P D F LIMITING FACTORS FOR THE

BIOREMEDIATION OF C0N"A'IXD SOILS AND ORGANIC WASTES

William Ney Hansard

Enviromental Management, Inc. 151 Brentwood Square

Nashville, Tennessee 37211 (615) 834-8257

Many soils and wastes contaminated with biodegradable organic compounds can be effectively treated using bioremediation technologies. Bioremediation, in many cases, is more cost effective than traditional incineration, physical, or chemical treatment technologies. By utilizing established biological principles, the kinetics and effectiveness of biodegradation processes can be significantly enhanced. Even bioresistant compounds and some concentrated organic chemical sludges can be biodegraded in special reactors in which environmental conditions for chemical breakdown, metabolic assimilation and cellular synthesis are optimized. By addressing Liebig's Law of the Minimum limiting factors and providing an environment conditioned to promote cellular metabolism, bioremediation technology is able to effectively treat a wide range of chemical contaminants.

INTRODUCTION

Bioremediation of contaminated soil and groundwater, and biotreatment of industrial wastes is becoming an increasingly popular environmental control technology. Until recently, biochemical treatment of wastes has been largely restricted to domestic and industrial wastewaters in this country. Also, until just a few years ago, the USEPA and state regulatory agencies have been reluctant to approve the use of biotechnology in contaminated site remediation projects. Today, some corporate and plant environmental managers continue to view the use of biotechnology with suspicion, as it is not a well established and proven alternative for the treatment of complex and toxic waste mixtures. This situation is improving steadily, however, and bioremediation technology is gaining wider acceptance as a useful and practical alternative to traditional remediation and treatment technologies.

The USEPA has launched the Bioremediation Field Initiative to evaluate the use of various biotechnology alternatives at contaminated sites. Over 140 pilot scale and full scale studies are currently planned or in operation for CERCLA/RCRA/UST sites [ll. The information developed will be used to

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c

evaluate the performance of selected field applications, provide technical assistance t o USEPA and state personnel with technical assistance in evaluating biotechnology alternatives, and develop a treatability database which will be available to the public through the Alternative Treatment Technologies Information Center (ATTIC). The USEPA launched the Superfund Technical Support Project (TSP) in 1988 to provide regulatory personnel with technical assistance in evaluating remedial alternatives for superhnd sites. During fiscal years 1988-1990, over half of TSP’s requests for assistance have been for the evaluation of biological technologies.

Additionally, the EPA and the University of Pittsburg Trust have formed the National Environmental Technology Applications Corporation (NETAC), a nonprofit corporation established in 1988 to participate in the development of new bioremediation protocols. In 1990 the EPA formed the Bioremediation Action Committee (BAC) to facilitate the safe use of bioremediation technologies to solve some of the nation’s pollution problems. The BAC is divided into six subcommittees: Pollution Prevention, Oil Spill Response, Research, Treatability Protocol, Education, and Data ID and Collection. All of the subcommittees report t o the assistant administrator of the USEPA’s Office of Research and Development, who functions as chair of the BAC. BAC subcommittee members are represented by industry and academia, as well as regulatory agencies.

At Purdue University’s 1991 Industrial Waste Conference, the keynote speaker, Ms. Rita Colwell stated that she believed that the use of biological technologies and the growth in environmental biotechnology industries would exceed all current estimates and projections. Ms. Colwell is a professor of microbiology at the University of Maryland in College Park, Maryland, and Director of the Maryland Biotechnology Institute.

The current optimism for environmental biotechnologies is well founded. There are, however, some potential problems associated with the misuse of biotechnology. Misleading claims are made by some involved in the industry, resulting in the loss of confidence in biotechnology. Probably a more important problem is the use of biotechnology in inefficient or improper applications, due to a lack of understanding of the limits of biotechnology. Biotechnologies have many limitations, depending on the technology and application under consideration. All biotechnologies have one factor in common: all depend on functional biochemical mechanisms in order to work. It has long been established that all living organisms are subject to Liebig’s Law of the Minimum which states that metabolic activity and growth are restricted by that environmental requirement or nutrient that is available in the minimum quantity.

This paper will explore limiting factors that affect cellular metabolism and the effectiveness and efficiency of biological technologies used in environmental applications. Limiting factors can be generally classified as falling under three headings: 1) Substrate Limiting Factors, 2)

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Environmental Limiting Factors, and 3) Biological Consortia Limiting Factors. Much of the data concerning limiting factors has been derived from the literature and the author’s experience. Since there is very limited data available for biological reactors treating contaminated ground water, soils and hazardous wastes, data for conventional, completely mixed activated sludge reactors has been used to illustrate the concept of limiting factors and to provide a baseline for evaluating them. Data values, therefore, presented in this paper should be used as guidelines only. Treatability studies should be employed to assess limiting factors on an application specific basis.

SUBSTRATE LIMITING FACTORS

The term substrate defines the food source that is presented t o microorganisms for degradation. For the purpose of this discussion organic chemical compounds are defined as substrate. Organic substrate must be biodegradable for biotechnology to be of any use. Some compounds, such as PCBs, are extremely bioresistant, but recent research has shown that certain organisms, such as the white rot fungus, may be able to successfully degrade PCBs. Toxicity is another important limiting factor. Microorganisms can be sensitive to both organic and inorganic toxic chemicals. In some cases toxicity can be controlled or completely overcome by waste dilution, pretreatment, carbon addition and environmental controls in the bioreactor. By carehlly regulating the addition of substrate to the appropriate biological reactors and monitoring and adjusting the reactor environment, problems associated with substrate limiting factors can often be controlled.

