5ppm Removed% removed

Runoff 5ppm Removed% removed

Ni 2.5ppm Removed% removed

Ni 5ppm Removed% removed

Zn 2.5ppm Removed% removed

Zn 5ppm Removed

% removed

Runoff 2.5ppm Removed

Foam

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

8 mesh

Plug

Algae?

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

No

Yes

Al

69.02-4.95-7.73

3.8331.33925.88

0.365-0.34-1497

0.271ndnd

0.302ndnd

0.3480.07918.56

72.3-8.23-12.8

5.731-0.56-10.8

0.377-0.35-1551

0.245ndnd

0.266nd

nd

0.1970.2353.9

67.18-3.11

Be

0.017950.0012

6.26632

0.00370.0019

33.9286

ndJfVALUE!

#VALUE!

ndLVALUE!nd

0.0003-0.0003#DIV/0!

0.00005LVALUE!0VALUE!

0.0180.001156.00522

0.004150.0014525.8929

nd#VALUEILVALUE!

0.00045-0.0004

-350

00

0DIV/O!

0.0016LVALUE!#VALUEI

0.017750.0014

Cd

0.01930.00157.2115

0.00580.001924.837

0.00010.000684.615

ndutsuuxttnd

0.00020.000466.667

ndttttttttttuttttmttttt

0.01860.002210.577

0.00620.001518.954

0.00020.000576.923

0.0006-4E-04

-200

0.0423-0.042-6942

0.0011-8E-04-214.3

0.01910.0017

Co

0.255350.0058

2.22095

0.22620.0059

2.54201

0.003#VALUE!tt VALUE!

0.0033-0.0023

-230

0.0046-0.0029-162.86

0.00185-0.0007-54.167

0.2634-0.0022-0.8616

0.23855-0.0065

-2.779

0.0008# VALUE!LVALUE!

nd#VALUE!#VALUE!

nd#VALUE!LVALUE!

0.00070.0005

41.6667

0.253750.0074

0.0004

0.00105

0.0023

Cr

0.03395-0.0006-1.7991

0.00150.0006530.2326

0.00010.0003

75

00.0004

100

0.001655E-O5

2.94118

ndLVALUE!LVALUE!

0.030550.00288.3958

0.00080.0013562.7907

0.000150.00025

62.5

0.0009-0.0005

-125

0.00060.0011

64.7059

0.00185-0.0005-37.037

0.030450.0029

0.0061

0.0032

0.0017

Cu

0.3281-0.128-64.27

0.06280.00395.7764

0.0769-0.069-837.2

0.0335-0.025-280.7

0.0705-0.07

-11642

0.0296-0.028-1810

1.0835-0.884-442.6

0.206-0.139

-209

0.1897-0.182-2213

0.1013-0.092-1051

0.1887-0.188

-31342

0.0911-0.09

-5774

0.18050.0192

nd

nd

nd

Fe

387.9-7.1

-1.86

10.46-0.63-6.42

0.0130.01960.13

0.0030.02689.31

0.0020.00978.18

ndtttttitttf

435.4-64.6-14.3

10.94-1.11-11.3

0.0030.02992.09

0.0060.02377.76

0.0050.00651.82

ndstunt*ntttttttt

402.3-21.5

0.7313

0.0256

0.0187

Ni

0.79870.03283.9447

0.58040.04417.0616

0.55280.217828.266

0.21130.558572.551

0.0176-0.011-151.4

0.0142-0.005-61.36

0.80580.02583.0968

0.5950.02964.7318

0.72930.04135.3598

0.72230.04766.1769

0.0216-0.015-207.9

0.0173-0.009-96.59

0.78820.0434

0.0102

1.313

1.3425

Zn

1.5725-0.004-0.255

1.3610.04

2.8551

0.0667-0.057-590.7

0.0318-0.021-186.5

1.04350.246S19.109

0.33981.042375.416

1.56350.005

0.3188

1.37050.0305

2.177

0.0428-0.033

-343-0.0335-0.022-201.8

1.3845-0.095-7.326

1.3060.076

5.4993

1.4820.0865

A-65

EXP7F.XLSWSRC-TR-96-0088

% removed

Runoff 5

ppm Removed% removed

Ni 2.5

ppm Removed% removed

Ni 5

ppm Removed% removed

Zn 2.5

ppm Removed% removed

Zn 5

ppm Removed% removed

Runoff 2.5

ppm Removed% removed

Runoff 5

ppm Removed% removed

Ni 2.5

ppm Removed% removed

Ni 5

ppm .Removed% removed

Zn 2.5

ppm Removed% removed

Zn 5

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

Plug

-

Plug

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

No

-4.85

5.479-0.31-5.96

0.311-0.29-1259

0.143nd

nd

0.181nd

nd

0.1560.27263.62

69.59-5.52-8.62

5.462-0.29-5.63

0.127-0.1

-454

0.104nd 'nd

0.074nd

nd

0.063

7.3107

0.00430.0013

23.2143

0.01435-0.0142-9466.7

0.0014-0.0013

-1300

0.00005-5E-O5

iCDIV/O!

0

LVALUE!#VALUE!

0.018250.0009

4.69974

0.03775-0.0322-574.11

0.0035-0.0034-2233.3

0.00025-0.0002

-150

0.0001-0.0001#DIV/0!

0.0001

8.1731

0.00630.001418.301

0.0138-0.013-2023

0.0009-7E-04

-325

ndtttiUUtiS

tittttttntt

ndtssntttiti

Mfitmtstt

0.01840.002511.779

0.0468-0.039-511.8

0.0046-0.004-607.7

0

0.0002100

ndtiftitttttttusuttttx

nd

2.83362

0.2331-0.001

-0.4308

0.0174#VALUE!LVALUE!

0.00245-0.0015

-145

0.0019-0.0002-8.5714

0.00090.0003

25

0.2741-0.013

-4.9588

0.297-0.0649-27.962

0.0152LVALUE!LVALUE!

nd

LVALUE!#VAIUE!

nd

#VALUE!LVALUE!

0.00095

8.69565

0.00080.0013562.7907

0.01475-0.0144-3587.5

0.0013-0.0009

-225

0.00120.0005

29.4118

nd

#VALUEILVALUE!

0.03030.003059.14543

0.0558-0.0537-2495.3

0.005-0.0046

-1150

0.00040

0

0.001050.0006538.2353

0.0023

9.6144

0.0550.011717.5&4

0.0191-0.011-132.9

0.00820.00066.8182

0.0029-0.002-383.3

0.0027-0.001-70.97

0.18570.01417.0356

0.0933-0.027-39.98

0.0115-0.003-39.63

0.00610.002730.682

0.0032-0.003

-425

0.0017

•5.63

10.46-0.636.37

nd

gtumntisttes

0.0170.01242.41

ndtstsxtstt

stmitti

ndXtttttttt

ttnssu

417

-36.2-9.49

10.5-0.67-6.78

9E-040.03197.15

nd

nsssstttntmt

ndttgmtnttnttttu

nd

5.2135

0.58880.03575.7166

0.71080.05987.7607

0.69740.0724

9.405

0.0165-0.01

•135.7

0.0169-0.008-91.48

0.79920.03233.8845

0.648-0.023-3.755

0.73430.03634.7109

0.73130.03855.0013

0.0256-0.019-265.7

0.0187

5.5148

1.3440.057

4.0685

0.0214-0.012-121.8

0.0176-0.006-58.11

1.27450.01551.2016

1.24650.13559.8046

1.54950.019

1.2113

1.456-0.055-3.926

0.0296-0.02

-206.2

0.01020.001

8.5586

1.313-0.023-1.783

1.3425

A-66

EXP7F.XLS

WSRC-TR-96-0088

ppm Removed% removed

Percentage Removal from pH 2.5 Runoff

8 +, algae8. no algae

Plug + algaePlug, no al

At

-7.7259-12.838

-4.8463-8.6156

Be

6.2663196.005222

7.3107054.699739

Percentage Removal from pH 5 Runoff

8 + algae8. no algae

Plug + algaePlug, no al

Al

25.8847-10.82

-5.9563-5.6275

Be

33.9285725.89286

23.21429-574.107

0.36485.21

Cd

7.21210.58

8.17311.78

Cd

24.8418.95

18.3-512

Percent Nickel Removal from pH 2.5 Ni standard

8 + algae8, no algae

Plug + algae 'Plug, no al

Ni

28.26555,35981

7.760694.71092

LVALUE!LVALUE!

Co

2.22095-0.8616

2.83362-4.9588

Co

2.54201-2.779

-0,4308-27.962

Percentage Nickel Removal from pH 5 Ni standardf

8 + algae •8, no algae

Plug + algaePlug, no al

Ni

72.5516.1769

9.4085.0013

Percentage Zinc Removal from pH 2. 5 Zn standard

8 + algae8, no algae

• • -

Rug + algaePlug, no al

Zn

19.109-7.326

1.2016-1.783

Percentage Zinc Removal from pH 5 Zn standard

Zn

ntttttt tintttwtttttt

Cr

-1.7998.3958

8.69579.1454

Cr

30.23362.791

62.791-2495

-

0.0002520.8333

Cu

-64.271-442.56

9.614427.03555

Cu

5.77644-209

17.5544-39.985

-0.001-70.37

Fe

-1.8645-14.325

-5.6329-9.4932

Fe

-6.4198-11.252

-6.3689-6.7759

-2E-04-9.677

Ni

3.94473.0968

5.21353.8845

Ni

7.06164.7318

5.7166-3.755

tttttttttt

utttttm

Zn

-0.260.319

5.5151.211

Zn

2.8552.177

4.069-3.93

-0.01-111.9

-

0.03952.8582

A-67

EXP7F.XLS

96-0088

8 + algae8, no algae

Plug + algaePlug, no al

75.41615.49928

9.804632.85818

A-68

T . . A „ WSRC-TR-96-0088rable A.6.8 Experimental procedures and results forExperiment #8

EXPERIMENT 8 Metal Removal using Cyanidium in Mini-Columns

I. PURPOSE:To investigate reasons for the poor performance of foam-Cyanidium aggregates

with coal runoff compared to standard metal solutions, we will attempt to treat bothrunoff (pretreated to reduce iron and Al content) and a mixture of metal standardscontaining Al, Be, Cd, Co, Cr, Cu, Fe, Ni, and Zn at levels similar to that of the runoff.Single-metal solutions of Ni and Zn will also be tested as a comparison to the mixturesand to the previous experiment. The experiment is expected to show whether theproblem relates to competition between metal ions or to some feature of the chemicalenvironment (such as organic content) specific to the actual runoff. Only pH 5 will beused this time, since metal removal is clearly superior at this pH. Similarly, based onthe previous experiment we have selected pulverized foam rather than plugs. The mini-columns will be modified to reduce the initial rapid flow rate observed previously,increasing average contact time of metal ions with the algal biomass. This is expectedto improve metal removal percentages.

Additionally, the entire experiment will be duplicated using Phaeodactylum andChlorella as a comparison with Cyanidium.

II. ALGAE:10 -14 - day old Phaeodactylum and thermophilic Cyanidium caldarium andChlorella vulgaris Harvested by centrifugation, washed once with Dl,resuspended at 3% by dry wt in Dl. Embedded at 6% in foam.

III. FOAM:8 mesh, 2 g/column.

IV. EFFLUENTS:D area coal basin runoff, collected from basin, adjusted to pH 5 andcentrifuged0.8 ppm Ni standard, pH 5, centrifuged1.5 ppm Zn standard, pH 5, centrifugedMixture of the following (pH 5, centrifuged)

Al standard, 64 ppmBe standard, 0.02 ppmCd standard, 0.02 ppmCo standard, 0.26 ppmCr standard, 0.03 ppmCu standard, 0.2 ppmFe standard, 380 ppm

A-69

Tabfe A.6.8 (Cont.)

WSRC-TR-96-0088

Ni standard, 0.8 ppmZn standard, 1.5 ppm

V. TEST APPARATUS (GENERAL DESCRIPTION):10 m! BioRad columns, 50 ml funnels. Modify to standardize and reduce

flow rates. Acid-washed.

VI. PROCEDURE:

A. Preparation of effluent

1. Collect surface water from D area coal pile runoff basin (1carboy). Measure pH.

2. Upon returning to lab, prepare a flask containing 1.5 litersof runoff.

3. Adjust pH to 5 using NaOH. Record initial and final pH valuesand volume of NaOH added (in drops; 1 drop = 0.042 ml).

4. Centrifuge to remove precipitates (10 min, 10000 rpm). Decantor pipet off supernatant so as not to disturb pellet.

5. Recheck and record pH value of supernatant.

6. Take two 30 ml samples of each, preserve (0.6 ml cone. HNO3)for analysis.

. B. Preparation of standards

1. Nickel, 100 ppm standard. Place 10 ml of a 1000 ug/ml NIST Nistandard (in 2% HNO3) in a 100 ml volumetric flask. Fill tomark with deionized water.

2. Nickel working standard, 0.8 ppm. Place 8 ml of 100 ppmstandard in a 1000 ml volumetric, dilute to 1 liter.

3. Check the pH of working standard. Adjust to pH 5.0. UseNaOH and/or HNO3 for these adjustments. Record initial and finalpH as well as drops of acid or base added.

4. Centrifuge standard and decant supernatant in the samemanner as for effluent.

A-70

Table A.6.8 (Cont.)WSRC-TR-96-0088

5. Take two 30-ml samples , preserve and send for analysis.

6. Zinc, 100 ppm standard. Place 10 ml of 1000 ug/ml NISTstandard (in 2% HNO3) into a 100 ml volumetric. Fill to markwith deionized water.

7. Zinc working standard, 1.5 ppm. Place 15 ml of 100 ppmstandard into a 1000 ml volumetric, dilute to 1 liter.

8. Check pH, adjust to pH 5, centrifuge, decant, and sample in thesame manner as for Ni standards (Steps 3-5).

9. Be, 100 ppm standard. Place 1 ml pf 10 mg/ml NIST standardinto a 100 ml volumetric. Fill to mark with deionized water.

10. Cd, 100 ppm standard. Dilute 10 ml of 1000 ug/ml NISTstandardising a 100 ml volumetric.

11. Co, 100 ppm standard. Dilute 1 ml of 10 mg/ml NIST standardusing a 100 ml volumetric.

12. Cr, 100 ppm standard. Dilute 10 ml of 1000 ug/ml NISTstandard using a 100 ml volumetric.

13. Cu, 100 ppm standard. Dilute 10 ml of 1000 ug/ml NISTstandard using a 100 ml volumetric.

14. Mixed metal solutions. Prepare a mixture of the following in aTOOt) ml volumetric:

Al - 64 ml of 1000 ug/ml NIST standardBe - 200 ul of 100 ppm standard.Cd - 200 ul of 100 ppm standardCo - 2.6 ml of 100 ppm standardCr - 300 ul of 100 ppm standardCu - 2 ml of 100 ppm standardFe - 380 ml of 1000 ug/ml NIST standardNi - 8 ml of 100 ppm standardZn - 15 ml of 100 ppm standard

Adjust pH to 5, centrifuge and decant.Take 2 X 30 ml samples, preserve as before.

A-71

Table A.6.8(Cont.)WSRC-TR-96-0088

G. Preparation of columns

1. Pack 8 columns with 2 g of particulate foam + Cyanidium (8mesh) each.

2. Pack 8 columns with 2 g particulate foam + Phaeodactylum (8mesh) each.

3. Pack 8 columns with 2 g plain particulate foam (8 mesh) each

D. Contacting biosorbent with effluent

1. Set up 8 columns containing ground foam + Cyanidium.Place funnels on top and acid-washed 125 ml flasksunderneath. Number columns GC1-8.

