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BioMed Research International

Bioremediation: An Overview on Current Practices, Advances, and New Perspectives in Environmental Pollution Treatment

Lead Guest Editor: Raluca M. HlihorGuest Editors: Maria Gavrilescu, Teresa Tavares, Lidia Favier, and Giuseppe Olivieri

Bioremediation: An Overview on CurrentPractices, Advances, and New Perspectivesin Environmental Pollution Treatment

BioMed Research International

Bioremediation: An Overview on CurrentPractices, Advances, and New Perspectivesin Environmental Pollution Treatment

Lead Guest Editor: Raluca M. HlihorGuest Editors: Maria Gavrilescu, Teresa Tavares, Lidia Favier,and Giuseppe Olivieri

Copyright © 2017 Hindawi. All rights reserved.

This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.

Contents

Bioremediation: An Overview on Current Practices, Advances, and New Perspectives in EnvironmentalPollution TreatmentRaluca Maria Hlihor, Maria Gavrilescu, Teresa Tavares, Lidia Favier, and Giuseppe OlivieriVolume 2017, Article ID 6327610, 2 pages

Recent Developments for Remediating Acidic Mine Waters Using Sulfidogenic BacteriaIvan Nancucheo, José A. P. Bitencourt, Prafulla K. Sahoo, Joner Oliveira Alves, José O. Siqueira,and Guilherme OliveiraVolume 2017, Article ID 7256582, 17 pages

Effect of Free Ammonia, Free Nitrous Acid, and Alkalinity on the Partial Nitrification of Pretreated PigSlurry, Using an Alternating Oxic/Anoxic SBRMarisol Belmonte, Chia-Fang Hsieh, José Luis Campos, Lorna Guerrero, Ramón Méndez,Anuska Mosquera-Corral, and Gladys VidalVolume 2017, Article ID 6571671, 7 pages

Identification of Multiple Dehalogenase Genes Involved in Tetrachloroethene-to-EtheneDechlorination in aDehalococcoides-Dominated Enrichment CultureMohamed Ismaeil, Naoko Yoshida, and Arata KatayamaVolume 2017, Article ID 9191086, 12 pages

Bioremediation of Mercury by Vibrio fluvialis Screened from Industrial EffluentsKailasam Saranya, Arumugam Sundaramanickam, Sudhanshu Shekhar,Sankaran Swaminathan, andThangavel BalasubramanianVolume 2017, Article ID 6509648, 6 pages

Effect of Hydraulic Retention Time on Anaerobic Digestion of Wheat Straw in the SemicontinuousContinuous Stirred-Tank ReactorsXiao-Shuang Shi, Jian-Jun Dong, Jun-Hong Yu, Hua Yin, Shu-Min Hu, Shu-Xia Huang,and Xian-Zheng YuanVolume 2017, Article ID 2457805, 6 pages

EditorialBioremediation: An Overview on Current Practices, Advances,and New Perspectives in Environmental Pollution Treatment

Raluca Maria Hlihor,1,2 Maria Gavrilescu,2,3 Teresa Tavares,4

Lidia Favier,5 and Giuseppe Olivieri6,7

1Department of Horticultural Technologies, Faculty of Horticulture, “Ion Ionescu de la Brad” University of Agricultural Sciences andVeterinary Medicine, 3 Mihail Sadoveanu Alley, 700490 Ias,i, Romania2Department of Environmental Engineering and Management, Faculty of Chemical Engineering and Environmental Protection,“Gheorghe Asachi” Technical University of Ias,i, 73 Prof. Dr. Docent D. Mangeron Street, 700050 Ias,i, Romania3Academy of Romanian Scientists, 54 Splaiul Independentei, 050094 Bucharest, Romania4Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal5Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, 11 Allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7, France6Department of Chemical, Materials and Industrial Production Engineering, Università degli Studi di Napoli Federico II,Piazzale V. Tecchio 80, 80125 Napoli, Italy7Bioprocess Engineering Group, Wageningen University and Research, Droevendaalsesteeg 1, 6708 AAWageningen, Netherlands

Correspondence should be addressed to Raluca Maria Hlihor; [email protected]