Many classes of organic compounds are or may be suitable for biodegradation. Table 1 identifies some classes of chemicals that may be so considered [21.

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TABLE 1

Classes of Chemicals that May Be Suitable for Bioremediation

Using Using Aerobic Anaerobic

Biodegradation Biodegradation Class Examunle Process Process

M o n o c h l o r i n a t e d n z e n e aromatic e

compounds and Benzene, toluene,xylene

Nonhalogenated 2-methyl phenol phenolics and cresols

e

Polynuclear Creosote, benzo-a- aromatic pyrene, anthracene 0

hydrocarbons

Alkanes a n d Fueloil alkenes

Polychlorinated Trichlorophenol biphenyls

e

e

Chlorophenols Pentachlorophenol 0

Nitrogen Pyridine heterocyclics e

Chlorinated solvents

Alkanes Chloroform

Alkenes Trichloroethylene

e

e

e

0

e

e

e

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Some of the compounds listed above are thought by some to be extremely bioresistant or not biodegradable at all. This may be the result of the inability to treat some of these compounds in conventional wastewater treatment reactors, which are not well suited to the treatment of many toxic o r bioresistant compounds. Other reasons may include the use of the wrong biological regimen (i.e. anaerobic, facultative, aerobic), or perhaps another limiting factor was at work in a particular attempt to test the efficacy of

Many wastes and contaminated media contain mixtures of chemicals, the combined effect of which is a resistance to biological treatment. Some wastes have been shown to be biodegradable only under laboratory conditions (PCBs for example), and whether or not biotechnology alone can effectively and economically degrade them has yet to be shown.

' biological treatment.

The biodegradability of many chemical compounds can often be indicated by a search of the scientific literature. Biotreatability investigations are needed to determine if biotechnology is applicable for a given waste stream, contaminated ground water or soil. The biodegradability of a compound or mixture depends primarily on its chemical structure, concentration, toxicity, and bioresistance. Far more compounds can be biodegraded than cannot.

O r g a n i C L o a d i n g R a t e

It is as important to control organic loading rates in biological reactors used to treat hazardous wastes and contaminated soil and ground water, as it is to control the same in conventional biological wastewater treatment plants. Acceptable organic loading rates should be determined experimentally in biotreatability investigations prior to system design. The organic loading rate represents the mass of substrate presented to the biological reactor to a certain mass of microorganisms during a specified period of time. The substrate represents food to the microorganisms, and the term Food-to- Microorganism Ratio (F/M) describes the rate of organic loading to a biological reactor. In the design of wastewater treatment plants the food or substrate concentration is usually measured by analyzing the waste for BODS, COD or TOC. F/M can be computed by dividing the food value (BODS, COD, TOC, or other value, in mg/l) by the concentration of biomass (MLVSS, mg/l) multiplied by the hydraulic detention time, in days: F/M = BOD5, mg/l / MLVSS, mg/l * DT, days.

On a BOD basis an F/M ratio range of 0.1-0.3 will normally provide the necessary degree of treatment. Higher loading rates are possible for some waste streams. Generally, F/M values greater than 0.3 result in partially treated substrate which can result in increased toxicity to the microbes, and a corresponding reduction in microbial diversity and population. F/M values of less than 0.1 can produce acceptable contaminant removals, even with the corresponding decline in microbial diversity and population. Basing F/M calculations and reactor volume on a BOD basis, however, is most useful for readily degradable wastes. For wastes that are bioresistant or inhibitory,

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organic loading rates should be calculated using COD, TOC or other organic loading indicator parameter and related to chemical removal efficiency.

Once a method for measuring organic loading rate has been established, the F/M ratio should be related to the respiration rate of the biomass. For aerobic systems the Specific Oxygen Uptake Rate (SOUR) measurement is a reliable measurement of oxygen utilization, or respiration. The test is conducted by placing a sample of the biomass in a BOD bottle, inserting an oxygen probe and plotting the consumption of oxygen over time, usually 15 minutes or less. MLVSS is determined and respiration is calculated as a function of grams of oxygen consumed grams of MLVSS per day. In aerobic systems a SOUR range of 0.1 to 0.4 g 02 / g MLVSS-day generally indicates a healthy system. SOUR rates of less than 0.1 g 02 / g MLVSS-day (but generally not less than 0.05 g 02 / g MLVSS-day) can indicate acceptable respiration for reactors operated in the extended, or low F/M, mode. SOUR rates of less than 0.05g 02 f g MLVSS-day generally indicates inhibition or loss of substrate.

For anaerobic systems the measurement of methane and CO2 gas production can be used to measure respiration rate. Measuring the rate of respiration and relating this rate to F/M and target compound residuals is important in the design of the biological system. It is also important as an operational tool to monitor system performance. This is particularly important for the remediation of contaminated soils, as the nature and concentration of contaminants in soils can be expected t o change as their excavation progresses. For routinely generated hazardous wastes and for groundwaters, their character and concentration may change somewhat or gradually over time, and the frequent measurement of respiration rate is less important.