2. Place 50 ml standard or effluent in each funnel as follows:

- Columns GC 1,2- Coal runoff, pH 5Columns GC 3, 4 - Ni standard, pH 5Columns GC 5, 6 - Zn standard, pH 5Columns GC 7, 8 - Mixed metal standards, pH 5

3. Wait until liquid stops coming out of columns. Remove 35 mlfrom each flask to a labeled centrifuge tube.

4. Centrifuge 10 min, 10000 rpm

5. Pipet off 30 ml from each tube to a labeled sample bottle.Preserve for analysis.

6. Set up 8 more columns containing ground foam +Phaeodactylum. Label them GP 1-8. Repeat Steps 2-5.

7. Set up 8 columns with plain ground foam (no algae). Labelthem G1-8. Repeat Steps 2-5.

VII. APPARATUS

Coal basin runoff, at least 3 liters1000 ppm Ni standard1000 ppm Zn standard1000 ppm Al standard

A-72

Table A.6.8 (Cont.)

10000 ppm Be standard1000 ppm Cd standard10000 ppm Co standard1000 ppm Cr standard1000 ppm Cu standard1000 ppm Fe standardHNO3, 1 NHNO3, 10 NNaOH, 1 NNaOH, 10 NHNO3, ca. 7 N for acid washingHNO3, ca 3 N for rinsing pH electrodeDeionized waterCyanidiumPhaeodactylumFoam as described in methods

Carboy for effluent

The following glass and plasticware should be acid-washed.

Bio-Rad columns and funnels, about 30 acid-washed24 125 ml flasks32 centrifuge tubesat least 32 sample bottles16 250 ml centrifuge bdttles (may have to rewash halfway through)7 100 ml volumetrics4 1000 ml volumetrics

. 4 2 liter flasks• 8 1 liter flasks

5 ml pipet tips, lots

VII. SAMPLE LABELS

The following labels will be used on samples sent to Analytical.

Label Description

8-1 Control - Effluent, pH 58-2

, 8-3 Control - Ni standard, pH 58-48-5 Control - Zn standard, pH 58-68-7 Mixed metal standard, pH 5

A-73

WSRC-TR-96-0088

Table A.6.8 (Cont)WSRC-TR-96-0088

8-88-98-108-118-128-138-148-158-168-178-188-198-208-218-228-238-248-258-268-278-288-298-30'8-318-32

SampleSampleSample

i t

i t

i t

i t

i t

n

f t .

i t

t i

M

tt

. II

I t

I f •

f t

I I

f f

t t

11

t l

I I

fromfromfrom

t t

i t

- t f

t i

t i -

t t

i f

I I

i t

I I

t i

t i

i t

I I

i t

i t

I I

t t

t t

i t

i t

GC1GC2GC3GC4GC5GC6GC7GC8GP1GP2GP3GP4GP5GP6GP7GP8G 1G2G3G4G5G 6G7G8

A-74

EXP8.XLSWSRC-TR-96-0088

Table A.6.8 ((:ont.)

Exp. 8- Mini-columns - 3-14-95

Effluent

Foam

Method

Notes

Results

Sample

8 18.2Mean

8 38 4Mean

8 58 6Mean

8 78 8Mean

8 98 10Mean

8 118 12Mean

8 138. 14Mean

8 158 16Mean

Effluent

CoalCoal

NiNi

Zn .Zn

Metal mixMetal mix

DIH2ODl H2O

CMP waterCMP water

CoalCoal

CoalCoal

0 coal runoff (pH 2.5 and adjusted to 5.0), collected 3-14-95cond = 2.760. turb = - 1 , DO = 8.36, temp 17.4, sal 0.13

CMP13 pit water 3-13-95, bottle 1252, pH 5.5210.8 ppm Ni standards, pH 5.01.5 ppm Zn standards, pH 5.0Mixed metal standard as described in Exp8pro.doc

Four foam types (made 3-11, ground up 3-13 Jwere used:Ground but unsieved, 11.03% Cyanidium. Used 1.7 g/column (.1875 g dw. algae)Ground but unsieved, 9.46% Phaeodactylum. Used 2 g/col (.1892 g dw algae)Ground but unsieved, 9.76% Chlorella. Used 1.9 g/col (.1854 g dw algae)Ground but unsieved, no algae

EXP8PR0.DOC

Adjusted runoff was actiually 4.95 after centrifugation, unmodified was 2.524Ni 5.0 took 0.42ml/! to adj. pHZn 5.0 took 0.59 mi/1Runoff 5.0 took 3.36 mlMixed metals took 5.29 ml/500 ml

pH

5

5

• 5

5.

55

-

55

55

5.525.52

2.52.5

5

5

Foam

NoneNone

None:

None

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

YesYes

Algae?

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

CyanidiumCyanidium

Al

3.1732.708

2.9405

0.2003nd

0.10015

nd -0.04620.0231

17.1917.3117.25

0.2243nd

0.11215

0.0855nd .0.04275

65.1765.7365.45

2.12.269

2; 1845

Be

0.02450.00770.0161

nd

0.0640.032

0.02590.00960.0178

0.01090.01850.0147

0.00590.O4370.0248

0.06810.029

0.0486

0.02680.01590.0214

ndnd

0

Qd

0.03770.01610.0269

0.00050.0686

0.O3455

0:0292'0 .0106

0.0199

0.05310.0169

0.035

0.00540.0403

O.02285

0.06280.0327

0.04775

0.02620.01280.0195

nd 1nd

0

1

Co

0.25620.23320.2447

0.00170.0623

0.032

0.02620.015

0.0206

0.2380.20920.2236

0.00180.04740.0246

0.06410.0374

0.05075

0.26380.244

0.2539

0.210310.21170.211

Cr

0.02670.00410.0154

nd0.05670.0284

0.03750.00980.0237

0.04540.00750.0265

nd0.05250.0263

0.06660.03750.0521

0.02590.02190.0239

ndnd

0

Cu

0.0780.05290.0655

nd

0.06860.0343

0.03040.00410.0173

0.09420.10660.1004

nd0.04630.0232

0.1030:05680.0799

0.20750.19550.2015

0.02160.02020.O209

Fe

3.6754.922

4.2985

0.26520.14730.2063

0.0409nd0.0205

79.981.4280.66

2.0461.143'

1.5945

nd

nd0

392.9398.1395.5

4.192.6823.436

Ni

0.5910.56960.5803

0.770.8340.802

0.03450.01

0.0223

0.58870.576

0.5824

0.00270.03560.0192

0.05850.03

0.0443

0.73660.73320.7349

0.50860.51990.5143

Zn

1.2711.262

1.2665

0.02250.08770.0551

1.441.4321.436

1.2161.179

1.1975

0.02750.03470.0311

0.0977

0.0638O.08075

1.4741.489

1.4815

1.2321.227

1.2295

A-75

EXP8.XLS WSRC-TR-96-0088

8 178 18Mean

8 198 20Mean

8 218 22Mean

8 238 24Mean

8 258 26Mean

8 278 28Mean

8 298 30Mean

8 318 32Mean

8 338 34Mean

8 358 36Mean

8 378 38Mean

8 398 40Mean

8 418 42Mean

8 438 44Mean

8 458 46Mean

Ni

Ni

ZnZn

Metal mixMetal mix

DIH2O01 H2O

CMP

CMP

CoalCoal

Ni

Ni

ZnZn

Metal mixMetal mix

0IH20Dl H20

CMP

CMP

CoalCoal

NiNi

ZnZn

Metal mix

Metal mix

55

55

55

55

5.55.5

55

55

55

55

55

5.55.5

55

55

55

55

YesYes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

YesYes

YesYes

YesYes

Yes

Yes

CyanidiumCyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

ChlorellaChlorella

ChlorellaChlorella

Chlorella

Chlorella

Chlorella

Chlorella

nd

nd0

nd

0.02230.01115

3.9523.7763.864

ndnd

0

ndnd

0

1.261.377

1.3185*

nd0.0299

0.01495

ndnd

0

0.7390.9005

0.81975

0.08160.0964

0.089

nd0.01220.0061

1.551.3961.473

0.099nd

0.0495

0.05760.1331

0.09535

2.0631.094

1.5785

nd

nd0

nd

nd0

nd

nd0

nd

nd

0

ndnd

0

nd

0

nd

nd0

0

nd0

0.00140.00150.0015

nd

nd

0

ndnd

0

0.00250.00250.0025

0.00280.00020.0015

0.00020

0.0001

0.00230.002

0.0022

ndnd

0

nd

nd0

0.00350.00230.0029

nd

nd0

ndnd

0

nd

nd0

ndnd

0

0.00120.00060.0009

0.01090.011

0.01095

0.00120.0007

0.00095

nd0.00060.0003

0.0050.0051

0.00505

0.0040.0017

0.00285

0.00150.0006

0.00105

0.00990.00910.0095

nd

nd

0

nd

nd0

0.16160.15940.1605

nd

nd0

ndnd

0

0.18140.1789

0.18015

nd

nd

0

0.0005nd

0.00025

0.16540.1686

0.167

0.00130.004

0.00265

nd0.0021

0.00105

0.20280.19480.1988

ndnd

0

nd

nd

0

0.13470.139

0.13685

nd

nd

0

nd

nd0

nd

nd0

nd

nd0

nd

nd0

nd

nd0

nd

nd0

0.0046nd0.0023

0.00290.00260.0028

0.00250.00290.0027

0.00290.00380.0034

0.00110.00120.0012

0.0082nd0.0041

0.00240.0016

0.002

nd

0.00210.0011

nd

nd

0

nd

nd0

0.02340.02630.0249

nd

nd0

ndnd

0

0.01280.01580.0143

nd

nd0

0.01160.0123

0.012

0.03360.03220.0329

0.0110.0089

0.01

0.01910.0189

0.019

0.03070.02970.0302

0.01440.00690.0107

0.0016nd0.0008

0.03030.02620.0283

nd

nd

0

nd

nd

0

12.619.665

11.138

nd

nd0

nd

nd

0

3.3933.376

3.3845

0.93060.94590.9383

0.98660.97440.9805

0.34740.41920.3833

0.8960.9960.946

1.0040.95520.9796

2.288• 2.252.269

0.07460.02510.0499

0.03870.02770.0332

5.0531.51

3.2815

0.63240.65340.6429

nd

nd0

0.43490.42640.4307

nd

nd0

nd

nd

0

0.42620.41890.4226

0.31770.28450.3011

0.00590.00910.0075

0.410.40570.4079

0.00430.00150.0029

0.00880.00330.0061

0.46570.45840.4621

0.12260.12850.1256

ndnd

0

0.39480.38630.3906

" 1

0.00970.01090.0103

1.2051.235

1.22

0.96010.94650.9533

0.00640.0019

0.00415

0.02130.02030.0208

0.97660.9707

0.97365

0.00550.00310.0043

0.44170.3438

0.39275

0.78710.79010.7886

0.01710.014540.01582

0.02250.01990.0212

1.071.059

1.0645

0.01340.0087

0.01105

0.23850.36010.2993

0.76340.76020.7618

A-76

EXP8.XLS WSRC-TR-96-0088

8 478 48Mean

8 498 60Mean

8 518 52Mean

8 538 54Mean

8 558 56Mean

8 578 58Mean

8 598 60Mean

8 618 62Mean

Dl H2ODl H20

CMP

CMP

CoalCoal

Ni

Ni

Zn

Zn

Metal mixMetal mix

01 H20Dl H20

CMP

CMP

55

5.55.5

55

5

5

55

5

5

55

5.55.5

Ves

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

YesYes

ChlorellaChlorella

ChlorellaChlorella

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

NoneNone

nd

0.27190.13595

nd

nd

0

2.3882.211

2.2995

0.1367nd

0.06835

nd

nd

0

12.3313.1712.75

0.0397nd

0.01985

nd

nd

0

0

nd

0

nd

nd

0

0.00320.00320.0032

nd

nd

0

nd

nd

0

0.00570.00580.0058

nd

nd

0

0.00050.00010.0003

0.00050.00090.0007

nd

0.00020.0001

0.00680.007

0.0069

0.00080.0015

0.00115

0.00050.00090.0007

0.01630.01670.0165

0.00110.0004

0.00075

0.00070.00110.0009

nd

nd0

nd

nd0

0.20950.207

0.20825

nd

nd

0

nd

nd0

0.17540.1731

0.17425

ndnd

0

ndnd

0

nd

0.00280.0014

0.00360.00090.0023

nd

nd

0

nd

nd0

0.00340.00340.0034

0.00460.00490.0048

0.0003nd

0.0002

nd

nd

0

nd

nd0

0.01210.00760.0099

0.04780.05080.0493

0.00840.0076

0.008

0.010.01050.0103

0.08380.08350.0837

0.01120.009

0.0101

0.02610.02650.0263

0.02560.02150.0236

0.02740.01810.0228

2.7233.2212.972

nd

nd

0

nd

nd

0

54.1756.62

55.395

ndnd

0

ndnd

0

nd

nd

0

nd

nd

0

0.52950.52080.5252

0.67240.66690.6697

0.0002nd

0.0001

0.50370.49850.5011

0.0014nd

0.0007

0.00380.00430.0041

0.00710.00970.0084

0.0130.0157

0.01435

1.1881.1781.183

0.01640.0139

0.01515

1.2521.245

1.248S

1.0641.0541.059

0.01170.0054

0.00855

0.02730.03710.0322

A-77

EXP8.XLSWSRC-TR-96-0088

8 63 Metal mix 5 Nonecentrif twice like column samples

Summary - Means

Effluent

Coal

Ni

Zn

Metal mix

Dl H20

CMP water

Coal

Coal

Ni

Zn

Metal mix

Dl H20

CMP

Coal

Ni

Zn

Metal mix

Dl H20

CMP

Coal

Ni

Zn

Metal mix

Dl H20

CMP

Coal

Ni

PH

5

5

5

5

5

5.52

2.5

5

5

5

5

5

5.5

5

5

5

5

5

5.5

5

5

5

5

5

5.5

5

5

Foam

None

None

None

None

None

None

None

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

None

Algae?

None

None

None

None

None

None

None

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Cyanidium

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Phaeodac

Chlorella

Chlorella

Chlorella

Chlorella

Chlorella

Chlorella

None

None

12.77

Al

2.9405

0.10015

0.0231

17.25

0.11215

0.04275

65.45

2.1845

0

0.01115

3.864

0

0

1.3185

0.01495

0

0.81975

0.089

0.0061

1.473

0.0495

0.09535

1.5785

0.13595

0

2.2995

0.06835

0.0066

Be

0.0161

0.032

0.0178

0.0147

0.0248

0.0486

0.0214

0

0

0

0

0

0

0

0

0

0.0015

0

0

0.0025

0.0015

0.0001

0.0022

0

0

0.0032

0

0.0172

Cd

0.0269

0.03455

0.0199

0.035

0.02285

0.04775

0.0195

0

0

0

0.0029

0

0

0

0

0.0009

0.01095

O.OOO95

0.0003

0.00505

0.00285

0.00105

0.0095

0.0007

0.0001

0.0069

0.00115

0.1902

Co

0.2447

0.032

0.0206

0.2236

0.0246

0.05075

0.2539

0.211

0

0

0.1605

0

0

0.18015

0

0.00025

0.167

0.00265

0.00105

0.1988

0

0

0.13685

0

0

0.20825

0

0.0032

Cr

0.0154

0.0284

0.0237

0.0265

0.0263

0.0521

0.0239

0

0

0

0

0

0

0

0

0.0023

0.0028

0.0027

0.0034

0.0012

0.0041

0.002

0.0011

0.0014

0.0023

0

0

0.085

Cu

0.0655

0.0343

0.0173

0.1004

0.0232

0.0799

0.2015

0.0209

0

0

0.0249

0

0

0.0143

0

0.012

0.0329

0.01

0.019

0.0302

0.0107

0.0008

0.0283

0

0.0099

0.0493

0.008

55.27

Fe

4.2985

0.2063

0.0205

80.66

1.5945

. 0

395.5

3.436

0

0

11.138

0

0

3.3845

0.9383

0.9805

0.3833

0.946

0.9796

2.269-

0.0499

0.0332

3.2815

0.0236

0.0228

2.972

0

0.5478

Ni

0.5803

0.802

0.0223

0.5824

0.0192

0.0443

0.7349

0.5143

0.6429

0

0.4307

0

0

0.4226

0.3011

0.0075

0.4079

0.0029

0.0061

0.4621

0.1256

0

0.3906

0

0

0.5252

0.6697

1.179

Zn

1.2665

0.0551

1.436

1.1975

0.0311

0.08075

1.4815

1.2295

0.0103

1.22

0.9533

0.00415

0.0208

0.97365

0.0043

0.39275

0.7886

0.01582

0.0212

1.0645

0.01105

0.2993

0.7618

0.0084

0.01435

1.183

0.01515

A-78

EXP8.XLSWSRC-TR-96-0088

Zn

Metal mix

Dl H20

CMP

5

5

5

5.5

Yes

Yes

Yes

Yes

None

None

None

None

0

12.75

0.01985

0

0

0.0058

0

0.0003

0.0007

0.0165

O.OOO75

0.0009

0

0.17425

0

0

0.0034

0.0048

0.0002

0

0.0103

0.0837

0.0101

0.0263

0

55.395

0

0

0.0001

0.5011

0.0007

0.0041

1.2485

1.059

0.00855

0.0322

A-79

EXP8.XLS WSRC-TR-96-0088

Metal mix 5 Nonecentrif twice like column samples

None 12.77

Metal Removal Compared to Controls (not passed through foam)