Received 7 October 2017; Accepted 10 October 2017; Published 1 November 2017

Copyright © 2017 Raluca Maria Hlihor et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Environmental pollution generated the need to search fornew environmentally friendly, low-cost, and more efficientenvironmental clean-up techniques for its removal or reduc-tion. Bioremediation, a branch of environmental biotechnol-ogy, is nowadays considered as one of the most promisingalternatives. This technology uses the amazing ability ofmicroorganisms or plants to accumulate, detoxify, degrade,or remove environmental contaminants. Bioremediation pro-vides the transformation and/or even removal of organicand inorganic pollutants, even when they are present atlow concentration. Continuous efforts are still made tounderstand the mechanisms by which microorganisms andplants remove or transform environmental pollutants. Thus,the purpose of this special issue was to explore differentvisions on bioremediation, while addressing recent advancesand new ideas in the perspective of efficient process scale-upin view of application at larger scales.

Authors’ contributions cover various topics with a rangeof papers including original research and review articlesspanning studies in remediation of different environmentswhich outline new findings in the biotechnology field. This

special issue contains five papers including one review articleand four original research articles. A brief description of thesefive manuscripts is detailed below.

During the treatment of wastewater with high ammo-nium concentrations, as is the effluent originating fromanaerobic digestion of pig slurry, the presence of free ammo-nia (NH

3or FA) and/or free nitrous acid (HNO

2or FNA)

can affect the performance of the partial nitrification process.Thus, in the paper titled “Effect of Free Ammonia, FreeNitrous Acid, and Alkalinity on the Partial Nitrification ofPretreated Pig Slurry, Using an Alternating Oxic/AnoxicSBR” by M. Belmonte et al., the authors applied a strategyallowing the use of organic matter to partially remove nitrite(NO

2

−) and nitrate (NO3

−) generated during oxic phases.Stable partial nitrification was achieved during the treatmentof the effluent of an anaerobic reactor fed with pig slurry.

In the paper titled “Identification of Multiple Dehalo-genase Genes Involved in Tetrachloroethene-to-EtheneDechlorination in aDehalococcoides-Dominated EnrichmentCulture,” M. Ismaeil et al. investigated a Dehalococcoides-dominated enrichment culture (designated “YN3”) that

HindawiBioMed Research InternationalVolume 2017, Article ID 6327610, 2 pageshttps://doi.org/10.1155/2017/6327610

https://doi.org/10.1155/2017/6327610

2 BioMed Research International

dechlorinates tetrachloroethene (PCE) to nontoxic ethene(ETH) with high dechlorination activity. The metagenomeof YN3 harbored 18 rdhA genes (designated YN3rdhA1–18)encoding the catalytic subunit of reductive dehalogenase(rdhA), four of which were suggested to be involved inPCE-to-ETH dechlorination based on significant increasesin their transcription in response to CE addition. Moreover,metagenome data indicated the presence of three coexistingbacterial species, including novel species of the genusBacteroides, which might promote CE dechlorination byDehalococcoides.

Thirty-one mercury-resistant bacterial strains were iso-lated from the effluent discharge sites of the SIPCOT indus-trial area in the paper of K. Saranya et al. titled “Bioremedi-ation of Mercury by Vibrio fluvialis Screened from IndustrialEffluents.” An interesting outcome of this study was thatthe strain V. fluvialis demonstrated, on one hand, a highbioremediation efficiency in the detoxification of mercuryfrommobile solutions and, on the other hand, a low resistanceagainst antibiotics. Hence, V. fluvialis can be successfullyapplied as a strain for the ecofriendly removal of mercury.

In the paper titled “Effect of Hydraulic Retention Time onAnaerobic Digestion of Wheat Straw in the SemicontinuousContinuous Stirred-Tank Reactors,” X.-S. Shi et al. selected arange of process parameters such as the biogas production,methane content, pH value, and volatile fatty acids (VFAs)component and demonstrate their influence on HydraulicRetention Time (HRT) in two operation modes of STR(Stirred-Tank Reactors). In addition, the degradation ofcellulose, hemicellulose, and crystalline cellulose in digestedwheat straw was also investigated. The obtained resultsindicated that HRT is an important parameter that affects theperformance and stability in the anaerobic digestion of wheatstraw.