Sudden changes in respiration rate is an indicator to the reactor operator that a change in the system has occurred. A sudden decrease in respiration rate may indicate a reduction in organic loading or the introduction of inhibitory chemicals. A sudden increase in respiration rate signals an increase in organic loading rate and alerts the reactor operator that control of F/M is being lost. A sudden cessation in respiration indicates that the biomass has been killed and a shutdown of the reactor is required until the offending limiting factor or toxic condition has been addressed. Gradual decreases in respiration rate with constant organic loading can indicate the accumulation of toxic compounds in the biomass or the introduction of less degradable substrate or inhibitory compounds. Gradual increases in respiration rate with constant organic loading can indicate improved system acclimation and biooxidation or the introduction of substrate which is easier to degrade.

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TOXIC CONCENTRATION LIMITING FACTORS

When evaluating biotechnology for a treatibility application, the concentration of the substrate compounds is a very important factor. The concentration of toxic chemicals can be viewed as a “dose” to a biological system. The dose determines whether the chemicals present represent a degradable substrate or a poison to the system. Sometimes the toxic concentration limiting factor can be overcome by dilution of the bioreactor feed, chemicaVphysica1 pretreatment of the waste, use of the appropriate biological regimen or combinations of them, substrate augmentation, carbon addition, bioaugmentation, or by addressing other degradation rate limiting factors. Some of these corrective measures may be relatively simple in the laboratory, but can be very difficult to implement in full scale operations.

Dation

Dilution, for example, is relatively easy for groundwater or light slurries. A slurry tank preceeds the bioreactor, and effluent, wastewater, or makeup water is proportioned into the feed tank to reduce influent feed concentrations. For treating contaminated soil or concentrated chemical sludges on a continuous basis, dilution is more difficult. Making slurries Erom soils requires experience in adapting solids handling, heavy equipment and making the appropriate slurry dilutions. Pumpable slurries are easier to dilute with the use of variable rate slurry pumps. Fortunately, most biological treatment of concentrated wastes and contaminated soils will be done on a batch basis, and the batch dilution of waste feedstocks is relatively easy. Dilution of a feedstock for toxic chemical concentration reduction has certain drawbacks. Increasing the volume of waste requires an increase in the size of the bioreactor. This in turn, increases requirements for mixing, aeration, temperature control and other environmental factors.

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PhysicaVChemcal Waste Pretreatment

Waste pretreatment for addressing the toxicity concentration liming factor is an option that may be useful in certain applications. Waste pretreatment could be used to partially degrade a compound, remove certain waste components or reduce toxic chemical concentrations to manageable levels. For contaminated ground water, pretreatment unit operations are in wide use, especially carbon adsorption and air stripping units for ground water treatment prior to discharge to publicly owned treatment works. For concentrated organic liquid wastes and contaminated soil, pretreatment unit operations are in limited use or are currently in the planning stages.

Conway and Ross 131 have investigated pollutant concentrations that may make pretreatment advisable, and data from their studies is presented in the following discussion. Table 2 summarizes their findings with regard to pretreatment requirements and pretreatment method for certain wastewater

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conditions. This information was developed for activated sludge wastewater treatment plants, and has direct application only for similar biological reactors. Inhibition levels for other types of biological reactors may be higher or lower, depending on the system design.

Excess sulfides can be air stripped or precipitated. For many biological treatment systems sulfide concentrations in excess of 100 mg/l require pretreatment. Anaerobic treatment systems can tolerate a maximum soluble sulfide concentration of 200 mgfl [41. High phenol concentrations are pretreated using solvent extraction, carbon adsorption or dilution. Phenol concentrations of from 70 to 300 mgfl can be toxic to biological treatment systems, depending on the type of phenolic compound involved. Excess ammonia is pretreated by dilution, ion exchange or air stripping. Ammonia concentrations in excess of 16,000 mg/l can be toxic to biological systems and generally require pretreatment. Ammonia concentrations of much less than 16,000 mg/l can be toxic to certain biological treatment systems. Dissolved solids or salts can inhibit biological activity by creating high osmotic pressure outside the cell membrane, causing transport difficulties for nourishment into the cell and the removal of wastes out of the cell. The limiting factor for dissolved solids is 10,000 mg/l (as TDS). High dissolved solids concentrations can be mitigated by dilution or pretreatment using ion exchange.

Heavy metals are absorbed by biological flocs and films and can accumulate to toxic concentrations. The tolerance for metals in biological systems varies widely, depending on the treatment system regimen, configuration, and waste mix. Wastes which contain both sulfides and metals, for example, often precipitate the metals before presentation to the biomass. Conway and Ross suggest that for aerobic biological treatment systems, that the limiting concentration for lead in influent wastewater 0.1 mgfl. Lead concentrations must be kept at 0.1 mg/l or less to avert toxic effects in the biomass due to lead accumulation. The limiting concentration for copper, nickel, and cyanide is 1.0 mgA; for hexavalent chromium and zinc it is 3.0 mgA; and for trivalent chromium the limiting concentration is 10.0 mgfl.