CYANlOIUM (.1875 g algae. 1.7 g foam/column)

Runoffppm Removed% removedmg/g algaemg/g foam

Nippm Removed% removedmg/g algaemg/g foam

Zn

ppm Removed% removedmg/g algaemg/g foam

Mixed metalsppm Removed% removedmg/g algaemg/g foam

Dl

ppm Removed% removedmg/g. algaemg/g foam

CMP waterppm Removed% removedmg/g algaemg/g foam

Al2.9405

0.75625.70990.2016

0.02224

0.100150.10015

100

0.026710.00295

0.02310.0119551.73160.003190.00035

17.2513.386

77.63.5696

0.39371

0.112150.11215

100

0.029910.0033

0.042750.04275

1000.0114

0.00126

Be0.01610.0161

100

0.0042930.000474

0.0320.032

1000.0085330.000941

0.017750.01775

100.0.0047330.000522

0.01470.0147

1000.00392

0.000432

0.02480.0248

1000.0066130.000729

0.048550.04855

1000.0129470.001428

Cd0.02690.0269

100

0.00717330.0007912

0.034550.03455

1000.00921330.0010162

0.01990.0199

1000.00530670.0005853

0.0350.0321

91.7.142860.00856

0.0009441

0.022850.02285

1000.00609330.0006721

0.047750.04775

1000.01273330.0014044

PHAEODACTYLUM {.1892 g algae. 2 g foam/column)

Runoffppm Removed% removedmg/g algaemg/g foam

Ni -ppm Removed% removedmg/g algaemg/g foam

Zn

ppm Removed

2.94051.622

55.16070.428650.04055

0.100150.0852

85.07240.022520.00213

0.02310.0231

0.01610.0161

1000.0042550.000403

0.0320.032

too0.008457

0.0008

0.017750.01775

0.02690.0269

1000.00710890.0006725

0.034550.03455

1000.00913050.0008638

0.01990.019

Co0.24470.033713.772

0.008990.00099

0.0320.032

1000.008530.00094

0.02060.0206

1000.005490.00061

0.22360.0631

28.220.016830.00186

0.02460.0246

1000.006560.00072

0.050750.05075

1000.013530.00149

0.24470.0645526.37920.017060.00161

0.0320.032

1000.00846

0.0008

0.02060.02035

0.0066

Cr

0.01540.0154

1000.00410.0005

0.02840.0284

1000.00760.0008

0.02370.0237

1000.00630.0007

0.02650.0265

1000.00710.0008

0.02630.0263

1000.007

0.0008

0.05210.0521- 100

0.01390.0015

0.01540.0154

1000.00410.0004

0.02840.0284

1000.00750.0007

0.02370.0214

0.0172

Cu0.065450.0445568.06720.011880.00131

0.03430.D343

1000.009150.00101

0.017250.01725

1000.0046

0.00051

0.10040.07555

75.2490.020150.00222

0.023150.02315

1000.006170.00068

0.07990.0799

1000.021310.00235

0.065450.0511578.15130.013520.00128

0.03430.0343

1000.009060.00086

0.017250.0053

0.1902

Fe4.29850.8625

20.06510.23

0.02537

0.206250.20625

1000.055

0.00607

0.020450.02045

1000.005450.0006

80.6669.5225

86.19218.53932.04478

1.59451.5945

1000.42520.0469

00

#DIV/0!00

4.29850.914

21.26320.241540.02285

0.20625-0.732

-354.91-0.1934-0.0183

0.02045-0.9601

0.0032

Ni0.58030.066

11.3820.01760.0019

0.8020.159119.8380.04240.0047

0.02230.0223

1000.00590.0007

0.58240.1517

26.050.04050.0045

0.01920.0192

1000.00510.0006

0.04430.0443

1000.01180.0013

0.58030.157827.1840.04170.0039

0.8020.500962.4560.13240.0125

0.02230.0148

0.085

Zn

1.26650.037

2.92140.00990.0011

0.05510.044881.3070.01190.0013

1.4360.216

15.0420.05760.0064

1.19750.244220.3920.06510.0072

0.03110.027

86.6560.00720.0008

0.08080.06

74.2410.016

0.0018

1.26650.292923.1230.07740.0073

0.05510.050892.1960.01340.0013

1.4361.0433

55.27

*

0.5478 1.179

A-80

EXP8.XLS

WSRC-TR-96-0088

% removedmg/g algaemg/g foam

Mixed metalsppm Removed% removedmg/g algaemg/g foam

01ppm Removed% removedmg/g algaemg/g foam

CMP waterppm Removed% removedmg/g algaemg/g foam

100

0.00610.00058

17.2516.430395.24784.342030.41076

0.112150.02315

20.6420.006120.00058

0.042750.03665

85.7310.009690.00092

1000.0046910.000444

0.01470.01323590.034010.0034980.000331

0.02480.0248

1000.006554

0.00062

0.048550.04855

1000.01283

0.001214

95.4773870.0050211

0.000475

0.0350.02405

68.7142860.00635570.0006013

0.022850.0219

95.8424510.00578750.0005475

0.047750.04745

.99.3717280.01253960.0011863

CHLORELLA (.1854 g algae. 1.9 g foam/column)

Runoffppm Removed% removedmg/g algaemg/g foam

Ni

ppm-Removed% removedmg/g atgaemg/g foam

Znppm Removed% removedmg/g algaemg/g foam

Mixed metalsppm Removed% removedmg/g atgaemg/g foam

Dl

ppm Removed% removedmg/g algaemg/g foam

SMP waterppm Removed% removedmg/g algaemg/g foam

2.94051.4675

49.90650.395770.03862

0.100150.0506550.57410.013660.00133

0.0231-0.0723-312.77

• -0.0195-0.0019

17.2515.671590.84934.2264

0.41241

0.11215-0.0238-21.222-0.0064-0.0006

0.042750.04275

1000.011530.00113

0.01610.0136

84.472050.0036680.000358

0.0320.0305

95.31250.0082250.000803

0.017750.01765

99.436620.00476

0.000464

0.01470.01255

85.374150.003385

0.00033

0.02480.0248

1000.0066880.000653

0.048550.04855

1000.0130930.001278

0.02690.02185

'81.2267660.0058927

0.000575

0.034550.0317

91.7510850.00854910.0008342

0.01990.01885

94.7236180.00508360.0004961

0.0350.0255

72.8571430.006877

0.0006711

0.022850.02215

96.9365430.00597360.0005829

0.047750.04765

99.7905760.01285060.0012539

98.78640.005380.00051

0.22360.0566

25.31310.014960.00142

0.02460.0219589.2276

0.00580.00055

0.050750.049797.931

0.013130.00124

0.24470.0459

18.75770.012380.00121

0.0320.032

1000.00863O.OOO84

0.02060.0206

1000.005560.00054

0.22360.08675

38:7970.0234

0.00228

0.02460.0246

1000.00663O.OOO65

0.050750.05075

1000.013690.00134

90.2750.00560.0005

0.02650.023789.6030.00630.0006

O.O263j0.023689.7140.00620.0006

0.05210.048793.5640.01290.0012

0.01540.014392.5320.00380:0004

0.02840.024385.5380.00650;OOQ6

0.02370.021791.5430.00580.0006

0.02650.0254

96.030.00690.0007

0.02630.024994.6670.0067•0.0007

0.05210,049895.6770.01340.0013

30.72460.0014

0.00013

0.10040.0675

67.23110.017840.00169

0.023150.0132

57.01940.003490.00033

0.07990.0609

76.22030,016090.00152

0.065450.0352553.85790.009510.00093

0.03430,0236568.95040.006380.00062

0.017250.0164595.36230.004440.00043

0.10040.0721571.86250.01946

0.0019

0.023150.02315

1000.006240.00061

0.07990.0700587.67210.018890.00184

-4694.6-0.2537-0.024

80.6680.276799.524821.21482.00692

1.59450.6485

40.67110.171380.01621

0-O.9796JKDIV/O!-0.2589-0.0245

4.29852.0295

47.21410.547330.05341

0.206250.1564

75.83030.042180.00412

0.02045-0.0128-62.347-0.0034-0.0003

80.6677.378595.9317

20.8682.03628

1.59451.57095

98.5230.423670.04134

0-0.0228#DIV/0!-0.0061-0.0006

66.2920.00390.0004

0.58240.174529.9650.04610.0044

0.01920.016384.8560.00430.0004

0.04430.038286.3280.0101

0.001

0.58030.118320.3770.03190.0031

0.8020.6765B4.3450.18240.0178

0.02230.0223

1000.006

0.0006

0.58240.191832.9360.0517

0.005

0.01920.0192

1000.00520.0005

0.04430.0443

1000.01190.0012

72.650.2757

0.0261

1.19750.408934.1460.10810.0102

0.03110.015349.1320.004

0.0004

0.08080.059673.7460.01570.0015

1.26650.202

15.9490.05450.0053

0.05510.044179.9460.01190:0012

1.4361.136779.1570.30660.0299

1.19750.435736.3840.11750.0115

0.03110.0227

72.990.00610.0OO6

0.08080.066482.2290.01790.0017

•

i •

A-81

EXP8.XLSWSRC-TR-96-0088

FOAM ONLY, 2 g/column

Runoffppm Removed% removedmg/g foam

Ni

ppm Removed% removedmg/g foam

Zn •ppm Removed% removedmg/g foam

Mixed metalsppm Removed% removedmg/g foam

Dl

ppm Removed% removedmg/g foam

CMP waterppm Removed

2.94050.641

21.7990.01603

0.100150.0318

31.75240.0008

0.02310.0231

100

0.00058

17.254.5

26.0870.1125

0.112150.0923

82.30050.00231

0.042750.04275

0.01610.0129

80.124220.000323

0.0320.032

1000.0008

0.017750.01775

100

0.000444

0.01470.00895

60.884350.000224

0.02480.0248

100

0,00062

0.048550.04825

0.02690.02

74.3494420.0005

0.034550.0334

96.6714910.000835

0.01990.0192

96.4824120.00048

0.0350.0185

52.8571430.0004625

0.022850.0221

96.7177240.0005525

0.047750.04685

0.24470.0364514.89580.00091

0.0320,032

100

0.0008

0.02060.0206

1000.00052

0.22360.0493522.07070.00123

0.02460.0246

100

0.00062

0.050750.05075

0.01540.0154

1000.0004

0.02840.0284

1000.0007

0.02370.020385.6240.0005

0.026S0.021782.0420.0005

0.02630.026199,4290.0007

0,05210.0521

0.065450.0161524.67530.0004

0.03430.0263

76.67640.00066

0.017250.007

40.57970.00018

0.10040.0167516.68330.00042

0.023150.0130556.37150.00033

0.07990.0536

4.29851.3265

30.85960.03316

0.206250.20625

100

0.00516

0.020450.02045

100

0.00051

80.6625.265

31.32280.63163

1.59451.5945

100

0.03986

00

0.58030.05529.50370.0014

0.8020.132416.5020.0033

0.02230.022299.5510.0006

0.58240.081213.9520.002

0.01920.018596.3450.0005

0.04430.0402

1.26650.08356.593

0.0021

0.05510.04

72.5050.001

1.4360.187513.0570.0047

1.19750.138511.5660.0035

0.03110.022672.5080.0006

0.08080.0486

.

.—• _ _

A-82

EXP8.XLS WSRC-TR-96-0088

% removedmg/g foam

1000.00107

99.382080.001206

Percent Removal From Runoff

Original ppm% Removal by:

CyanidiumPhaeodactylumChlorellaFoam only

Al

2.9405

25.709955.160749.9065

21.799

Be

0.0161

100

100

84.4720580.12422

Percent removal from Ni solution

Original ppm% Removal by:

CyanidiumPhaeodactylumChlorellaFoam only

0.10015

100

85.072391S0.57413931.752371

0.032

100

100

95.3125

100

Percent removal from Zn solution

Original ppm% Removal by: -

CyanidiumPhaeodactylumChloretla -••

Foam only

0.0231

51.731602100

-312.7706100

0.01775

100

10093.4366197

100

Percent Removal From Metal Mix

Original ppm% Removal by:

CyanidiumPhaeodactylumChlorellaFoam only2ndcentrifugation

1

17.25

77.695.247890.8493

- 26.08725.971

Percent removal from Dl

Original ppm% Removal by:

CyanidiumPhaeodactylumChlorellaFoam only

0.11215

100

20.642-21.22282.3005

0.0147

10090.0340185.3741560.8843555.10204

0.0248

100

100100

100

Percent removal from CMP water

Original ppm% Removal by:

CyanidiumPhaeodactylumChlorellaFoam only

0.04275

100

85.731100

100

0.04855

100

100100

99.38208

98.1151830.0011713

Cd

0.0269

100

100

81.22676674.349442

0.03455

100

100

91.75108538

96.67149059

0.0199

100

95.47738794.7236180996.48241206

• 0.035

91.71428668.71428672.85714352.85714350.857143

•

0.02285

100

95.84245196.93654396.717724

0.04775

10099.37172899.79057698.115183

100

0.00127

Co

0.2447

13.77226.379218.757714.8958

0.032

100

100

100

100

0.0206

100

98:7864100

100

0.2236

28.2225.3131

38,79722.070714.9374

0.0246

100

89.2276100100

0.05075

10097.931

100100

100

0.0013

Cr

0.0154

100

100

92.532100

0.0284

100

100

-SS.S3792

too

0.0237

100

90.27591.5433485.62368

0.0265.

100

89.60396.03

82.04287.902

0.0263

100

89.71494.66799.429

0.0521

10093.56495.677

100

67.08390.00134

Cu

0.06545

68.067278.151353.857924.6753

0.0343

100

100

68.950437

76.676385

0.01725

100

30.724695.36231940.57971

0.1004

75.24967.231171:862516.683315.3386

0.02315

100

57.0194100

56.3715

0.0799

100

76.220387.672167.0839

J»DIV/0!0

Fe

4.2985

20.065121.263247.214130.8596

0.20625

100

-354.909175.830303

100

0.02045

100

L-4694.6-62.34719

100

80.66

86.19299.524895.931731.322831.4778

1.5945

100

40.671198.523

100

0

#DIV/0!#DIV/0!#DIV/0!#DIV/0!

90.8470.001

Ni

0.5803

11.38227.18420.3779.5037

0.802

19.83862.45684.34516.502

0.0223

100

66.292100

99.55056

. 0.5824

26.0529.96532.93613.9525.9329

0.0192

100

84.856100

96.345

0.0443

10086.328

10090.847

60.1240.0012

Zn

1.2665

2.921423.12315.9496.S93

0.0551

81.30672

92.1960179.9455572.SO4S4

1.436

15.04272.65

79.15713.057

1.1975

20.39234.14636.38411.5661.5449

0.0311

86.65649.13272.99

72.508

0.0808

74.24173.74682.22960.124

•

*

— — —

^

— .