Recent approaches using low sulfidogenic bioreactors toboth remediate and selectively recover metal sulfides fromacidic mine drainage are reviewed in the paper of I. Nan-cucheo et al. The manuscript titled “Recent Developmentsfor Remediating Acidic Mine Waters using SulfidogenicBacteria” also highlights the efficiency and drawbacks of thesetypes of treatments for metal recovery and points to futureresearch for enhancing the use of novel acidophilic and acid-tolerant sulfidogenic microorganisms in AMD treatment.

We hope that this collection of papers provides to thereaders a valuable scientific source and support addressingcurrent practices, advances, and new perspectives applicablein the treatment of environmental pollution and we hope itcan also help specialists in the field of biotechnology towardssustainable scale-up.

Acknowledgments

We would like to extend our gratitude to all the authors whosubmitted their work for consideration in our special issueand to reviewers for their critical feedback. Contributionsof Raluca Maria Hlihor and Maria Gavrilescu to this specialissue were supported by a grant of the Romanian NationalAuthority for Scientific Research, CNCS-UEFISCDI (Projectno. PN-III-P4-ID-PCE-2016-0683, Contract no. 65/2017).

Teresa Tavares’ contribution is supported by the PortugueseFoundation for Science and Technology (FCT) under thescope of the research project PTDC/AAG-TEC/5269/2014,the strategic funding of UID/BIO/04469/2013 unit andCOMPETE 2020 (POCI-01-0145-FEDER-006684), andBioTecNorte operation (NORTE-01-0145-FEDER-000004)funded by the European Regional Development Fund underthe scope of NORTE 2020 (Programa Operacional Regionaldo Norte).

Raluca Maria HlihorMaria Gavrilescu

Teresa TavaresLidia Favier

Giuseppe Olivieri

Review ArticleRecent Developments for Remediating Acidic Mine WatersUsing Sulfidogenic Bacteria

Ivan Nancucheo,1 José A. P. Bitencourt,2 Prafulla K. Sahoo,2 Joner Oliveira Alves,3

José O. Siqueira,2 and Guilherme Oliveira2

1Facultad de Ingenieŕıa y Tecnologı́a, Universidad San Sebastián, Lientur 1457, 4080871 Concepción, Chile2Instituto Tecnológico Vale, Rua Boaventura da Silva 955, 66055-090 Belém, PA, Brazil3SENAI Innovation Institute for Mineral Technologies, Av. Com. Brás de Aguiar 548, 66035-405 Belém, PA, Brazil

Correspondence should be addressed to Ivan Nancucheo; [email protected] Guilherme Oliveira; [email protected]

Received 27 March 2017; Revised 31 July 2017; Accepted 23 August 2017; Published 3 October 2017

Academic Editor: Raluca M. Hlihor

Copyright © 2017 Ivan Nancucheo et al.This is an open access article distributed under theCreativeCommonsAttribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acidic mine drainage (AMD) is regarded as a pollutant and considered as potential source of valuable metals. With diminishingmetal resources and ever-increasing demand on industry, recovering AMD metals is a sustainable initiative, despite facing majorchallenges. AMD refers to effluents draining from abandoned mines and mine wastes usually highly acidic that contain a varietyof dissolved metals (Fe, Mn, Cu, Ni, and Zn) in much greater concentration than what is found in natural water bodies. Thereare numerous remediation treatments including chemical (lime treatment) or biological methods (aerobic wetlands and compostbioreactors) used for metal precipitation and removal from AMD. However, controlled biomineralization and selective recoveringof metals using sulfidogenic bacteria are advantageous, reducing costs and environmental risks of sludge disposal. The increasedunderstanding of the microbiology of acid-tolerant sulfidogenic bacteria will lead to the development of novel approaches to AMDtreatment. We present and discuss several important recent approaches using low sulfidogenic bioreactors to both remediateand selectively recover metal sulfides from AMD. This work also highlights the efficiency and drawbacks of these types oftreatments formetal recovery and points to future research for enhancing the use of novel acidophilic and acid-tolerant sulfidogenicmicroorganisms in AMD treatment.

1. Introduction

Metal mining provides everyday goods and services essentialto society. However, this activity has at times caused extensiveand sometimes severe pollution of air, vegetation, and waterbodies [1]. Streams draining active or abandoned mines andmine spoils are widely considered as hazardous to humanhealth and the environment, but on the other hand, they mayalso be alternative potential sources of valuable metals [2, 3].