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TABU3 2

Concentration of Pollutants That Make Prebiological or Primary Treatment Advisable*

Pollutant or Limiting Concentration System Condition Kind of Treatment

Suspended solids >50 to 125 mgfliter

Oil or grease

Toxic ions Pb Cu + Ni + Cn Cr+6 +An Cr+3

PH

A1 kalini ty

Acidity

Organic load variation

Sulfides

Phenols

Ammonia

Dissolved salts

>35 to 50 mgfliter

10,l mgAiter 21 mgAiter 13 mgkter 110 mgAiter

c6, >9

0.5 lb alkalinity as CaCOdb BOD removed

Free mineral acidity

>2:1 to 4:l

>lo0 mg/liter

>70 to 300 mg/liter

>1.6 gAiter

>10 to 16 gfliter

Sedimentation, flotation, or lagooning

Skimming tank or separator

Precipitation or ion exchange

Neutralization

Neutralization for excessive alkalinity

Neutralization

Equalization

Precipitation or stripping with recovery

Extraction, adsorption, or internal dilution

Dilution, ion exchange, pH adjustment, or stripping

Dilution or ion exchange

Temperature 13" to 38°C in reactor Cooling or steam addition

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Other pretreatment technologies not yet fully developed may have potential future applications. Pretreating contaminated soils is a significant challenge to investigators today. Solvent addition to a feedstock can render certain wastes amenable to biodegradation. Certain petroleum sludges, high in oil and grease, can be rendered more degradable with the addition of solvents. The solvents, themselves, are then subject to biodegradation. Wilson [SI is currently investigating solvent extraction as a method of removing PCBs from contaminated soils, using hexane and sodium dodecylsulfate as extraction solvents. This technique may have application for sites contaminated with PCBs and other contaminants. Supercritical extraction is another emerging technology that may have future application. Chemical dechlorination of compounds with high chlorine concentrations may also be useful. PCBs, for example, have been shown to be subject to chemical dechlorination using sodium hydroxide, leaving the easily degradable biphenyl for subsequent biological treatment. Dechlorination with quicklime or other caustic agent may also be possible.

Carbon Addition

Powdered activated carbon (PAC) can be added to a biological reactor t o reduce the toxic effect of the reactor influent and to retain contaminants in the system to promote biodegradation. PAC adsorbs both toxic pollutants and oxygen. Biomass attaches to the PAC particles to which it has available both oxygen and the substrate for metabolism. PAC can also adsorb some toxic metals, thus accumulating and concentrating them in the system. By adsorbing toxic organic pollutants and being incorporated into the biomass, the pollutants are physically retained in the biomass and are kept in contact with the biomass longer. This can be an extremely important attribute €or PAC systems because while motile microbial cells may be able to use their motility to maintain contact with substrates, non-motile cells which cannot are able to grow around and encapsulate contaminants in close contact with them, facilitating their degradation.

BIODEGRADABlLlTY OF TOMC CHEMICALS

The term ‘toxic organic’ compound refers to both the chemical compounds found on the EPA Emuent Guidelines Division List of Priority Pollutants and to other chemicals not on the list which exert toxic effects to biological systems. Other lists of ‘toxic organic’ compounds have been developed by the EPA Contract Laboratory Program for Superfund program work, lists of compounds regulated under the RCRA and CERCLA programs and lists of compounds developed by state regulatory agencies. For the purpose of this discussion we will refer to a ‘toxic organic’ compound as one which exerts a toxic effect on any biological system. The majority of the EPA listed priority pollutants can be removed through convention biological treatment. Table 3 provides a comparison of the biodegradability of toxic pollutants as predicted by an EPA study to the average results of priority pollutant removals from five treatment plants [41.

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TABLE 3

Comparison of Biodegradability of Toxic Organic Pollutants as Predicted by EPA Study

To the Five Plant Study Results

EPA Five-Plant Study: Volatile Compound Degradation, %t Removal, %$

Acrylonitrile Benzene Bromomethane Bromodichloromethane Carbon tetrachloride Chlorobenzene C hloroe t hane Chloroform Dibromochloromethane l,l,-Dichloroethane 1,2-Dichloroethane 1,l-Dichloroethene t- 1,2-Dichloroethene 1,2-Dichloropropane 1,3-Dichloropropane Ethylbenzene Methylene chloride 1,1,2,2-Trichloroethane Tetrachlorethene l,l,l-Trichloroethane 1,1,2-Trichloroethane Trichloroethene Toluene Vinyl chloride

100 100 48 67 100 100 NA 100 56 100 100 100 100 92 100 100 100 36 100 100 59 100 100 NA

99 100 100 89 100 w 91 99 100 7% 99 100 48 97 48 99 75 93 27§ 3% 72§ 40§ 100 100

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TABLE 3 (cont'd)

Comparison of Biodegradability of Toxic Organic Pollutants as Predicted by EPA Study

To the Five Plant Study Results

EPA Five-Plant Study: Basemeutral Compound Degradation, %t Removal, %$.