•

— —

A-83

WSRC-TR-96-0088

Table A.6.9 Experimental procedures and results forExperiment #9

EXPERIMENT #9 Comparing Metal Removal from well water by foamembedded with algae packed in the DCB-4A water using in the modifiedFrisby test bed. 11/7/95

I. PURPOSE:Compare bioremoval efficacy of four algal species by passing metalcontaminated ground water through columns containing foam-algae aggregates.Metals to be measured will include Al, Cr, Fe, & Ni,

II. ALGAE:Cyanidium (33 days old)Mastigocladus (33 days old)Phaeodactylum (21 days old)Chlorella (21 days old)

AH harvested by centrifugation.

III. FOAM:8 mesh containing 10% algae by dry weight

IV. Effluent-

Monitoring Well DCB-4A

V. PROCEDURE:

A. Preparation of algae

Set up water baths for the growth of 16 4-I bottles of algae. (8 for thermophilesand 8 for non-thermophiles). Make media for growing 4 bottles of of each of thefollowing 4 species: Cyanidium caldarium, Mastigocladus laminosus,Phaeodactylum tncornutum, and Chlorella vulgaris. Fill each bottle with 2 litersof media and autoclave. Set bath apparatus for light conditions and 0.1% CO2bubbling as before. Inoculate each bottle with with a 25ml aliquot taken fromthe most recent culture transfer,'following homogenization. Measure dry wt ofeach bottle after 5 days and 10 days. Harvest by centrifugatipn (10 min, 10000rpm). Wash cells with DI water and resuspend at 5% by dry wt. with Dl.

A-84

Table A.6.9 (Cont.)

WSRC-TR-96-0088

B. Preparation of foam/algae aggregates

Take algal suspensions to Frisby for embedding. Have them use the best combinationof surfactant, prepolymer and biomass as determined for minimal "washout" in G-4experiments. Following embedding, the samples should be ground and sieved to 8mesh. Plan to use about 2 g of granulated foam per column per test run.

C. Preparation of effluent

1. Collect water from D area monitoring well DCB-4AMeasure pH and other parameters with a Horiba ASAP aftercollection.

D. Preparation of columns in Test Bed

1. Pack 2 columns with 2 g of particulate foam +algae for each ofthe four species

E. Contacting biosorbent with effluent in Test Bed1. flush apparatus with DI water and drain2. Fill Reservoirs with 1 liter of untreated runoff each3. start pumps and adjust flow rates to 2.5gal/hr4. Run for 8 hrs = 53 bed volumes5. collect samples after 8 hrs by opening effluent valves

and collecting 30 +ml of treated water in acid-wasKed 60ml vials6. preserve with 0.5 ml cone. HNO3

VI. Supplies neededCarboy for Coal pile runoff basin water68 acid cleaned 60 ml vials for samples to be taken to ADSHNO3 concentrated for preserving samplesHNO3, 1N (for adjusting pH)HNO3, 10 N "NaOH, 1 NNaOH, 10 N "HNO3, ca 3 N for rinsing pH electrodeDeionized water

A-85

ITable A.6.9 (Cont.)

WSRC-TR-96-0088

VII. SAMPLE LABELS

The following labels wiil be used on samples sent to ADS:

Label9-1 a&b9-2 a&b9-3 a&b9-4 a&b9-5 a&b9-6 a&b9-7 a&b9-8 a&b

DescriptionChlorellaCyanidiumMastigocladusPhaeodactylum

ChlorellaCyanidiumMastigocladusPhaeodactylum

A-86

EXP9.XLSWSRC-TR-96-0088

Table A.6.9"Bioremova! in Modified Test Rig #1{Cont.)

Experiment 9 -Modified Frisby Packed Bed Rig 11-7-95 .

Effluent

Foam

Method

Notes

Results

AlgaeCont/6Cont/6Coht/6Cont/7Cont/7Cont/7Chlor.Chlor.Chlor:Chlor.Chlor:Chlor.Cyan. 'Cyan.Cyan.Cyan.Cyan.Cyan.Mast.Mast.Mast.Mast.Mast.Mast. .Phaeo.Phaeo.Phaeo...Phaeo.Phaeo.Phaeo.

Well DCB-4Acond = 0.290 mS/cm , turb = 1 , DO = 7.4 mg/1, temp = 20.4 , sal = 0.01%

Four algae, Chlorella, Cyanidium, Mastigocladus, and Phaeodactylum wereembedded in foam. All had 10 % algae by dry weight in the final foam.

EXP9PRO.DOC

Percent removal by the algae calculated relative ot metal concentrations in untreated wastewater

At10.5410.5910.5710.9110.8110.864.604.674.643.223.233.233.543.363.453.2.13.253.232.943.123.035,765.815.79

.4.324.46

: 4.39: 5.91

6.025.97

Fe0.270.260.270.210.210.210.110.120.120.120.120.120.14

.••Oil 30.140.180.170.180.160.160.160.170.18O.180.170.170.170.170.170.17

Cr

- 2-52

2.251.91.9

1-90.4

0:30.35

0.8

0.60.70.50.50.50.60.6.0.6

; .21.81.9

2.42.4

2.4

1.81.7

1.752.22.5

2.35

Ni .676365

i- 6 4

6263

63

616 2

71

.6568

6667

66.5676 8

67.5.. 74

72" 73

6868686968

68.5676968

% AlRem.,

1.376896149

-1.376896149

56.7327888

69.8949825

67.79463244

. 69.84830805

71.71528588

45.99766628

59.01983664

44.31738623

% Fe Rem.

-11.57894737

11.57894737

51.57894737

49.47368421

43.15789474

26.31578947

32.63157895

26.31578947

28.42105263

28.42105263

% Cr Rem.

"- -8.43373494

8.43373494

83.13253012

66.26506024

75.90361446

71.08433735

8.43373494

-15.6626506

15.6626506

-13.25301205

% Ni Rem.

-1.5625

1.5625

3.125

-6.25

-3.90625

-5.46875

-14.0625

-6.25

-7.03125

-6.25

A-87

WSRC-TR-96-0088

Table A.6.10 Experimental procedures and results forExperiment#10

EXPERIMENT #10 Follow-up comparison of metal Removal from well water by foamembedded with algae packed in the DCB-4A water using in the modified Frisby testbed. 11/28&29/1995

I. PURPOSE:Repeat conditions of Experiment 9, except add controls to replace Phaeodactylumwhich did the poorest in previous test. Compare bioremoval efficacy of three algalspecies by passing metal contaminated ground water through columns containingfoam-algae aggregates. Metals to be measured will include Al, Cr, Fe, & Ni,

II. ALGAE:Cyanidium (33 days old at harvest)Mastigocladus (33 days old)

Chlorelfa (21 .days old) '<

All harvested by centrifugation.

III. FOAM:8 mesh containing 10% algae by dry weight

IV. Effluent:

Monitoring Well DCB-4A

V. PROCEDURE:

I.Use same foam/algae aggregates as in Experiment 92. For preparation of effluent, collect water from D area monitoring well DCB-

4A. Measure pH and other parameters with a Horiba ASAP after collection.

3. Prepare columns in Test Bed by packing 2 columns with 2 g ofparticulate foam +algae for each of the three algae. Pack the other twoColumns with plain foam and no foam, respectively.

4. To contact biosorbent with effluent in test bed:

1. flush apparatus with Dl water and drain2. Fill Reservoirs with 1 literof untreated runoff each3. start pumps and adjust flow rates to 2.5gal/hr4. Run for 8 hrs = 53 bed volumes

A-88

Table A.6.10 (Cont.)WSRC-TR-96-0088

5. collect samples after 8 hrs by opening effluent valvesand collecting 30 +ml of treated water in acid-washed 60m! vials

6. preserve with 0.5 ml cone. HNO3

VI. Supplies neededCarboy for wastewater18 acid cleaned 60 ml vials for samples to be taken to ADSHNO3 concentrated for preserving samples

VII. SAMPLE LABELS

The following labels will be used on samples sent to ADS:

Label10-110-210-310-410-510-610-710-8

a&ba&ba&ba&ba&ba&ba&ba&b

DescriptionChlorellaCyanidiumMastigocladuswastewater only - no foamChlorellaCyanidiumMastigocladusplain foam

A-89

EXP10JCLS

WSRC-TR-96-0088iDie A.b.i

Experiment 10

Effluent

Date

- Bioremoval using the Modified Frisby Packed Bed Rig

11/28/95

Well DCB-4A

cond = 1.14 mS/cm . turb = 0, DO =

Algae - Chlorella, Cyanidium, Mastigocladus,

Foam

Method

Results

AlgaeControl/1Control/1Control/1Control/2Control/2Control/2ChlorChlorChlorChlorChlorChlorCyan.Cyan.Cyan.Cyan.Cyan.Cyan.Mast.Mast.MastMast.Mast.Mast.No FoamNo FoamNo FoamPlain FoamPlain FoamPlain Foam

10%algae

Al52.1551.0961.6251.9851.4451.7144.4845.3944.9448.9450.3349.6449.6648.6349.1549.8949.6749.7849.75

-49.3649.5649.2749.14

- 49.2152.7552.2062.4847.7147.5347.62

Iby dry weight in final foam

EXP10PRO.DOC

Fe0.110.090.100.080.090.080.110.090.100.090.110.100.110.100.110.080.050.060.080.070.080.130:120.120.060.030.050.100.050.08

Cr1.501.501.601.501.601.651.701.601.650.600.600.600.400.400.400.300.300.300.500.500.500.601.10

^0.850.800.700.760.500.500.60

Ni260260260260275268260255258245235240230230230230230230230220225220250235225225225225220223

KAlRem.

0.09

4.09

13.03

3.93

4.88

3.65

4.08

4.76

-1.67

7.83

=7.12mg/l,temF

% Fa Rent.

-3.97

8.97

-11.37

-13.33

-21.82

27.97

15.67

-36.91

47.92

m

14.33

> = 20.7 . sal = 0.05

%CrRem.

1.64

-1.64

•3.20

60.66

73.77

80.33

67 .21

44.26

60.82

67.21

%NIRem.

1.42

-1.42

2.37

9.00

12.80

12.80

14.69

10.90

14.69

15.64

A-90

WSRC-TR-96-0088

Table A.6.11 Experimental Procedures and Results for Experiment #11

EXPERIMENT 11(2/8/96-2/15/96)

Metal Removal using foam embedded withCyanidium, Chlorella andMastigocladus following various pretreatments

I. PURPOSE:Evaluate effects of pretreatment of biomass prior to embedding in foam for

three species of algae on metal removal

/ II. ALGAE:33-day old cultures of:Cyanidium caldariumChloreHa sp.Mastigocladus laminosus

AH Harvested by centrifugation,

III. PRETREATMENTS:

Acid wash (0.2N HCL)Organic solvent wash (O.2N Acetone)Heat killing (110°C for 4hrs)salt shock (0.2N NaCI)NaOH (0.2N) *Dlwash

IV. FOAM:8 mesh containing 10% algae by dry weight

V. EFFLUENT:Monitoring well DCB-4A

V1 TEST APPARATUS:

Modified Frisby packed bed bioreactor

VH. PROCEDURE:

A. Preparation of effluent

A-91

Table A.6.11 (Cont)WSRC-TR-96-0088

1. Collect water from pump at well near D-Area coal pile runoffbasin

2. Upon returning to lab measure Horiba parameters(pH, etc.)

B. Preparation and Use of Bioreactor

1. Flush test bed with several bed volumes of Dl water2. Make three 8-hr runs with the test rig (one for each alga)

3. Fill columns with 2 g of foam using the following strategy:Column 1 - HCL .Column 2 - NaOHColumn 3 - NaCIColumn 4 - HeatColumn 5 - AcetoneColumn 6 -D lColumn 7 - Plain FoamColumn 8 - no foam (raw wastewater)

Also prepare samples of untreated (not run through test bed) wastewater for controls

4. fill test rig with 1 liter of wastewater for each column assemblyand run in recirculating mode for 8 hrs at a flow rate of 2.5 gal/hr

5. Collect 30ml samples for chemical analyses irUEPA-certified acidcleaned bottles containing 0.5 ml concentrated HNO3.

6. Take samples to ADS for the following chemical analyses:Cr & Ni by ICP-MSFe & Al by ICP-ES

A-92

Table A.6.11 (Cont.)

VIII. SAMPLE LABELSWSRC-TR-96-0088

The following labels were used on samples sent to ADS:

Label11CY-1A&B11CY-2A&B11CY-3A&B

11CY-5A&B11CY-6A&B11CY-7A&B11CY-8A&B11 Cont 2/8-A&B

11CH-1A&B11CH-2A&B11CH-3A&B11CH-4A&B11CH-5A&B1-1CH -6A&B11CH-7A&B11CH-8A&B11 Cont 2/13 -A&B

11M-1A&B11M-2A&B-11M-3A&B11M-4A&B11M-5A&B11M-6A&B11M-7A&B11M-8A&B11 Cont 2/15 -A&B

DescriptionCyanidium, HCLCyanidium, NaOHCyanidium, NaCICyanidium , Heat killedCyantdium, acetoneCyanidium, Dl washplain foamtreated control - no foam or algaeuntreated control

Chiorella, HCLChorella, NaOHChlorella, NaCIChiorella, Heat killedChlorella, acetoneChloreila, Dl washplain foamtreated control - no foam or algaeuntreated control

Mastigocladus, NaCIMastigocladus, heat killed ^Mastigocladus, acetoneno foamDl washNo foamplain foamno foamuntreated control

A-93

EXP1TC.XLSWSRC-TR-96-0088

Table A.6.11 (Cont.)

Experiment 11 Bioremoval by three algae using six pretreatments

Date 2/8, 2/13, & 2/15/96

Wastewater

WQ

Rig

AlgaCyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.:CyarihCyan:.Cyan,,Cyan.Cyan:Cyan.Cyan.Cyan.Cyan.Cyan:Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.

Well DCB-4A

pH- 3.96. Cond. - 1.05ms/cm, Turb -0,

Modified packed Bed

TreatmentHCIHCIHCINaOHNaOHNaOHNaCINaCtNaCIHeatHeatHeatAcetoneAcetoneAcetoneDlDlDlPlain FoamPlain FoamPlain FoamNo FoamNo FoamNo FoamControl 2/8Control 2/8Control 2/8HCIHCIHCINaOHNaOHNaOHNaCINaCINaCIHeatHeat

AI349.8549.8949,8748.8449.1348.98548.6148.8448.72548.2649.91 j49.08549.8949.2749.5849.7750.2650.01550.18, .50.1950.18551.8651.4851.6752.7152.4152:5647.9447.947.9249.3848.648,9948:0949.1748.6347.0949.02

Temp. 17.6, DO 6.

2.5gal/hr -for 8hr. in recirc. mode

Fe20.11140.06430.087850.1105 -0.08280.096650.07770.08860.083150.11830.09450.10640.10460.J5760.13110.05750.12180.08965'0.1669;0.10030.1336:0.08610.07360.079850.12430.0870.105650.09580.11430.105050.10420.2490.17660.10320.10980.10650.07740.06

Cr3.43.43.43.843.993,66,34.654.64.24:44-34:64.64.66.85.46.154:84.95.45.45.44.84.64.78.84.86.84.84.84.85.45.2

Ni320330325330330330350330340340340340350360355360360360370370370370370370390380385340330335352340346340340340340340

IAl

5.12

6.80

7.30

6.61

5.67

4.84

4.52

1.69

0:00

7.84

5.78

6.47

56. Sal. <

3ercentFe

16.85

8.52

21.30

-0.71

-24.09_ "

15.14

-26.46

24.42

0.00

-28.42

T115.89

-30.20

).O4

removalCr

L37.04

27.78

-16.67

11.11

20.37

14.81

-12.96

9.26

0.00

6.93

-34.65

4.95

ft

Ni

15.58

14.29

11.69

11.69

7.79

6.49

3.90

3.90

0.00

6.94

3.89

5.56

A-94

EXP11C.XLS

WSRC-TR-96-0088

Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor,Chlor.Chlor.Chlor.Chlor.Chlor.Chlor^Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast,Mast.MastMast-Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast;Mast.Mast.Mast.