Currently, millions of tons of ores are processed everyyear by the mining industry and are disposed in the formof waste rocks and mine tailings. As higher-grade ores arediminishing, the primary ores that are processed by miningcompanies are of increasingly lower grade (metal content)and the growing amount of waste material produced bymining operations is consequently significant. The use oflower grade ore was made possible by the development of the

flotation technique in the late 19th century, which allowed theseparation of metal sulfide minerals from gangue mineralsthat have no commercial value [4]. As a result of selectiveflotation, about 95 to 99% of the ground primary ores endup as fine-grain tailings, in the case of copper ores. Thecomposition of tailings is directly dependent on that of theore, and therefore they are highly variable, though pyrite(FeS2) is frequently the most reactive and dominant sulfide

mineral present in tailings wastes [4–6].Pyritic mine tailings therefore have the potential to

become extremely acidic when in contact with surface water.Under oxidizing conditions, pyrite-bearing wastes producesulfuric acid. The acidic water further dissolves other metalscontained in mine waste, resulting in low pH water enrichedwith soluble sulfate, Fe, Al, and other transition metals,known as acid mine drainage (AMD) (Figure 1) [7, 8].


https://doi.org/10.1155/2017/7256582


(a) (b)

(c) (d)

(e) (f)

Figure 1: Illustration of streams of acidicwaters draining fromactive or abandonedmines andmine spoils. (a)AMD froma coppermine in theState of Pará, Brazil, that has been remediated with limestone treatment, (b) acidic water released from abandoned undergroundmetalliferousmine in the Republic of South Africa (reproduced fromAkcil and Koldas [9]), (c) acidic mine water draining from an abandoned sulfurmine,northern Chile, (d) AMD discharge in the Lomero-Poyatos mine, Spain (reproduced from España et al. [10]), (e) acidic water draining fromCoal mines, Jaintia Hills, and (f) AMD originated from mine tailings, Canada, (reproduced from Burtnyski [11]).

BioMed Research International 3

2. Remediation of Acidic Mine Water

Waters draining from abandoned metal mines and minewastes are often acidic (pH < 4) and contain elevated concen-trations of dissolved metals and metalloids and high osmoticpotential associated with concentration of sulfate salts [14]. Inmost cases, active chemical treatment and passive biologicaltreatment can provide effective remediation of AMD [15](details and literature of the advantages and disadvantagesof these treatment and others are presented in Table 1). Amajor drawback to both approaches is that the immobilizedmetals are contained in “sludge” (chemical treatment) orwithin spent compost (biological treatment) and need to bedisposed in specially designated landfill sites, precluding theirrecovery and recycling. Changes in redox conditions duringstorage can lead to remobilization of metals (and metalloidssuch as arsenic) in both sludge and spent composts. Inaddition, potentially useful and valuable metal resources arenot recovered using conventional approaches for remediatingmine waters [3, 16].

A radically different approach for remediating AMDwhich, like compost bioreactors, derives from the abilities ofsome microorganisms to generate alkalinity and to immo-bilize metals, is referred to generally as “active biologicaltreatment.”Microbiological processes that generate alkalinityare mostly reductive processes and include denitrification,methanogenesis, and dissimilatory reduction of sulfate, ferriciron, and manganese (IV), which tend to be limited inAMD. Considering that AMD usually contains elevatedconcentrations of both ferric iron and sulfate, the ability ofsome bacteria to use these compounds as terminal electronacceptors suggests that these reactions can be highly usefulfor mine water remediation. Acidic environments in whichsulfur or sulfide minerals are subjected to biologically-accelerated oxidative dissolution characteristically containlarge concentrations of soluble sulfate [17]. Therefore, micro-bial sulfate reduction might be anticipated to occur withinanaerobic zones in both acidic and nonacidic environments.Biological sulfidogenesis generates hydrogen sulfide as aresult of a reductive metabolic process using sulfate reducingbacteria (SRB). Biological sulfidogenesis has the additionalbenefits of being a proton-consuming reaction, allowing theincrease in pHof theminewater treated contributing towardsmitigation and remediation. The hydrogen sulfide generatedcan be used in controlled situations to selectively precipitatemany potentially toxic metals (such as copper and zinc) oftenpresent in AMD at elevated concentrations [3, 18]. Activebiological treatment has many advantages over alternativestrategies for treatingmine waters, one of the most importantbeing its potential for recovering metals that are commonlypresent in AMD.