Acenaphthene Acenaphtylene Anthracene Benzo (a) anthracene Benezo (b) fluoranthene Benzo (a) pyrene Bis (2-ethylhexy1)phthalate Butylbenzylphthalate Dibenzo (a, h) anthracene Di-n-butylphthalate 1,3-Dichlorobenzene 1,2-Dichlorobenzene Diethylphthalate Dimethylphthalate Dioctylphthalate Fluoranthene Fluorene Isophorone Naphthalene Nitrobenzene Pyrene 1,2,4-Trichlorobenzene

100 98 92 35 100 NA 95 100 NA 100 35 29 100 100 94 100 77 100 100 100 100 24

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TABLE 3 (cont'd)

Comparison of Biodegradability of Toxic Organic Pollutants as Predicted by EPA Study

To the Five Plant Study Results

EPA: Five-Plant Study: Acid Compound Degradation, %t Removal, %$

%Chlorophenol

!2,4-Dichlorophynol

2.4-Dimethylphenol 2,4-Dinitrophenol

2-Nitrophenol

Pentachlorophenol Phenol 2,4,6-Trichlorophenol

100 100 100

100 100

100

100 100

41

91 100

84

T7

36 98 45

t Includes volatilization losses at 25°C and initial concentration of 5 mgh. 4 Based on arithmetic means. 8 Influent concentration less than 40 ppb. NA = not available.

468

Removal of organic compounds in activated sludge systems as well as other biological reactors is through air stripping, adsorptiononto the biomass and biodegradation. Only a small portion of the pollutant removal is attributed to adsorption (generally less that 1%). Air stripping is an important removal mechanism for volatile organic pollutants and some chemicals can be completely removed by stripping alone. When air stripping is not acceptable or applicable, many biological reactors can be adapted to minimize volatilization losses.

Table 4 illustrates specific removal mechanisms and efficiencies of some priority pollutants [61 for a conventional activated sludge system. Similar removals can be expected for aerobic systems treating contaminated ground water. Lower stripping efficiency and similar adsorption and biodegradation efficiencies can be expected for the treatment of concentrated hazardous wastes.

As stated earlier, a majority of the priority pollutants and other toxic organics will biodegrade, although some will degrade much more slowly than others. Appropriate biological treatment system design, directed toward the removal of specific compounds, can provide the needed level of treatment. Biological treatment systems, such as the bioslurry reactor, are designed for the removal of a number of specific chemical compounds--not for BOD, TSS and ammonia removal which typically governs the design of wastewater treatment systems.

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TABLE4 Specific Removal Efficiencies of Priority Pollutants

Compounds PercentTreatment Achieved

Stripping Adsorption Biodegradation

Nitrogen Compounds

Phenols Acrylonitrile 99.9

Phenol 2,4-DNP 2,4-DCP PCP

Aromatics 1,2-DCB 1,3-DCB Nitrobenzene Benzene Toluene Ethylbenzene

Halogenated Hydrocarbons Methylene Chloride 1,2-DCE l,l,l-TCE 1,1,2,2-TCE 1,BDCP TCE Chloroform Carbon Tetrachloride

21.7

2.0 5.1 5.2

8.0 99.5

100.0 93.5 99.9 a. 1 19.0 33.0

0.58

0.02 0.19

99.9 99.3 95.2 97.3

78.2

97.8 97.9 94.9 M.6

-_--

91.7 0.50

0.83 1.19 1.38

33.8 78.7 64.9

Oxygenated Compounds

Polynuclear Aromatics

99.9 Acrolein

Phenanthrene Naphthalene

Bis (2-Ethylhexyl) Phthalates

Ethyl Acetate

98.2 98.6

Phthalates

Other

76.9

98.8

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E"lMENTAL LIMITING FACTORS

Microbes live and reproduce in a wide variety of environments. Bacteria have been found in the deep ocean, underground, and in the polar ice caps. Some require a highly specialized environment, like the sulfate reducing bacteria which live in the vicinity of active vents along the ocean floor. Some can live and function well in almost any environment that can offer only minimal support for life. The microbes that are most useful to the environmental scientist or engineer, however, require that certain environmental factors and sources of nutrition me carefully maintained, if they are to function as we wish. The careful maintenance of environmental limiting factors is vital to maintaining the optimal rate of pollutant degradation.

Ti?mp€WtltUt-e

Temperature is a very important environmental limiting factor. Temperature affects the rate of cellular metabolism and the solubility of substrate. An individual microbial species has an optimum and maximum temperature for growth. At high temperatures extracellular enzymes can be denatured, or cell death may occur. At low temperature metabolic activity decreases significantly. Psychrophiles microbes grow best at temperatures below Z O O C. Mesophiles do best at 15" C to about 45" C. Most microbes are grouped into this category. Thermophiles grow at temperatures above 45°C. The microbes of interest to us are the Mesophiles. Most biological reactors are operated at about 20" C. Since metabolic reaction rate can double for each 10" C increase in temperature, there can be some advantage to operating a biological reactor at temperatures higher than those permitted by ambient conditions. Temperature should be maintained at no less than 10" C and no more than 35" C to 40" C. The cost of pumping calories into a reactor to raise temperature can be prohibitive unless a cheap source of steam or other heat source is availible at the site. Heat in biological reactors comes primarily from influent feed temperature, mechanical energy input, solar energy and metabolic activity. Heat losses result primarily from aeration and heat loss through reactor walls. For relatively small reactors the aeration system can be designed to minimize heat loss, and the reactor can be insulated. For large lagoon-sized bioremediation projects, it is rarely possible to control reactor temperature, except to cool the biomass by spray cooling or aeration. For in-situ applications temperature cannot be controlled, and below ground temperatures are quite low. This means that in-situ bioremediation will proceed at relatively slow rates and will require longer time frames before site remediation can be completed.