HeatAcetoneAcetoneAcetoneDlDlDlPlain FoamPlain FoamPlain FoamNo FoamNo FoamNo Foam .Control 2/13Control 2/13Control 2/13NaCINaCINaCIHeatHeatHeatAcetoneAcetoneAcetoneNo FoamNo FoamNo FoamDlDlDlNo FoamNo FoamNo FoamPlain.FoamPlain FoamPlain FoamNo FoamNo FoamNo FoamControl 2/15Control 2/15Control 2/15

NT. = No treatmentNF = No FoamPF = Plain foam

48.05550.0549.649.82549.8448.7949.31551.2549.450.32550.0751.2550.6652.3651.6351.99550.651.4751.03550.9851.4451.2150.8451.0350.93552.1252.452.2651.149.7850.4451.3952.351.84550.3147.448.85551.4552.3551.951.8752.2152.04

0.06870.09980.11010.104950.0840.09280.08840.16050.10160.131050.17890.07710.1280.07940.08420.08180.3370.14190.239450.17160.08670.129150.08880,05120.070.1330.09640.11470.08310.05760.070350.14060.08260.11160.12430.09150.10790.10730.11480.111050.14410.09280.11845

5.34.74.64.655.15.45.254.94.94.94.64.94.7555.15.054.84.84.855.15.055.15:45.255.35.15.25.14.9555.15:055.35.35.35.155.055.15.15.1

340330340335340350345350350350350350350360360360370350360380380380370380375380370375380380380380380380380380380370370370380380380

7.58

4.17

5.15

3.21

2.57

0.00

1.93

1.59

2.12

-0.42

3.07

0.37

6.12

0.27

0.00

16.01

-28.30

-8.07

-60.21

-56.48

0.00

-102.15

-9.03

40.90

3.17

40.61

5.78

8.91

6.25

0.00

-4.95

7.92

-3.96

2.97

5.94

0.00

5.88

0.98

-2.94

-1.96

1.96

0.98

-3.92

0.98

0.00

5.56

6.94

4.17

2.78

2.78

0.00

5.26

0.00

1.32

1.32

0.00

0.00

0.00

2.63

0.00

A-95

WSRC-TR-96-0088

Table A.6.12 Experimental Procedures and Results for Experiment #12

EXPERIMENT 12 (3/13&15/96)

Metal Removal using foam embedded withCyanidium and Mastigocladususing packed bed and static mixer bioreactors

I. PURPOSE:Evaluate effects of treatment with two different bioreactor types using twospecies of algae for metal removal

II. ALGAE:35-day-old cultures of:Cyanidium caldariumMastigocladus laminosuChlorella pyrenoidosa

All Harvested by centrifugation,

III. PRETREATMENTS:

Dlwash

IV. FOAM:8 mesh containing 10% algae by dry weight

V. EFFLUENT:Monitoring well DCB-4A

V1 TEST APPARATUS:

Modified Frisby packed bed bioreactor and Static Mixer bioreactor

VII. PROCEDURE:

A. Preparation of effluent1. Collect water from pump at well near D-Area coal pile runoff

basin

2. Upon returning to lab measure Horiba parameters(pH, etc.)

B. Preparation and Use of Bioreactors

• Flush each with several bed volumes of Df water

A-96

TableA.6.12(Cont.)

WSRC-TR-96-0088

1. Static Mixer:Flow rates = 2.5 gal/hr for Mastigocladus,

19.6gal/hr for Cyanidiumalgae+foam/wastewater ratio = 160g/8liters wastewaterRun for 8 hours pulling samples each hour

2. Packed Bedrun as before

• Make one 8-hr run along with the. 1st static mixer run.

• Also prepare samples of untreated (not run through testbed) wastewater for controls

• fill test rig with 1 liter of wastewater for each column assemblyand run in recirculating mode for 8 hrs at a flow rate of 2.5 gal/hr

• Collect 30ml samples for chemical analyses in EPA-certified acidcleaned bottles containing 0.5.ml concentrated HNO3.

• Take samples to ADS for the following chemical analyses:Cr & Ni by ICP^MSFe & Al by ICP-ES

VIII. SAMPLE LABELS

The following labels were used on samples sent to ADS:

Label Description12PB-1A&B Mastigdcladus packed bedT2PB-2A&B Cyanidium12PB-3A&B Chiorella12PB-4A&B Plain foam12PB-5A&B Mastigocladus "12PB-6A&B Cyanidium12PB-7A&B, Chiorella "12PB -8A&B treated control - no foam or algae12Cont - A&B untreated wastewater control

12SM -1 A&B-M Mastigocladus 1 hr12SM -2A&B -M Mastigocladus 2hr12SM -3 A&B-M Mastigocladus 3hr12SM -4A&B -M Mastigbcladus 4hr12SM -5A&B-M Mastigbcladus 5hr12SM-6A&B-M Mastigocladus 6hr

A-97

TableA.6.12(Cont.)WSRC-TR-96-0088

12SM-7A&B-M Mastigocladus 7hr12SM -8A&B-M Mastigocladus 8hr

12SM -1 A&B -CY Cyanidium 1 hr12SM-2A&B-CY Cyanidium 2hr12SM -3 A&B- CY Cyanidium 3hr12SM -4A&B -CY Cyanidium 4hr12SM -5A&B -CY Cyanidium 5hr12SM -6A&B-CY Cyanidium 6hr12SM-7A&B-CY Cyanidium 7hr12SM -8A&B -CY Cyanidium 8hrCont. .A&B untreated wastewater controls

A-98

EXP12A.XLSWSRC-TR-96-0088

TableA.6.12 Cont;

Experiment 12 Bioremoval by two algae using two test rigs

Date

Effluent

Water Q

Test Rig

AlgaCyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan,Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Cyan.Mast.

3/13/96 {run #1) 3/15/96 (run #2)

Well DCB-4A

pH 3.79, Cond. 1.11ms/cm, DO 7.27, Temp 21.3, Sal 0.05%

IModified packed bed bioreactor and static mixer bioreactor

Rig/Col.PB/2PB/2PB/2PB/6PB/6PB/6SM 'SMSM

SM

SM

SMSM

SM

SMSM

SMSM

SM

SMSM

SMSM

SM

SMSMSM

SMSMSM

SM

SMSM

PB/1

1Metal concentrations-

Time8 Hr8 H r8 Hr8 H r

8 Hr8 Hr0 HrOHrOHr1 Hr1 Hr1 Hr2 Hr2 Hr2 H r

3 H r3 H r

3 H r4 H r

4 Hr4 H r5 Hr5 Hr5 Hr6 H r6 Hr6 H r

7 Hr7 H r7 Hr8 Hr8 Hr8 Hr8 Hr

Al(ppm)62.02762.12162.07461.59161.51

61.550564.94764.81364.8857.0857.2

57.1459.26459.177

59.220559.19259.089

59.140559.312

59.1559.23159.14259.183

59.162558.56859.123

58.845559.24259.107

59.174558.45858.36658.41261.198

Fe(ppm)0.1260.123

0.12450.1250.1170.1210.0530.054

0.05350.1910.189

0.190.368

0.360.3640.51

,0.5130.5115

0.6560.641

0.64850.7950.7910.7930.9740.9080.9411.0050.9971.0011.0481.0481.0480,153

Cr(ppb)2.7

2,92.8

3.6

3.23;44.7

4.6

4.6540.5

42

41.2599

87.793.35

133

127,2^ 130.1

147.8155.9

151.85204

209.9206.95

242.3224.6

233.45257

256.3256.65

292.7289.8

291.253

Nifppb)360

390375

420

390405449

480

464.5365

369

367

426

' ,3784 0 2

431: 408

419.5

402

430416

487496

491.5

537503

520

531537534

581

580580.5

380

Percent removal *Al

-0.959

-0.107

0

11.93

8.723

8.846

8.707

8.812

9.301

8.794

9.969

Fe

-8.261

-5.217

0

-255.1

-580.4

-856.1

-1112

-1382

-1659

-1771

-1859

Cr

6.6667

-13.333

0

-787.1

-1907.5

-2697.8

-3165.6

-4350.5

-4920.4

-5419.4

-6163.4

Ni

8.5366

1.2195

0

20.99

13.455

9.6878

10.441

-5.813

-11.95

-14.96

-24.97

A-99

EXP12A.XLSWSRC-TR-96-0088

Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast. -Mast.Mast.Mast. 'Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast.Mast. .Mast.Mast.Mast.Mast.Mast.Chlor.Chlor.Chlor.Chlor.Chlor.Chlor.P FP FP F

N FN FN F

PB/1PB/1PB/5PB/5PB/5SMSMSM

SMSMSM

SM

SMSM

SM .SM

SMSM

SM

SMSM

SMSM

SM

SMSMSM

SM

SM

SMSM

SM

PB/3 -PB/3PB/3PB/7PB/7PB/7PB/4PB/4PB/4PB/8PB/8PB/8

* No data

8 Hr8 Hr8 Hr8 Hr8 Hr0 HrOHr0 Hr1 Hr1 Hr1 Hr2 H r

2 Hr2 H r

3 Hr .3 Hr3 Hr4 Hr4 Hr4 Hr5 Hr5 Hr5 Hr6 H r

6 Hr6 H r7 Hr .7 Hr7 Hr8 Hr8 Hr8 H r

8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr8 Hr

61.29361.2455

61.22461.205

61.214561.35861.611

61.484557.12257.183

57.152556.846

56.5656.703

. 56.74556.75156.74856.01456.521

56.267556.24556.62556,435

56.9256.78456.85256.60857.261

56.934556.90757.096

57.001561.20861.74461.47661.29361.332

61.312562.15361.936

62.044561.58561.312

61.4485

0.1190.1360.1020.118

0.110.1010.1290.1150.3750:365

0.370.3630.3730.3680.3980.3840.3910.4090.391

0.4

0.440.443

0.44150.44

0.4690.4545

0.520.559

0.53950.5580.5660.562

0.110.1140.1120.1390.128

0.13350.1160.113

0.11450.1290.112

0.1205

3

3

2.8

2.9

2.8533

347

47

47

56

5555.5

64

64

64

72

73

72.588

89

88.59899

98.5100

110

105

110110

110

2.9

3

2.95

3.33.3

2.93.1

33.13.1

3.1

380

380j

390

400

395

410

410410

490

5004 9 5

500

490495

500

520

510

520

520

520530

540

535

540

550

545

550

550

550560

560

560

380

390

385*

4 0 0400

390

400395

400410

405

0.389

0.439

0

7.046

7.777

7.704

8.485

8.213

7.534

7.4

7.291

0.014

0.28

-0.911

0.059

-18.26

4.348

0

-221.7

-220

-240

-247.8

-283.9

-295.2

-369.1

-388.7

2.609

-16.09

0.435

-4.783

0

5

0

-1466.7

-1750

-2033.3

-2316.7

-2850

-3183.3

-3400

-3566.7

1.6667

-10

0

-3.3333

7.3171

3.6585

0

-20.73

-20.73

-24.39

-26.83

-30.49

-32.93

-34.15

-36.59

6.0976

2.439

3.6585

1.2195

A-100

WSRC-TR-96-0088

Table A.6.13 (Cont.) Experimental Procedures and Results for Experiment#13

EXPERIMENT 13 (3/25-29/96)

Bioremoval of Tc-99 using foam embedded with nine types of biomasscompared to removal by ion exchange resin

I. PURPOSE:Evaluate alga! and non-algal biomass for radionuclide removal. Use

methodology routinely used by Donna Beales for Tc-99 spike experiments, i.e.1g filter media packed in bio-rad columns , 500 ml spiked solution (river waterand Dl), gravity flow, etc. to comparatively evaluate removal efficacy ofbiomass/foam aggregates and ion exchange resins.

II. Biomass:Alga#1 = Mastigocladus laminosus/l/ga#2 = Cyanidium caldahumA!ga#3 = Nostoc Sp.Bacteria#1 -Strain G-4Bacteria#2 = Pseudomonas aeruginosaFungus#1 = Yeast strain #R14Fungus#2 = Yeast strin R-42Plant#1 = Datura (Gypsum weed)Plant#2 = Azpfla ,Ion exchange resin = TEVA resin

III. BIOMASS PRETREATMENTS:

Dl wash

IV. FOAM:

8 mesh containing 10% biomass by dry weight

V. EFFLUENT:Tc-99 spiked DI water and Tc-99 spiked Dl water

V1 TEST APPARATUS:Bio-Rad Columns

VII. PROCEDURE:

A-101

TableA.6.13Cont.) WSRC-TR-96-0088

A. Preparation of effluent

1. Add 16nCi Tc-99 to 500 ml of water (Dl or River)

B. Preparation and Use of Bio-rad Poly prep colunnMake two runs , one w/ Tc-99-spiked Dl water, and one w/ Tc-99 spiked riverwater. Set up duplicate columns for each adsorbent material for each run asfollows:

Column arrangements for Run #1 Dl water and Run #2 River water

Col.1 and 12 - algae 1 (Mast.)Col.2 and 13 - algae 2 (Cyan)Col.3 and 14 - Bad 1 (G-4)Col.4 and 15- Bad. 2 (P. aerug.)Col.5 and 16 - Fungi 1 (yeast #14)Col.6 and 17 -Fungi 2 (Yeast #42)Col.7 and 18 - Plant seed 1. (Azolla)Coi.8 and 19 - plant seed 2 ( Datura)

. Col 9 and 20 - ion exchange resinCol 10 and 21 - plain foam controlCol 11 and 22 - Control - no foam or resin

Total number of columns used = 44 (22 columns per run X 2 runs)Total number of samples = 88 (duplicate samples taken from each column)

2. Add 1.00-1.05 gram of material to each column.

A-102

EXP13.XLS

WSRC-TR-96-0088TableA.6.13Cont.) —

Experiment #13 Tc-99 extraction using foam embedded with nine types of biomass/resin

Date 3/25-28/96

Test apparatus/technique: Bio-Rad polyprep columns and overnight batch extraction

Percent of Tc-99 extracted from solution

BiomassMast.Mast.Cyan.Cyan.G-4G-4P. aerug.P. aerug.Yeast #14Yeast #14Yeast #28Yeast #28AzollaAzollaDaturaDaturaTEVA resinTEVA resiriPlain foamPJain foamNo foamNo foam

Amount0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml0-250ml '250-5OOml0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml0-250ml250-500ml

* sample lost

Deionized waterTesti

2.10

4.80.83.6

*0.90.54.90.56.51.13.4

03.10.5

99.899.85.50.30.30.3

Test 24.50.1

25.19.3

10.13.2

00

0.30

6.90

0.80

24.1**

99.8100.3

11.22.5

00

** Column plugged, did not pass whole sample

Unfiltered river waterTesti

5.60

16.40.7

5000

0.9

1,1120

1.3000

84.695.9

6.700

2.4

Test 22.9

018.3

03.5

00.3

00

*-*1.7

00000

96.797.613.40.30.6

0

Deionized water/batchAliquot 1

3.4

33.8

3.4

1.3

0

5.6

0

0.6

95.5

17.4

0

Aliquot 23.2

37

3

0

1.5

5.9

0

0

97.1

21.2

0

A-103

WSRC-TR-96-0088

Table A.6.14 Experimental Procedures and Results for Experiment #14

EXPERIMENT 14 (3/26-28/96)

Bioremoval of metals using foam embedded with nine types of biomass using thepacked bed bioreactor

I. PURPOSE:Evaluate algal and non-algal biomass for metal removal

II. Biomass:Alga #1 = Mastigocladus laminosusAlga #2 = Cyanidium caldariumAlga #3 = Nostoc Sp.Bacteria #1 = Strain G-4Bacteria #2 = Pseudomonas aeruginosaFungus #7 = Yeast strain #R14Fungus #2 = Yeast strain R-42Plant #1 = Datura (Gypsum weed)Plant #2 = Azolla

III. PRETREATMENTS:

Dl wash

IV. FOAM:

8 mesh containing 10% biomass by dry weight

V. EFFLUENT:Monitoring well DCB-4A

VI TEST APPARATUS:

Modified Frisby packed bed bioreactor

VII. PROCEDURE:

A. Preparation of effluent

1. Collect water from pump at well near D-Area coal pile runoffbasin

2. Upon returning to lab measure Horiba parameters(pH, etc.)

A-104

Table A.6.14 (Cont.)1 V WSRC-TR-96-0088

B. Preparation and Use of Bioreactor

1. Flush test bed with several bed volumes of Dl water

2. Make two 8-hr runs with the test rig

3. Fill columns with 2 g of foam using the following strategy:

Run#1Column 1 MastigocladusColumn 2 -CyanidiumColumn 3 - G-4Column 4 - P. aeruginosaColumn 5 -R-14 (yeast)Column 6 - R-42 (yeast)Column 7 - Plain FoamColumn 8 - no foam (raw wastewater)

Also prepare samples of untreated (not run through test bed) wastewater for controls

Run#2Column 1 DaturaColumn 2 -AzollaColumn 3 - Nostoc-DColumn 4 - Nos.toc - NDColumn 5 -not usedColumn 6 - not usedColumn7 -Plain FoamColumn 8 - no foam (raw wastewater)

4. fill test rig with 1 liter of wastewater for each column assemblyand run in recirculating mode for 8 hrs at a flow rate of 2.5 gal/hr

5. Collect 30ml samples for chemical analyses in EPA-certified acidcleaned bottles containing 0.5 ml concentrated HNO3.

6. Take samples to ADS for the following chemical analyses:Cr & Ni by ICP-MSFe & Al by ICP-ES

A-105

WSRC-TR-96-0088

VIII. SAMPLE LABELS

The following labels were used on samples sent to ADS:

Label

14 -1A&B14 -2A&B14-3A&B14 -4A&B14 -5A&B14-6A&B14-7A&B14-8A&B14 Cont.