There have been few successful applications of SRB-mediated active AMD treatment systems, even though thispossibility has long been appreciated. One major reason forthis is that SRB happens preferentially between pH 6 and8 [19], whereas AMD generally has a pH between 2 and 4and commonly pH < 3 [20]. Under these circumstances, aneutralization step is necessary beforeAMDeffluents are sub-jected to bacterial sulfate reduction or, alternatively, “off-line”

systems need to be used. The latter is necessary by the factthat current systems use neutrophilic SRB or sulfur reducingbacteria, and direct exposure to the inflowing acidic solutionbeing treated would be lethal to these microorganisms.Therefore, a separate vessel in which sulfide generated bythe bacteria is contacted with the acidic, metal-laden wastewater, is required [16, 21]. Examples of this technology arethe Biosulfide and Thiopaq processes (Figure 2) operatedunder the auspices of two biotechnology companies, BioTeq(Canada) and Paques B. V. (The Netherlands), which arecurrently in operation in various parts of the world.

The Biosulfide process has two stages, one chemicaland the other biological. Metals are removed from AMDin the chemical stage by precipitation with biogenic sulfideproduced in the biological stage by SRB under anaerobiccondition. In this system, hydrogen sulfide is generated bythe reduction of elemental sulfur, or other sulfur source, inthe presence of an electron donor, such as acetic acid. Thegas is passed to an anaerobic agitated contactor in whichcopper can be precipitated as a sulfide, usually without pHadjustment and without significant precipitation of otherheavy metals present in the water. The end result is a highvalue copper product, usually containing more than 50% ofthe metal. Other metals such as nickel, zinc, and cobalt canalso be recovered as separate high-grade sulfide products,although pH control using an alkali source is usually requiredto selectively precipitate the metal as a sulfide phase. Thehigh-grade metal sulfide precipitate is then recovered byconventional clarification and filtration to produce a filtercake which can be shipped to a smelter [12].

TheThiopaqprocess uses another system that involves theuse of two biological continuous reactors connected in series(I) to an anaerobic upflow sludge blanket (UASB) reactorfor the reduction of oxidized sulfur species. In this reactor,ethanol or hydrogen is utilized by the SRB as electron donor,producing sulfide (mostly HS−) for the precipitation of metalsulfides (which can proceed in the same reactor depending onthe toxicity of the wastewater), and (II) an aerobic submergedfixed film (SFF) reactor where the excess sulfide is oxidizedto elemental sulfur, using sulfide-oxidizing bacteria. In thisprocess, metals such as Zn and Cd can be precipitated downto very low concentrations [22].

The Paques B. V. process has been successfully imple-mented at an industrial scale at the gold mine Pueblo Viejo,located in the Dominican Republic. A copper recovery plantinstalled in 2014 based on sulfide precipitation is used torecover the copper liberated from the gold extraction process.The sulfidogenic bioreactor generates H

2S to recover up to

12,000 ton of copper per year generating value and reducingthe amount of copper sent to the tailing dam [23]. Applicationof this process has also been demonstrated on a pilot-scaleat the Kennecott Bingham Canyon copper mine in Utah,where >99% of copper present in a pH 2.6 waste stream wasrecovered [22, 24, 25].

Sulfate reduction activity has been reported in low pHecosystems, for example, in acidic lakes, wetlands, and acidmine drainage [19, 26, 27]. However, few acidophilic/tolerantSRB have been cultured [16, 26, 28–30]. A major potentialadvantage of using acidophilic sulfidogens would be to allow


Table1:Summaryof

thevario

ustypeso

ftreatmentfor

AMD

(com

piledfro

mSaho

oet

al.[15],Gazea

etal.[36],Trum

m[37],T

aylore

tal.[38],R

oyCh

owdh

uryet

al.[39],John

sonand

Hallberg[22],Skousen

[40],Skousen

etal.[41],andSeervietal.[42]).