471

Most of the microbes of use in bioremediation applications are obligate aerobes which use aerobic respiration to obtain energy. For in-situ bioremediation projects oxygen can’ be supplied t o the subsurface environment by aerating recirculation water, or by injecting solutions of hydrogen peroxide or ozone. For surface or marine spills atmospheric oxygen is availible and supplemental sources are not needed.

The use of strictly anaerobic regimens to treat hazardous wastes, contaminated soils or groundwater will be limited to a few specialized, substrate-specific applications. Anaerobic degradation of pollutants occurs but the rate of degradation is much slower than that of aerobic degradation. Facultative anaerobes can exist in environments in which anaerobidaerobic conditions alternate, by sequentially substituting oxygen with nitrate as the chemical electron acceptors. It is becoming apparent that for some applications (like those for the degradation of chlorinated aliphatic compounds) a combination of aerobic and facultative biological regimens is needed, and precise oxygen control is essential. Aerobic/facultative biological treatment systems can be designed by separating the reactor into separate cells for sequential facultative o r aerobic treatment. The aerobidfacultative regimen can also be maintained in a single reactor in which soil particles or carbon are added as growth media and dissolved oxygen levels are kept low (about 0.5 mg/l). Biological floc grows around the media particle and the microbes attached to or nearest to the particle become covered by subsequent growth. This outer growth is exposed to the oxygen in solution while the inner growth has limited exposure to oxygen. By keeping the dissolved oxygen content low, the driving force of the oxygen into the floc is reduced significantly, compared to saturated conditions. For the treatment of liquid wastes or contaminated groundwater, recirculating biological contactors, trickling filters, suspended growth biological filters, and other biological systems provide the aerobidfacultative interface very efficiently.

Most bacteria (neutrophiles), protozoans and other microbes grow best at a neutral pH. Acid producing bacteria (acidophiles) are adapted to environments at lower pH value. Some bacteria (basophiles) are adapted to alkaline environments. Fungi can be grown in environments of extreme pH better than most bacteria. Biological reactor pH is maintained in the 6-8 pH value range by adjusting reactor feed pH. The optimum pH for biological reactors is believed to be in the middle of this range Anaerobic biological reactors often go “sour” when acid producing bacteria gain predominance, resulting in biomass pH values of 5 or less. Careful monitoring of pH is needed for these reactors, and the addition of caustic neutralizing agents and sodium bicarbonate can help maintain pH control. For biological treatment of surface or marine spills of petroleum products or other organic chemicals, pH control in surficial soils is usually not required except in salt marshes

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and other places where acid soils are present. For in-situ treatment applications involving highly acid or alkaline ground water, biological treatment may not be a viable option.

Nutrients

An inadequate supply of nutrients can severely limit the growth and metabolism of microorganisms, thus limiting the rate of substrate removal. Nitrogen and phosphorus are recognized as being needed in the greatest quantity for cell synthesis and metabolism. Some industrial wastewater treatment plants add phosphoric acid and liquid ammonia to provide these essential nutrients. In addition to nitrogen and phosphorus other nutrients are needed by microbes. These include sulfur, magnesium, potassium, calcium, and metallic elements including gold, boron, iron and other metals in trace quantities. Fertilizer mixtures can be added to biological reactors to provide needed nutrients. Nitrogen, phosphorus and other nutrients are injected to the subsurface for in-situ bioremediation applications. Nutrient addition in activated sludge treatment plants is provided at a B0D:N:P ratio of 100:5:1. For landfarming operations a C:N:P ratio of 100:2:0.2 has been found to be successful.

Essential micronutrients such as calcium, iron, metals and others are often assumed to be present and available in the subsurface environment. This may be the case in many locales, but should not be assumed to be the case everywhere. It would be safer to assume that micronutrients are not present a t a given site, and add them to promote optimum biological growth. For biological treatment of surface or marine spills of petroleum products or other organic chemicals, nutrients are applied to the spill site to stimulate biodegradation of the pollutants. Approximately 110 miles of beaches in Prince William Sound, Alaska were treated with nitrogen- and phosphorus- rich fertilizers to accelerate the biodegradation of crude oil released to the environment as a result of the Exxon Vuldez oil spill of March 1989. Researches report that the rate of biodegradation was acellerated by 200-400% as a result of nutrient addition 173.

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BIOLOGICAL CONSORTIA LIMITING FACTORS

The success of a given bioremediation application depends on the factors mentioned previously and the development, acclimation and growth of a biological regimen capable of degrading the target substrates. The factors discussed below relate to the biomass itself; it’s regimen, diversity, consortia development,and acclimation.