14-1C&D14 -2C&D14-3C&D14 -4C&D14-7C&D14-8C&D14Cont. C&D

Description

MastigocladusCyanidiumG-4P. aeruginosaR-14R-42plain foamtreated control - no foam or algaeuntreated control

DaturaAzollaNostoc-DNostoc-NDplain foamtreated control - no foam or algaeuntreated control

A-106

EXP14.XLS

WSRC-TR-96-0088

Table A.6.14 (Cont.)

Bioremoval of metals using foam embedded with nine types of biomassusing the packed bed bioreactor

Date

Effluent

Water Qual.

Test Rigs

BiomassMast.Mast.Mast.Cyan,Cyan.Cyan.G-4G-4G-4P. aer.P. aer.P. aer.#14#14#14#42#42#42Plain Foam 3/26Plain Foam 3/26 •Plain Foam 3/26No Foam 3/26No Foam 3/26No Foam 3/26Control 3/26Control 3/26Control 3/26Gypsum WeedGypsum WeedGypsum WeedAzollaAzollaAzoliaNostoc - DNostoc - DNostoc - D

3/26/96 (run #1) 3/28/96 (run #2)

Well DCB-4A (same water as Exper. #12)

pH 3.79, Cond. 1.11ms/cm, DO 7.27, Temp 21.3, Sal 0.05%

Modified packed bed bioreactor

Column111222333444555666777888

111222333

Metal concentrationsAl(ppm)

64.12264.689

64.405563.87

63.94863.90963.49463.219

63.356563.51563.13563.32562.68562.444

62.564561.77661.977

61.876563.09863.36463.23163.16563.256

63.210564.39764.00164.19962.35662.504

62.4361.40461.493

61.448562.78462.81462.799

Fe(ppm)0.09

0.0640.0770.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.1250.112

0.11850.1150.1070.1110.0640.0640.064

Cr(ppb)3

2.82.92.93.2

3.053

3.13.05

3.13.53.33.13.8

3.454.2

44.1.3.93.8

3.853.63.5

3.553.23.3

3.252.12.2

2.152.62.5

2.552.42.3

2.35

Ni(ppb)440440440460480470480480480490510500510530520580580580550550550550550550560580570380390385400400400400390395

Percent removal *Al

-0.32166

0.45172

1.31233

1.36139

2.54599

3.61766

1.50781

1.53974

0

1.90055

3.44283

1.32072

Fe

-20.31

0

0

0

0

0

0

0

0

-85.16

-73.44

0

Cr

10.77

6.154

6.154

-1.538

-6.154

-26.15

-18.46

-9.231

0

4.444

-13.33

-4.444

Ni

22.807

17.544

15.789

12.281

8.7719

-1.7544

3.5088

3.5088

0

8.3333

4.7619

5.9524

A-107

EXP14.XLS

WSRC-TR-96-0088

Nostoc - NDNostoc - NDNostoc - NDPlain Foam 3/28Plain Foam 3/28Plain Foam 3/28No Foam 3/28No Foam 3/28No Foam 3/28Control 3/28Control 3/28Control 3/28

444777888

62.75862.359

62.558562.633

62.662.616562.12262.481

62.301563.85263.427

63.6395

0.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.0640.064

2.32.2

2.252.52.52.52.42.22.32.22.3

2.25

410400405410400405410410410420420420

1.69863

1.60749

2.10247

0

0

0

0

0

0

-11.11

-2.222

0

3.5714

3.5714

2.381

0

A-108

WSRC-TR-96-0088APPENDIX 6B

BIOREACTOR SYSTEMS DEVELOPED FOR USE IN EVALUATINGBIOREMOVAL CAPABILITIES OF FOAM-EMBEDDED MICROBES

Bioreactor System #1Bioremoval Evaluation System Test stand (B.E.S.T)(pages 110-115)

Bioreactor System #2Bioremoval Evaluation System Test stand (B.E.S.T)REV. 1 (pages 116-134)

Bioreactor System #3Static Mixer Contracter(pages 135-143)

Bioreactor System #4Biofiltration Equipment for Test and Research (B.E.T.R.)(pages 144-157)

A-109

WSRC-TR-96-0088

I — .^TJTTT1 € C H H O I O G ' £

i • M r J - J

OPERATING INSTRUCTIONS

FOR

THE FRISBY TECHNOLOGIES

BIOREMOVAL EVALUATION SYSTEM TESTBED

DELIVERED UNDER

CONTRACT NO. AA07217N

PREPARED FOR:

WESTINGHOUSE SAVANNAH RIVER COMPANY

SAVANNAH RIVER SITE, AIKEN, SC

NOVEMBER 29,1994

Prepared By:

Approved By:

Approved By

jichael Browning, Test Engineer

Paul Hermann, Principal Investigator

1TU4

Dougjas McCrosson, Program Manager

Frisby Technologies, Inc3635 Whiskey Road

Aiken, SC

A-110

WSRC-TR-96-0088

H N O

n r

TO FILL BIOREMOVAL EVALUATION SYSTEM TESTBED

1 Locate Uuee-way valve at the bottom of panel Turn the handle to fill. (The arrow should be facing straight

up.) This shuts the flow off.

2. Locate air bleed valve at the top of the filter. Turn the handle to open. (The handle should be vertical.) This

allows the trapped air to bleed into the reservoir while the filter is being filled.

3 Remove the cover of the reservoir. Fill reservoir with the fluid being tested. Replace cover and make sure that

the air bleed and return lines are secured in the tank cover.

4. Turn the pump on by the switch located at the top of the testbed. There may be a surging sound coming from

the pump. This is caused by air in the feed line. The switch may have to be turned off, wait a few seconds,

then turned back on. If this does not clear the line the pump may have to be lifted so the pump head is higher

than the reservoir to release the air pocket created. Replace pump and try again.

5 Set the Batch Meter to zero, make sure the meter is on. Hold down the DISPLAY button for three seconds

until zeros appear. Release the button.

6. After the filter fills with liquid, allow the trapped air to circulate back into reservoir.

7. Close the air bleed valve (handle horizontal), locate the flow control valve at the top of the panel and close

Open the three way valve to either DRAIN or RUN, depending on the test being run.

8 Slowly open the flow control valve to desired flow rate.

A - l l l

WSRC-TR-96-0088

E C H N O l O C i f S

TO DRAIN FILTER

1 Turn off the pump by the switch located at the top of the testbed

2 Open the air bleed valve, pull the air bleed line out of the fluid in the reservoir, and allow the filter to drain If

the filter does not completely drain, switch the 3-way valve to drain and dram remaining fluid

TO CHANGE FILTER MEDIA

1. Make sure the filter is empty.

2. Loosen the top and bottom unions

3 Remove the filter, cover the inlet (a #3 rubber stopper, found in chemistry labs, will work), and turn upside

down.

4. With the tool supplied, loosen the screen housing of the filter. Remove the screen housing, and remove the

existing media

5 Clean the stainless steel screen, fill with new media and replace screen housing.

6 Tighten the screen housing, and reinstall the filter, being careful to only tighten the unions hand tight

A-112

COcooo

I

en

HCJ

en

PLUMBING SCHEMATIC

BLEED VALVE

TOCARBOY

3-WAY VALVE

GAUGE

CONTROL VALVE

CO

PUMP

RESERVOIR

FOR EACH INDIVIDUAL UNIT

CO

H

o

n

o

n

sn

s

m oo

125 V-

WSRC-TR-96-0088

o

o

00o

>

O

A-114

T E C H N O L O G I E S

WSRC-TR-96-0088

417 SOUTH MAIN STREET . FREEPORT. NEW YORK 11520 • 516 378016? . FAX 516 378-0262

PREPARED BY MICHAEL B BROWNING

A-115

WSRC-TR-96-0088

TO FILL BIOREMOVAL EVALUATION SYSTEM TESTBED

Rev. 1

Flow Rates of .04 GPM to .36 GPM

Valve positions

Low flow shut off (black handle) open

Control valve (orange handle) open

Bleed air valve (gray handle) open

Three way valve (white handle) close

Drain valve (located in back of testbed) close

1. Fill the reservoir with water.

2. After the pump has filled with water, locate the control valve at the top of the testbed (orange handle) and

close. This will close the high flow rate circuit off. Locate the low flow shut off valve (black handle) also at

the top of testbed and close.

3. Turn the pump on by the switch located at the top of testbed

4. Slowly open the low flow shut off valve (black handle) so the fluid will not "hammer" the rotameter and

damage the float.

5. As the rotameter and filter fill with water there will be air bubbles present. As the air passes through the

rotameter it will cause some surging.

6. After the air bubbles dissipate, adjust the flow by the black knurled knob located at the base of the rotameter.

* This valve is a high turn metering valve and caution should be taken to never fully seat the valve.

A-116

WSRC-TR-96-0088TO CHANGE FILTER MEDIA

1. Make sure the filter is empty of water

2. Loosen the top and bottom unions.

3. Remove the filter, and turn upside down and remove media.

4. Clean the stainless steel screen, by flushing with water.

5. Refill with new media, and reinstall the filter, being careful to only tighten the unions hand tight

TO DRAIN SYSTEM

1. With the supplied male disconnect insert into the mating disconnect located at the inlet of the pump. Drain

remaining fluid into a suitable container.

2. Open the drain valve located in the back of the test stand Drain remaining fluid into a suitable container.

3. Because of the small lines and the high turn metering valve this part of the system will drain slowly. Take the

top of the small filter and lay over the front of the test bed. Open the control valve (orange handle) and this

will introduce more air into the lines allowing the fluid to drain faster.

A-117

WSRC-TR-96-0088

TO FELL BIOREMOVAL EVALUATION SYSTEM TESTBED

Rev. I

Flow Rates of .3 GPM to 1.8 GPM

Valve positions check list:

Low flow shut off (black handle) closed

Control valve (orange handle) open

Bleed air valve (gray handle) open

Three way valve (white handle) close

Drain valve (located in back of testbed) close

1. Locate three-way valve (white handle) at the bottom of panel. Turn the handle to fill (The arrow should be

facing straight up.). This shuts the flow off.

2. Locate air bleed valve (gray handle) at the top of the filter. Turn the handle to open (The handle should be

vertical.) This allows the trapped air to bleed into the reservoir while the filter is being filled

3. Locate low flow ball valve (black handle) at the top of the panel above the control valve (orange handle) and

turn to the off position (vertical). This shuts off the water flow to the low flow rate circuit

4. Remove the cover of the reservoir. Fill reservoir with the fluid being tested. Replace cover and make sure that

the air bleed and return lines are secured in the tank cover

5. To set the Batch Meter to zero, make sure the meter is on. Hold down the DISPLAY button for three seconds

until zeros appear. Release the button.

6. Turn the pump on by the switch located at the top of the testbed.

7. After the filter fills with liquid, allow the trapped air to circulate back into reservoir

8 Close the air bleed valve (handle horizontal), locate the flow control valve (orange handle) at the top of the

panel and close. Open the three way valve to either DRAIN or RUN, depending on the test being run.

9 Slowly open the flow control valve (orange handle) to desired flow rate.

A-118

WSRC-TR-96-0088

TO DRAIN FILTER

1. Turn the three way valve (white handle) to drain

2. When the flow meter is reading zero ,tum off the pump by the switch located at the top of the testbed.

3. Open the air bleed valve, and allow the filter to drain. The totalizer has already taken this amount into effect

TO CHANGE FILTER MEDIA

1. Make sure the filter is empty.

2. Loosen the top and bottom unions.

3. Remove the filter, cover the inlet (a #3 rubber stopper, found in chemistry labs, will work), and turn upside

down.

4. With the tool supplied, loosen the screen housing of the filter. Remove the screen housing, and remove the

existing media.

5. Clean the stainless steel screen, fill with new media and replace screen housing.

6. Tighten the screen housing, and reinstall the filter, being careful to only tighten the unions hand tight.

A-119

WSRC-TR-96-0088

TO DRAIN SYSTEM

1 With the supplied male disconnect insert into the mating disconnect located at the inlet of the pump. Drain

remaining fluid into a suitable container.

2 Open the drain valve located in the back of the test stand. Drain remaining fluid into a suitable container.• • " : <

3. To drain the remaining fluid from the return line going to the reservoir, turn the three way valve to run wait aa<

few seconds and then turn back to drain.

. JJ;

A-120

CO00oo

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PLUMBING SCHEMATIC

BLEED VALVE

TO CARBOY

3-WAY VALVE

LARGE RESERVOIR

SMALL RESERVOIR

DIGITAL FLOW METERHIGH FLOW RATES

GAUGE

CONTROL VALVEHIGH FLOW RATES

FILTER

METERINGVALVE

SHUT OFFVALVE

M

ROTAMETERLOW FLOW RATES

DRAIN

T.'f '.