Biological Regimen

Biological regimens for biotreatibility applications are divided into anaerobic, facultative, and aerobic systems. Aerobic systems are able to effectively treat many waste mixtures but are subject to toxic concentration effects for others. For many biotechnology applications it is becoming evident that anaerobic or facultative systems, or combinations of them with aerobic systems, can be used to degrade toxic or bioresistant compounds. In the wastewater treatment field, combinations of biological regimens are widely employed to enhance contaminant removal rates in sequential biologocal reactors in which different biological regimens are maintained. Use of the appropriate biological regimen or combination of regimens is crucial to successful biotechnology applications.

U d b Microorganisms have evolved their capability t o degrade natural organic compounds over a period of millions of years. Biodegradable man-made compounds, which began to appear in the environment within the last 100 years, also provide a rich source of organic carbon and energy needed for growth. The activity and growth of microorganisms in a biological reactor or in the environment is largely governed by their ability to produce enzymes to catalyze metabolic reactions. Lack of an appropriate enzyme-or the species of microbe that produces it-can mean a failure to break down a parent compound or the inability to completely degrade daughter compounds. Hydrolytic enzymes are secreted by the cells into the biological medium. These enzymes break higher molecular weight compounds into lower molecular weight compounds. Some compounds and ions can pass through the cytoplasmic membrane into the cell by free diffusion, which is dependent on the concentration gradient of the compound between the inside and outside of the cell membrane. Certain proteins exist inside the cytoplasmic membrane which couple substrate transport to an energy-yielding process. This mechanism, called active transport, is the principal means of cell entry for most substrates and ions. These proteins can be very specific for a particular substrate, even t o the exclusion of very similar or chemically related substrates. A biological consortia with a high diversity provides a biological system with a larger variety and concentration of enzymes and metabolic systems capable of metabolizing a larger number of substrates.

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Biological diversity, therefore, is an important factor in developing the biological consortia to be employed for a given application. Addressing potential limiting factors thoroughly ensures that the maximum system diversity possible is available.

Starting the Biological Consortia .

Municipal sewage treatment plant mixed liquor or waste activated sludge is most often used to seed biological reactors. This source of microorganisms is often sufficient for starting aerobic or facultative reactors. Anaerobic digester sludge is used as seed for anaerobic reactors and to supplement facultative systems. The optimum biological consortia for a given project will almost always consist of a population of microbes with moderate to high diversity. Municipal wastewater treatment plant biomass (especially biomass treating industrial wastewaters as well as sewage) provides a good basis for culturing a biological consortia. For most applications municipal treatment plant biomass will be suficient. For some applications, however, the biological consortia needs to be supplemented with additional substrate, nutrients or microorganisms for optimum substrate degradation. Other sources of microorganisms include contaminated soil, horse manure, and cultured microorganisms.

Most surficial organic soils contain up to 1 x 106 microbial cells per gram of soil. Some contaminated soils will contain microbes already adapted to the degradation of a contaminant or mixture of contaminants. These can be excavated and cultured into a biological consortia which is already adapted and acclimated for the degradation of the target compounds.

There is increasing interest in providing specific groups of microbes-those best suited t o provide the needed degree of treatment-for a specific treatment application. Highly specialized biological consortia have been cultured and produced on a commercial scale for the treatment of specific classes of chemicals or wastes. Microbial cultures have been developed for the treatment of phenolic and cyanide compounds, aromatic hydrocarbons, petroleum products and spills, for the nitrification of ammonia to nitrate, and for other specific purposes. These cultures are available in powder, freeze dried and liquid forms. Bioaugmentation, the use of supplemental cultured microorganisms, may be useful in some applications. One problem with bioaugmentation is that in many cases the biological supplement must be continuously added. The bacteria generally work well in degrading a given substance, but the beneficial trait that gives the strain(s) an advantage in degrading a particular compound sometimes disappears after the mutation of a number of generations.

Biological supplements are in wide use for the breakdown of oil and grease in a wide variety of applications ranging from the degreasing of sewage pumping station wet wells to the cleaning of bilge tanks of ocean going vessels. Biological supplements can sometimes prove superior to naturally

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ocurring organisms, but not always. Researchers investigating bioremediation alternatives during the Exxon Vuldez oil spill cleanup, however, were unable to demonstrate any advantages to bioaugmentation for the products tested [7].

Acclimation

After seeding a biological system a period of acclimation is needed to condition the microbes to the substrate. The microbes need time to adapt to the substrate and develop the metabolic pathways needed for them to exploit substrate carbon and energy. Some compounds can require several weeks for acclimation to occur. Benzidine, for example, has been shown to require up to six weeks for acclimation to occur. Figure 1 represents an acclimation curve for benzidine for an activated sludge application.

Acclimation can be accomplished on either a batch or continuous basis. Waste is added gradually to a biological reactor at an organic loading rate a t an F/M of less than 0.1 (BOD basis) for readily to moderately biodegradable compounds. For bioresistant or toxic compounds, batch laboratory studies can be used to develop acclimation feed rates. It is important not to shock the microorganisms during the acclimation process as i t would then possibly be necessary to reseed the system and start over again. Acclimation can be observed by examining bacterial growth by measuring MLVSS concentrations, using culture and counting methods, analyzing the biomass for adenosine triphosphate concentrations, by measuring biomass respiration rate, and by measuring emuent or filtered mixed liquor target compound concentrations.