FOR EACH INDIVIDUAL UMT

PARTS LIST FOR THEB.E.S.T

PARTS LIST FOR B.E.S.T TEST STAND

WSRC-TR-96-0088

ITEM # DESCRIPTION

2 3/4 x1/2 HEX BUSHING SCH 80 PVC

QUANTITY

16

1/2 xC NIPPLE SCH 80 PVC 144

1/2x 3 NIPPLE SCH 80 PVC

1/2 NPT TEE SCH 80 PVC 40

6 1 /2 NPT BULKHEAD SCH 80 PVC 56

1" DIA. SS TYPE 304 30 MESH SCREEN

8 1/2 x 1/4 SS HEX BUSHING 32

2" PVC CLEAR TUBE CUT TO 6" LONG

10 3/4 DOUBLE UNION PVC BALL VALVE BODYCUT IN HALF & MACHINE PER SKETCHES

12 1/4 PVC LAB COCK 24

13 3- WAY 1/2 NPT BALL VALVE SCH 80 PVC

14 1/2 NPT 1/2 PLASTIC HOSE NIPPLE 56

15 1/2 NPT ELBOW SCH 80 PVC 56

16 DIGITAL TOTALIZER

17 1 x 1/2 HEX BUSHING SCH 80 PVC 16

A-122

PARTS LIST FOR THEB.E.S.T

ITEM # DESCRIPTION20 3/8 OD TUBE x 1/4MPT PARFLEX ADAPTER

WSRC-TR-96-0088

QUANTITY24

21 3/8 OD TUBE x 1/2 MPT PARFLEX ADAPTER

29 1/2 Y-STYLE STRAINER SCH 80 PVC

31 316 SS UNPERFORATED WORM DRIVE HOSE CLAMP 88

37 1/2 TUBE OD 1/2 NPT MALE ELBOW POLYPROPYLENE 32

41 0-30 PSI 316 SS 1/4 MPT GAUGE 16

42 LITTLE GIANT 5 GALLON TANK & PUMP ASSEMBLY

43 CHEMTROL 1/2 TRU-UNION BALL VALVE

44 FABRICATED STAND (THERMCRAFT)

50 5/8 OD STAINLESS STEEL WORM GEAR CLAMPS

51 3/8 OD TUBE x 1/4MPT PARFLEX TEE

52 1/4 ID TUBE x 1/4MPT BARBED HOSE FITTING

53 1/4 ID TUBE x 1/2MPT BARBED HOSE FITTING 16

54 SMALL FILTER ASSEMBLY

55 SMALL RESERVOIR

56 1/2 MPT COUPLING INSERT

57 1/2 TUBING COUPLING INSERT

A-123

PARTS LIST FOR THEB.E.S.T

ITEM # DESCRIPTION58 1/2 TUBING COUPLING BODY

WSRC-TR-96-0088

QUANTITY8

59 1/4 FPT 2 WAY SS PANEL MOUNT BALL VALVE

60 22 GPH ROTAMETERS

3/16ID x 1/2 OD POLYETHLYNE TUBING 16

3/8 VINYL CLEAR TUBING 22

1/4 VINYL CLEAR TUBING 24

1/2 ID 3/4 OD VINYL CLEAR TUBING 76

1/4ID x 3/8 OD POLYETHLYNE TUBING 24

A-124

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A-133

WSRC-TR-96-0088

INSTRUCTION MANUAL

STATIC MIXER CONTACTER

AT E C H N O L O G I E S

3635 WHISKEY ROAD • AKEIM. SC 23303642-0296 • FAX: BO3 &aS-1

CORPORATE HEAOOUARTERS•317 SOUTH tvIAf^ STREET • FReEPOFrr. isJY 1152O

S16 378-0162 • FVXX: S16 37B-O26a

MATRIX R & D CORPORATION21 Fieldstone DriveDover, NH 03820

A-134

10/0.9/1995 -16:14 1-603-742-3826 MATRIX R&D PAGE 02

WSRC-TR-96-0088

STATIC MIXER CONTACTER

The unit consists of two separate flow circuits as shown on theschematic 'A' and 'B1. In both circuits the effluent in reservoir gravityfeeds to the input of pump and is pumped through 'A' mixer circuit or'B1 filter circuit

The filter media to be tested will be added to reservoir and mustbe continuously mixed by stirrer supplied by client.

CIRCUIT 'A' STATIC MIXERS

The static mixers ensure complete contacting and plug flowconditions which will maximize contact of effluent and media. Eachmolecule of effluent will be in contact with media at some point alongthe length of mixer tubes.

CIRCUIT ' B ' FILTER STAND

The filter stand is provided to collect and separate media fromeffluent after it has been determined that the media has been incontact with effluent for its desired length of time. The filter standcontains a flat disc filter in top plate to filter media from effluent as flowin from bottom progresses through unit.

A-135

MATRTX R&D CORPORATION21 Fiejdstone OriveDover, NH 03820

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A-137

10/39/1995 16:14 1-683-742-3826 MATRIX R&D WSRC-TR-96-0•AGE 05

SAMPLING PORTS

Sampling ports are provided by means of valves 2, 4 and 5.The individual valve functions are as follows:

POSITION

Front Exit Tube

VALVE NO.

down position

FUNCTION

Empty Reservoir orSample prior to mixer

Right Side Exit Tube 4pointing left

Sample of Effluentwithout media after

mixer

Left Side Exit Tube 5down position

Sample of Effluentand media aftermixer or purge ofentire system

POSITION

Right Side Exit Tube

CIRCUIT B

YALYEJU0

4pointing left

A-138

Sample of effluentwithout media

after filter

18/99/1S95 16:14 1-683-742-3826 MATRIX R&D 06

WSRC-TR-96-0088

STATIC MIYER COISTflCTER ©

a 4-

OUT t t t tTHRU

8 t tt t

TlfftO PUMP

OUT /o t t tt t t t t t

t t t t t

MATRIX R&D CORPORATION21 Fieldstone Drive

A-139 Dover, NH 03820

10/00/1005 10:11 1 COD 712 DO2C MATRIX

SET UP AND RUN

Prior to any test the reservoir should be rinsed-with a compatiblecleaning solution and the system should be flushed. To accomplish aflush, proceed as follows:

1. Remove reservoir and filter2. Empty any residual material and clean both.

Refill reservoir with appropriate cleaner.

3. Replace reservoir on stirrer, place valve 6 in off

position. Connect Quick disconnect and install stopper.

4. Reinstall filter and connect quick disconnects.

5. Reset position of valving:

NOTE: DO NOT START MOTOR AT ANY TIMEWITHOUT CHECKING VALVE POSITIONS

VALVE 5 6 2 1 3 4down up up up up up

6. Place container under SS tube exiting stand on leftside of unit to catch flow of cleaning solution

7. Turn speed on potentiometer to zero. Apply AC powerto unit (swiches on). Slowly advance speedpotentiometer towards maximum. Flow will startand reverse flush system through mixers and out lefthand purge tube.

8. Stop motor by turning off power switch. Swap valves 1and 3 from up position to down position. Restart powerand flow will flush through filter out left side purge tube.

A-140

10/09/1995 1-683-742-3826 MATRIX R&D PAGE 88WSRC-TR-96-0088

9. Stop power. Swap Valve 5 to center off position. SwapValve 4 to center sample position. Place containerunder SS tube exiting right side standStart power and clean Valve 4 and strainer.

9.1 During flush of Valve 4, remove stopper fromreservoir. Place tube into waste container cmomentarily swap Valve 5 from^owrTnosition toup position to flush leg from Va/ve~trtfifoughstopper.

10. Stop power. Place all Valves in UP position withexception of Valve 2 which should point down.Place container under front purge tube.

11. Start power and empty reservoir through purge tubeon front of machine.

NQTJE; Steps 1-11 may be repeated with distilled water toremove cleaner from system. In any case the system willremain full of liquid cleaner or water. To remove the residualliquid proceed as follows:

12. Using low pressure air line^P'psi or lower. Insure ailvalves are in up position and drain valve on pump outletis open. Catch flow out of 1/4 line exiting pumpoutlet valve. Gently blow air into tube entering stopper toair flush back through mixer circuit. Momentarily swapValve 4 to down position and clean strainer and Valve 4.Stop air flow and close pump outlet drain valve.

13. Reset Valve 3 to down position and-Qoen valve onbottom of filter. Gently blow air into<stippj^ tube and catchfluid out of valve on bottom of filter, ^ ^

NOTE: The filter may contain full volume of liquid. Leg from reservoiroutlet to pump input will still contain water.

A-141

10/89/1995 16:14 1-603-742-3826 MATRIX RSD PAGE 09

WSRC-TR-96-0088

7

RUN MACHINE

To run machine the reservoir must be filled with effluent andmedia mix of choice. The unit is provided with quick disconnect onoutlet of glass reservoir and stopper, so as to be able to removereservoir for filling or cleaning. The media and effluent must be stirredand a magnetic stirrer is suggested to be provided by client which willkeep media in suspension. Media should be 20-8 mesh(.033-.093) in size to prevent plugging.

As shown on schematic CIRCUIT A through mixers, requires allvalves in up position pointer towards black lettering.

CIRCUIT B through filter requires Valves 3 and 1, pointing downto red lettering.

NQIEL_Shut off power to swap valves and check valve positioningbefore restarting pump. In A or B runs, sequence valves as above andstart pump.

jo

A-142

WSRC-TR-96-0088

PREPARED BY MICHAEL B. BROWNING

A-143

WSRC-TR-96-0088

OPERATING INSTRUCTIONS

FOR THE

BIOFILTRATION EQUIPMENT for TEST and RESEARCH

B.E.T.R

CAUTIOM Before operating the testbed read all owners manuals !! Make sure that the air supply

regulator is set to the correct pressures.(Not to exceed 30 PSI) Failure to do so could result in

serious injury and damage to the equipment

TO FILL FILTER

1. Close the ball valves to the inlet of the air tanks. These valves (yellow handles) are located on each

tank at the air supply inlet.

2. Turn the effluent and nutrient valves to the off position.

3. Separate the quick disconnect assemblies located at the filter. There are three sets of disconnects

for each filter.

4. Disassemble the filter by removing only the white nylon wing nuts located at the top of the filter

assembly

5. Remove the aluminum top and top gasket. Fill with media to be tested.

6. Place the gasket on top of the glass making sure the glass and gasket align , then place the

aluminum top on filter assembly, tighten wing nuts finger tight only.

A-144

WSRC-TR-96-0088

TO RUN SYSTEM

1. Turn the effluent and nutrient valves to the off position.

2. Turn the recirculate / carboy valve to fill.

3. Open the filter bleed valve (located on top of the filter assembly).

4. Open the air supply valves (located at the inlet of each tank).

5. Turn the effluent and nutrient valves to the run position.

6. Adjust the flow rate by the black knob (marked with an L) located at the bottom of the flow meters.

To read the flow meters center the float on the scale reading. The scale reading correlates to a

calibration sheet furnished by the manufacturer. It is important that the right flow meter is matched

with the right calibration.

7. When the filter assembly is full, close filter bleed valve, turn the recirculate / carboy valve to the

operation to be performed.

TO RECIRCULATE FLUID

1. Turn the effluent and nutrient valves to the off position.

2. Turn the recirculate / carboy valve to recirculate.

3. Set the peristaltic pumps to desired flow rate, (see operating manual)

TO SEND FLUID TO CARBOY

1. Turn the effluent and nutrient valves to the run position

2. Turn the recirculate / carboy valve to carboy. The fluid will now flow into the carboy.

A-145

WSRC-TR-96-0088

TO SAMPLE FLUID

1. To sample any of the fluids turn the corresponding valve to sample.

TO PURGE SYSTEM

Since there are check valves through out the system, purging the system with air is the only method of

draining.

1. Turn the air supply off to the tanks by closing ball valves located at the inlet of each tank.

2. Bleed air out of the pressure tanks by opening the tank bleed valves.

3. Remove any existing fluid.

4. Reseal tanks, close tank bleed valves.

5. Turn the effluent and nutrient valves to the run position

6. Turn the recirculate / carboy valve to carboy.

7. Open air supply valve and purge remaining fluid into carboys or suitable containers.

A-146

00CO©o

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EFFLUENTRESERVOIR

PLUMBING SCHEMATIC

EFFLUENTFLOW

EFFLUENTSAMPLE

PERISTALTICPUMP

RECIRCULATEDSAMPLE

NUTRIENTFLOW

NUTRIENTSAMPLE

BLEEDVALVE

<

AIRIN

CARBOY

NU.TRfENTRESERVOIR

v,f

PLUMBING IS IDENTICAL FOR LEFT AND RIGHT SIDE OF TESTBED.BOTH SIDES SHARE THE EFFLUENT & NUTRIENT RESERVOIR.

PARTS LIST FOR THEB.E.T.R.

PARTS LIST FOR B.E.T.R TEST STAND

ITEM # DESCRIPTION

1 DASH 8 STAINLESS STEEL TUBING

2 STATIC MIXERS

3 DASH 8 to 1/4 MPT ADAPTER

4 1/4 x 1/8 FPT SS COUPLING

5 1/8 x CLOSE SS NIPPLE

6 1/8 MPT X1/4OD TUBE ADAPTER

7 1/8 MPT x1/4 ID TUBE BARBED FITTING

8 1/8 MPT SS TEE

9 1/8 MPT SS CROSS

10 CHECK VALVE 1/3 PSI 1/8 MPT

11 1/8 FPT SS COUPLING

12 CHECK VALVE 25 PSI 1/8 MPT

13 4 1/2" 30PSI SS GAUGE

14 ROTOMETER

15 1/8 3WAYSS VALVE

16 1/4 TUBING COUPLING BODY

17 1/4 TUBING COUPLING INSERT

18 GLASS COLUMN

19 TFE GASKET

20 ALUMINUM PLATES

21 5/16-18 THREADED ROD

22 5/16-18 NYLON WING NUT

23 5/16-18 SS LOCK NUT

WSRC-TR-96-0088

QUANTITY

2

8

4

8

4

38

26

8 .

2

6

4

2

4

4

8

6

6

2

4

2

8

8

8

A-148

ITEMS24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

PARTS LIST FOR THEB.E.T.R.

DESCRIPTION5/16-18 SS NUT

1/8 FPT SS STREET ELL

1/8 NPTSS PLUG

1/8 2WAY SS BALL VALVE

1/4NPT 2WAY BRASS BALL VALVE

REGULATOR

BACK PRESSURE REGULATOR

1/4 x CLOSE SS NIPPLE

1/4 NPT SS STREET ELL

2" 160 PSI GAUGE

2 1/2" 30 PSI GAUGE

MANUAL RELIEF VALVE

1/4 MPT x1 MOD TUBE TEE

5GALSS PRESSURE VESSEL

1/4 SS PLUG

1/4 x 3 SS NIPPLE

1 GAL SS PRESSURE VESSEL

PERISTALTIC PUMP AND DRIVER

#12 RUBBER STOPPER

CARBOY

WSRC-'

QUANTITY8

2

2

2

2

2

2

5

2

2

2

2

2

1

2

1

1

2

2

2

A-149

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STATIC MIXER ASSEMBLY

6

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27

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4 310

7

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16

22.

17 717

19

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25

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2 ASSEMBLIES NEEDED

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2 ASSEMBLIES NEEDED

WSRC-TR-96-0088APPENDIX 7

POLYURETHANE FOAM IMMOBILIZATION OF TCE-DEGRADINGBACTERIA: ENTRAPMENT EFFECTIVENESS AND INFLUENCE ON

METABOLIC ACTIVITY

A-159

Appendix 7.1 WSRC-TR-96-0088

Peptone Tripticase Yeast Glucose (PTYG) Agar

Deionized waterGlucose (dextrose)Yeast extractTripticase (tryptone)MgSO4 • 7 H2OCaCl2 - 2 H2OAgar, purified

Heat to boiling while stirringAutoclaveCool to 50°CPour plates such that 1 I makes

1 liter10g10g5g0.60 g0.070 g15g

35-40 plates

A-160

WSRC-TR-96-0088

Appendix

Pseudomonas

Solution 1

Deionized waterK2HPO4KH2PO4(NH4)2SO4MgSO4•7H2OTrace elementspH to 7.2Heat to boilingAutoclave

7.2

Medium

895 ml12.5 g7.2 g1.0 g0.1 g5.0 ml

Let cool, then add glucose (100 ml/I)

Solution 2. Trace Elements

H3BO3ZnSO4 • 7H2OFeSO4•7H2OC0CI2 • 6H2ONa2MoO4 - 2H2QCuS04-5H2OMnSQ4•4H2O

Solution 3. Glucose

Deionized waterGlucose (dextrose)

Sterile filter

0.232 g0.174 g0.082 g0.069 g0I)04g0.008 g0.008 g

100 ml14.4 g

A-161

WSRC-TR-96-0088

Appendix 7.3

Basal Salts Medium (BSM) and Yeast-Glucose Medium (YGM)

So//?. 1. BSM Stock (20X) 2000 ml 4000 ml

K2HPO4 170 g 340 gNaH2PO4 • H2O 40 g 80 gNH4CI 80 g 160 g

pH to 7.2 before adjusting to final volume

Use 50 ml/lliter final volume in Dl to make BSM or YGM

Soln.2. Trace Metals (20X)

Nitrilotriacetic acid, trisodium salt 5.377 g 10.754MgSO4-7H2O 8.0 g 16.0 gFeSO4 - 7H2O 0.48 g 0M gZnSO4-7H2O 0.12 g 0.24 gMnSO4-H2O 0.12 g 0.24 gStore cold

Use 50 ml/lliter final volume in Dl to make BSM or YGM

Soln. 3 Glucose-Yeast Extract (for YGM only)

Deionized water 100 mlGlucose (dextrose) 10 gYeast Extract 5 gSterile filterAdd to BSM at 100 ml/liter final volume to make YGM

A-162

WSRC-TR-96-0088

Appendix 7.4Summary of Washout, Viability, and GC Experiments

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

6/29/95N/APR131NB vs Pseudomonas mediumGrowth Yield vs Glucose Content and Medium TypeN/AN/APseud medium better than NB, 20 g/l glucose enough.MA

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam if, Description:

Notes:

Filenames:

7/13/95-7/14/957/13/95PR131Pseudomonas mediumWashout and viability8.1% and 17.7% in Pseud medium#1-13. Orig. prepolymer (Bipol 6B; NCO 6). Varied foamformulation as described in file.Surfactant type & amount and slurry density were mostimportant. This exp. was 50 ml DI through 2 g foam induplicate. 10% formalin, AODC. Viability very low.WASHOUT.XLS, WASHOUT.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:

Filenames:

7/20/957/13/95PR131Pseudomonas mediumLarger volume washout of Foams 10-138.1% and 17.7% in Pseud medium#10-13. Bipol 6B. Varied foam formulation as described in file.This exp. was 1000 mi DI through 2 g foam induplicate. Collected 50 ml at 50,150, 250, 400, 550, 700, 850,1000 mlWASHOUTB.XLS, WASHOUT.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:

Filenames:

7/20/957/13/95PR131Pseudomonas mediumLarger volume washout of Foams 10-138.1% and 17.7% in Pseud medium#10-13. Bipol 6B. Varied foam formulation as described in file.This exp. was 2000 ml DI through 2 g foam in

duplicate. Collected 1st 50 ml, then 950, then 1500 mlWASHOUTB.XLS, WASHOUT.PPT

A-163

WSRC-TR-96-0088

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:

Filenames:

8/16/958/15/95PR131Pseudomonas mediumViability and Washout (Bipol 3; NCO 3), effect cold embedding6.8% and 13.4% in Pseud medium#14-18. New prepolymer (81195 = Bipol 3; NCO 3) vs original.50 mi washout in duplicate, 2 g foam. Viability low again.

Washout still low.NEWPOLVB.XLS, WASHOUT.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

8/31/958/31/95PR131. #14, Chlorobenzene degraders, 01-bPseudomonas mediumViability (prepolymer 350). effect heat & pH shift on viability6.8%PR131,5.4%#14 in Pseud medium#21-25. Prepolymer 350; NCO unknown but thought < Bipol 3Did plates and MPN*sNEWPOL3.XLS, WASHOUT.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

9/18/959/17/95PR131Pseudomonas mediumViability (plate &MPN)6;4% PR131 in Pseud medium#26-31 Prepolymer 350Did plates and MPN'sPOL3EM2:XLS

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foamt, Description:Notes:Filenames:

10/3/95-10/4/9510/3/95PR131Pseudomonas mediumViability 10/3 and TCE 10/4, prepolymer 3506.4% PR131 in Pseud medium#32 - 33 controls, 34-37 PR131, prepol 350Plate counts, TCE on foam. MSB medium for TCE exp.? Have printout, no TCE removal. POL3EM3.XLS.

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment

10/18/9510/18/95PR131, P. aeruginosaPseudomonas mediumViability

A-164

WSRC-TR-96-0088

Slurry Description:Foam #, Description:

Notes:Filenames:

6.9% PR131, 0.4% P. aer in Pseud medium#38 Polymer 350 control, #39 Polymer 802 (NCO unknown,thought < prepol 350) control, #40 350 PR131. #41 802 PR131,#42 802 P. aer.Plate counts, compare old & new plates.POL3 4.XLS

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam if, Description:Notes:Filenames:

11-1-95N/A18d. 2d-b, 01-b. G4YGMBenzene degradation by culturesN/AN/ANot worth plotting110895JR.ALL, EXE. SUM or variants

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:Filenames:

11/3/95N/APR131.G4 (phenol)YGMTCE removal by culturesN/AN/AWorked!110695JR.ALL, EXE, SUM or variants

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:Filenames:

11/9/95N/AG4(phenoi)YGMTCE removal, cultures. PM vs YGM. vary TCE levelN/AN/A

111395JR.ALL, EXE. SUM or variants. VIDED.PPT

Experiment Date:Embedding Date:Organismfs):Growth Medium:Type of Experiment

Slurry Description:Foam #, Description:

Notes:

11/14/9511/14/95G4 (phenol)YGMTCE removal by foam & slurry, induced in culture. BSM vsYGMG4 2.4% in BSM, 2.2% in YGM71 BSM, 72 YGM. 73 G4/BSM. 74 G4/YGM. Orig. prepolymer(Bipol 6B) from now on.Worked! BSM better than YGM. Remade standards 11/25/95

A-165

Filenames:

WSRC-TR-96-0088111795JR, 112795.ALL, EXE, SUM or variants. VIDEP.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

11/16/9511/14/95G4 (phenol)YGMBenzene removal by foam & slurry. BSM vs YGMG4 2.4% in BSM. 2.2% in YGM73 G4/BSM. 74 G4/YGM

112095JR.ALL, EXE, SUM or variants

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment

Slurry Description:Foam #, Description:Notes:Filenames:

12/5/95-12/6/9512/5/95G4 (phenol)YGMTCE removal by foam & slurry. Induced w/ phenol, benzeneafter embedding (foam) or slurry prep (slurry). Uninducedcontrols. TCE. viability 12/5/95. Washout 12/6/95.G4 5:8% in BSM75 BSM, 76 G4Contaminated. Don't use.120795JR.ALL, EXE. SUM or variants.

Experiment Date:Embedding Date:brganlsm(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:Filenames:

12/13/9512/13/95G4 (phenol or. benzene)YGMRepeat previous expiG4 8.9% in BSM77 BSM, 78 G4 (cups mislabeled 96, 97) :

Slurry made previous day.121595JR.ALL, EXE, SUM or variants. VIDEO.PPT.VIG41213.XLS

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam if, Description:Notes:

Filenames:

12/19/9512/19/95G4 (phenol added to slurry)YGMTime course TCE removalG4 in BSM, 5.6% induced, 5.5% uninduced79 BSM. 80 induced G4, 81 uninduced G4Induced in slurry form. Uninduced controls. Slurry madeprevious day.122095JR.ALL. EXE, SUM or variants.

A-166

WSRC-TR-96-0088

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:Filenames:

1/5/961/5/96G4 (phenol added to cultures)YGMRibosomal probes/growth stimulation5.5% G4 in BSM82 BSM, 83 G4

NA

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam it, Description:.Notes:Filenames:

1/11/95-1/12/961/11/96G4 (uninduced)YGMBenzene removal by foam & slurry 1/11/95 Viability 1/12/96G4 5.0% in BSM84 BSM control. 85 G4

011696B.SUM. ALL. EXE, VIDEO.PPT

Experiment Date:Embedding Date:drganismfs):Growth Medium:Type of ExperimentSlurry Description:Foam #f Description:Notes:Filenames:

1/18/951/18/96G4 (induced in culture)YGMTCE removal, spiked landfill waterG4 5.1% in BSM86 BSMcontrol, 87G43 TCE levels, spiked BSM controls012196JR.SUM, ALL, EXE. VIDEO.PPT

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:Foam #, Description:Notes:Filenames:

1/25/951/25/96G4 (induced in culture)YGMTCE removal. M area waterG4 5.2% in BSMCHECK NUMBERS3 wells, spiked BSM controls012896JR.SUM,ALL. EXE. VIDEO.PPT

A-167

WSRC-TR-96-0088

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

2/6/962/6/96G4 (uninduced), PR131YGMViability, respirometry4.2% G4. 4.6% PR131 in BSM117 BSM. 118 G4, 119PR131

VIAB2_4.XLS (wrong date in name)

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam if, Description:Notes:Filenames:

2/1S/962/16/96G4 (phenol-induced in culture)YGMEff. slurry density, C source on TCE removal5.1% and 2.5% (nominal) G4 in BSM120 BSM. 121 G4 (hi), 122 G4 (lo)Compared BSM with BSM + 0.1 g/l glucose. 0.05 g/I YE0220SUMB.XLS

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of Experiment:Slurry Description:Foam #, Description:Notes:Filenames:

2/19/962/19/96G4 (phenol-induced in culture) Old material from 2/16.YGMDuration of induction (used same foam as last time)5.1% and 2.5% (nominal) G4 in BSM120 BSM. 121 G4 (hi), 122 G4 (lo)Compared BSM with BSM + 0.1 g/I glucose. 0.05 g/l YE022696J.ALL, EXE. SUM, variants

Experiment Date:Embedding Date:Organism(s):Growth Medium:Type of ExperimentSlurry Description:FoamU, Description:Notes:Filenames:

2/23/962/23/96G4 (induced after embedding)YGMInduction after embedding5.5% G4 in BSM123 BSM. 124 G4Overnight vs 4 h induction022796J.ALL, EXE, SUM, variants

A-168

WSRC-TR-96-0088APPENDIX 8

DEGRADATION OF TRICHLOROETHYLENE AND BENZENE BYEMBEDDED BACTERIA

A-169

WSRC-TR-96-0088

Appendix 8.1 Characteristics of M Area Groundwaters

WELL MSB 25AMEASUREMENTS CONDUCTED IN. THE FIELD

Sample dale: 01/27/95 Time: 14:14Depth to water 148.41 ft (4SJ4 m) below TOC pK 5.4Water elevation: 217.99 ft (66.*4 m) msl AHujIWty. 1 mg/LSp. conductance: 23 f/Stan Water temperature: 19.7XTurbidity. 1 MTU A«r temperature: 13.1'CWater evacuated before sampling: 140 Qal

LABORATORY ANALYSES

£ Analyte Result Mog UnK U

0 Aluminum, total recoverable t «330 Arsenic,' total recoverable <3.30 Barium, total recoverable S.60 Beniene <840 8romodicrtloromethane «840 Bromoform <640 Bromometrtane <840 Cadmium; total recoverable <3.30 Carbon tetrachlpride <840 Chlorobenzene *640 Crdoroethane -aA

0 CNoroethene (Vinyl chloride)0 2-Chloroethyt vinyl ether0 Chloroform0 Chloromethane0 Chloromethane0 Chromium, total recoverable0 Cyanide0 Cyanide0 Dtbromochlorometharte0 1.1-l)ichloroetnar>e0 1.2-Dichloroetnane0 1.1-OicMorbetriylene0 trans-1J2<}ict>loroetriylene0 Dicnforornethane0 1^-Oichloropropane

0 cis-1.3:OicfUoropropenefj trans-1.3~0ichloropropene0 Efftvlbenzene0 ton. local recoverable0 Lead, total recoverable0 - Litf 'J"* total recoverable0 Manganese, iota! recoveraoie0 Mercury, total recoverable0 Nickel, total recoverable0 Nitrate-nitrite as nitrogen0 Selenium, total recoverable0 Saver, total recoverable0 Suttate0 1.1.2.2-Tetrachtoroetnane2 TetracWoroelhylene0 Toluene0 Total organic carbon2 Total oroanic halogens0 1 1.1-Tnchtoroethane0 1.1^-Trichloroethane2' TricriforoeChytene0 Tricrtloronuoromethane0 Zinc, total recoverable0 Gross alpha0 Nonvolatile beta0 Radium, total alprta-emttling0 Tritium

<84<84<84•c84<6.7

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1.720<84<J.3

1 1E-09jS.4E-109.8E-10i6.8£-104.0E-10*4.0£-10

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: GPGPGPGP

A-170

WSRC-TR-96-0088

WELL MSB 75BMEASUREMENTS CONOOCTED IN THE FIELO

Sample date: 01/20/95Oepth to water. 11S.79 ft (35.29 m) Below TOCWater elevation: 210.91 tl (64.29 m) mslSo. conductance: 61 //StonTuroifty: 7KTU

Water evacuated before sampling 110 gal

LABORATORY ANALYSES

F Analyte

I000

0000000000Q000000000000000

000000000002002000000

Aluminum, total recovee^bteArsenic, lotal recoverable.Banum. totaf recoverableBenzene

BromodichloromethaneBromoformBromomethane.Cadmium, totaf recoverableCarbon tetrachlorideCNorobenzeneChloroethaneCNoroethene (Vinyl chloride)2-Chloroethyl vinyl ethwCnloroformChlorornethaneChromium total recoverableCyanideDtbromocnloromeCiane1.1-Oichloroethanet .2-^icnloroetharte1.1-Dichloroetfiylenetrans-1.2-OichloroethyleneOtchforometrone1.2-Oichtorppropanecis-t .3-0icn/oropropenetrans-t.3-0ichkxopropeneEthytbenzeneIron, total recoverableLead, total recoverableLithium, total recoverableManganese, total recoverableMercury, total recoverableNickel, total recoverableNitrate-nitrite as nitrogenPhenolsSelenium* total recoverableSitver. total recoverableSutfate1.1.2.2-TetrachlorbethaneTetrachloroethyleneTolueneTotaf organic carbonTotal organic halogens1 11-Tncnforoethane1.1^-TrichloroethaneTrichforoethyleneTnchtorofluoromettianeZinc, total recoverableGross alphaNonvolatile betaRadium, total alpha-emittingTritium

44<3.3

24<8.4

<8.4X8.4<8 4<3.3< 8 4<8.4<8 4<8.4<8 4<8 4<8.4<6.7<8.3<8.4<8 4<8.4<8.4<8.4<8.4<8.4<8.4<a.<<B 4

49<S.O<8.3

27O.33<6.7

3.420<8.3<3.3<3.3

3.000<8.4<8 4<8.4< 1.670

62<6.4<8.4

1.110<8.4

3.01.5E-09t7.3E-102.4E-09±9.3E-101.1E-O9t6.0E-t01-2E457t3.SE-07

Time. 14:17PH:6.2Aflcalinity: 11 mg/LWater temperature: 18.6-CAir temperature: 9 1-C

GEGEGEGE

,/oA

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GEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEG£GEGEGP

//CiftnL GP

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rgA.-

A-171

WSRC-TR-96-0088

WELL MSB 34AMEASUREMENTS C O N O U C T E O I N THE FIELD

Sample flaw 01WV9S

Oeptfi 10 water 164 18 n (SO 04 m) t>elo~ IOCWater elevation 219.82 ft (67 00 m) mslSp conductance: 20 (/StemTurbidity 1 NTUWater evacuated before sampling 182 pa'

Alkalinity 1 mQA.

tut lemperature* 7 1*C

LABORATORY ANALYSES

f

00000000000000000000000000000000000000000002002000000

Analyte

Ahjrrunurrt. total recoverableArsenic, total recoverableBarium, total recoverableBenzeneBromoo*ichlort)metharieBrornoformBromomethaneCadmium, total recoverable fCatt>oe\ tetrachlorioeChlorobenzeneChloroetnaneChloroethene (Vinvl chloride)2-Chloroethyt vinyi effectChloroformCWorometKaneChromium total recoverableCyanideOfbromochlorornethar>et.i-Oichloroemane1,2-Oichloroetnane1,1 chloroethytenetrans A .2 -OicNoroeihyteneOicriloromethane1,2-Oichloropropanecis -1,3-Dichlcropropeoetrans -1.3-OichtoropropeneEfflyffjertzenekon. total recoverableLead. Iota! recoverableLithium, total recoverableManganese, total recoverableMercury, total recoverableNicfcet total recoverableNitrate-nitrite as nitrogenPhenolsSefeniurn. total recoverableSilver.-total recoverableSuKate1.1.2^-TetrachloroethaneTetrachloroelnyteneTolueneTotal organic carbonTotal orpanic carbonTotal organic halogens1.1.1-Trichloroerhane1.1.2-TricWoroe<haneTnchloroethyleneTrKf^oroOuorocnethaneZinc, total recoverableGross alphaNonvolatile betaRadium, total alpha-emittingTritium

Result

23<3 3

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917<8.3<3.3< 3 3

1.390<)67<167<167<t 670< 1.670

537<167<167

2.720<167

f*7E'.,Ol53e .l0- 1 4£.1O*67E-lO

7.0E-10tS.OE-lO• T IE-07,3 4E-07

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Unit

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A-172