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WEEKS OF ACCLIMATION

FIGURE 1 ACCLIMATION FOR THE DEGRADATION OF BENZIDINE

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Figure 2. Bacterial Growth Curve. A, lag or acclimation phase; B, logarithmic growth phase; C, stationary phase; D, declining phase (endogenous phase); E, surviving population.

The bacterial growth curve (Figure 2) illustrates bacterial growth in a batch system. The biological system starts out with a nominal seeded population. Substrate is introduced and initially there is no change in population. This is the lag or acclimation phase. Upon acclimation to the substrate, the bacteria enter a period of unrestricted multiplication called the log or exponential growth phase. Under optimal conditions a bacterial cell can divide every 15 minutes. The increase in microbial numbers can be observed by using the methods described above. When a nutrient or carbon source becomes a limiting factor, or when toxic or inhibitory compounds accumulate, the biomass enters a stationary phase characterized by no net growth. The bacteria then enter a stage of endogenous respiration and declining population and diversity. With no new carbon and energy available, the cells metabolize their own protoplasm until death occurs. When death occurs the cells lyse, releasing nutrients and food to the system which is consumed by other cells. In this way a surviving population of microorganisms can exist for some time. In continuous feed systems the population will increase until substrate concentration or another limiting factor governs the rate of growth of new cells. The rate of cell death corresponds to the rate of cell growth.

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For both batch and continuous feed systems it is important to monitor the biomass to determine the health, activity and growth phase of the system. Having successfully acclimated the system, feed rate should be increased to the point of maximum cell growth rate, without overdosing or shocking the system.

During acclimation of a system intended to treat toxic o r bioresistant compounds, it is often the practice to supplement the target compound with a more degradable food source, enzymes, folic acid or other supplement. This is done to provide optimal support for the microbes while they mutate strains that can exploit the toxic or bioresistant substrate. Also during acclimation, it is a common practice to provide a surplus of nutrients to the system. When the microbes enter the maximum growth phase and the system stabilizes, nutrient addition is scaled back.

Biomass acclimation is an important phase in the development of a biological system. All of the limiting factors discussed previously should be addressed during acclimation to ensure the optimum growth and operation of the system.

Substrate Augmentation

Substrate augmentation involves the addition of supplemental organic substrate to promote the viability and growth of biomass in order to degrade target organic compounds. Substrate augmentation, in some cases, will enhance the rate of biodegradation of target contaminants. Substrate augmentation chemicals are added to bioreactors t o provide carbon to a system in which carbon as a source of energy is a limiting factor and/or, to stimulate the microbial production of enzymes or other metabolites needed to degrade a particular contaminant.

Many contaminated aquifers do not contain sufficient readily degradable carbon to support an active biomass, and for these applications substrate augmentation is necessary. With supplemental carbon the biomass can grow to concentrations and diversity with sufficient energy to biodegrade the target contaminants.

Wilson and Wilson 181 have experimented with the addition of methane to an unsaturated soil column in the laboratory to stimulate the production of monooxygenase to promote the degradation of TCE. They showed that methane oxidizing bacteria (methanotrophs) can degrade TCE via the phenomena known as cometabolism. Methanotrophs excrete monooxygenase, a metabolic enzyme, for the initial step in the oxidation of methane, which the organisms use for energy and growth. Monooxygenase also is able to oxidize other hydrocarbons, and appears to cause the epoxidation of chlorinated alkenes [91 such as TCE, TCA and others. Chlorinated alkene epoxides hydrolyze in water to a number of compounds which are quite readily mineralized by other bacteria.

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McCarty, et. al., proposed a biological treatment system combining in-situ and above ground biological systems which feature the injection of methane and oxygen to the subsurface environment to initiate and suppport cometabolism and mineralization of chlorinated aliphatic compounds. Adding methane in this manner consitutes substrate enhancement, and illustrates the potential usefulness of this substrate enhancement technique.

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References:

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U.S. Environmental Proctection Agency, Bioremediation in the Field, EPA/540/2-91/007, No. 2, March 1991.

US. Environmental Proctection Agency, Understanding Bioremediation, EPA/540/2-9 1/002

Conway, R.A, and R.D. Ross. 1980. Handbook of Industrial Waste Disposal Van Nostrand Reinhold Co., New York.

W. Wesley Eckenfelder, Industrial Water Pollution Control, 2nd ed., McGraw-Hill Book Company

Wilson, David J., Soil Clean Up by in-situ Surfactant Flushing, Separation Science and Technology, 23(11), pp.863-892,1989.

Kincannon, D. F. and Stover, E.L., Determination of Activated Sludge Biokinetic Constants for Chemical and Plastic EPA DraR Report, CR-806843-01-02,1982

Wastewaters,

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9.

U.S. Congress, Office of Technology Assessment, Bioremediation for Marine Oil Spills-Background Paper, OTA-BP-0-70 (Washington, DC:U.S. Government Printing Office, May 1991).

Wilson, John T. and Wilson, BArbara, H., Biotransformation of Trichloroethylene in Soil, Applied and Environmental Microbiology, Jan. 1985, pp.242-243.

McCarty, P.L., Semprini, L. and Roberts, P.V., Methodologies For Evaluating The Feasibility On In-Situ Biodegradation Of Halogenated Aliphatic Groundwater Contaminants By Me thano trophs.

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