Syste

mtype

Applicability

Supp

ortm

aterials

Mechanism

sLimitatio

nBiologica

l

Aerobicw

etland

(AeW

)Mod

eratea

cidity,netalkalin

emined

rainage

Organicmatter,soil,lim

estone

gravel

Oxidatio

n,hydrolysis,

precipitatio

nRe

quire

dlonger

detentiontim

eandhu

gesurfa

cearea

Anaerob

icwetland

(AnW

)Net-acidicw

ater

with

high

Al,Fe

andDO

Organicmatter,such

ascompo

st,sawdu

st,hay,andlim

estone

gravel,

Sulfateredu

ction,

metal

precipitateas

sulfides,microbial

generatedalkalin

ityRe

quire

dlong

resid

ence

time

Verticalflo

wwetland

(VFW

)Net-acidicw

ater

with

high

Al,Fe

andDO

Limestone,organicmatter

SulfateandFe

redu

ction,

acid

neutralization

Highcapitalcost,po

tentialfor

armoringandplug

ging

with

hydroxides

Sulfateredu

cing

bioreactor

(SRB

)Sm

allfl

owso

rtosituatio

ns,very

acidicandmetalric

hwater

Organicsubstrates

uchas

hay,

alfalfa,saw

dust,

paper,

woo

dchips,crushed

limestone

andcompo

stor

manure

Microbialsulfateredu

ction

Highcapitalcost,extre

mely

low

pHseverelyim

pactthee

fficiency

ofSredu

cing

bacteria

Pyrolusitelim

estone

beds

Mod

eratep

Handwhere

majority

ofacidity

isrelated

toMn

Limestone,organicsubstrate,

aerobicm

icroorganism

Hydrolysis

ofMn

Not

suitablefor

drainage

which

contains

high

Fe,high

maintenance

Perm

eabler

eactiveb

arrie

rs(PRB

)Groun

dwater,low

DO

Organicmatter,lim

estone,zero

valent

iron

Sulfateredu

ction,

sulfide

precipitates,

neutralization

Iron

-oxidizing

bioreactor

Acidicwater

Fe-oxidizing

bacteriaand

archaea

Feoxidation

Phytorem

ediatio

nAny

AMD-im

pacted

sites

Metaltolerant

plantspecies

Phytoextractionand

phytostabilization

Successd

epends

onthep

roper

selectionof

the

metal-hyperaccumulator

plant


Table1:Con

tinued.

Syste

mtype

Applicability

Supp

ortm

aterials

Mechanism

sLimitatio

nGeochem

ical

Ano

xiclim

estone

drain(A

LD)

Acidicwater

with

lowAl,Fe,D

OLimestone

gravel,

compacted

soil

Limestone

dissolution,

raise

pH,

precipitatio

n

Fe-oxide

armoringlim

estone

limitperm

eabilityandcause

plug

ging

Alkalinity

prod

ucingsyste

m(A

PS)

Acidicwater

Organicmatter,lim

estone

Ano

xicc

onditio

n,neutralization,

precipitatio

n

Openlim

estone

channel(OLC

)Re

quire

dste

epslo

pes,net-a

cidic

water

with

high

Al,Fe

andDO

Limestone

Limestone

dissolution,

neutralization

Arm

oringor

thec

oatin

gof

the

limestone,large

amou

ntis

needed,decreases

the

neutralizingcapacity

Limestone

leachbed(LLB

)Lo

wpH

andmetal-fr

eewater

Limestone,

Limestone

dissolution,

neutralization

Arm

oringwith

Fehydroxides

Steel-slagleachbed(SLB

)Highlyacidicandmetal-fr

eewater

Steelslag

Raise

alkalin

ity,neutralization

Not

suitablefor

metal-la

den

water

Limestone

diversionwe

lls(LDW)

Sitesthato

ffera

suitable

topo

graphicalfall

Crushedlim

estone

aggregate

Hydraulicforce,hydrolysis,

and

neutralization

Requ

iredrefillin

gwith

limestone

every2–4weeks

Limestone

sand

Stream

flowwater

Sand

-sized

limestone

neutralizingacid

Coatin

gof

limestone

Low-pHFe

oxidationchannels

Shallowchannels

Limestone

orsand

stone

aggregate

Feoxidation,

adsorptio

nand

coprecipitatio

n

Itremoves

someF

e,bu

trem

oval

efficiency

hasn

otbeen

determ

ined

Sulfide

passivation/microencapsulation

Pitw

allfaces,sulfid

ebearin

gwastesrocks

piles

Inorganicc

oatin

g:ph

osph

ate,

silica,fly

ash,lim

estone;organic

coating:hu

micacid,lipids,

polyethylene

polyam

ine,

alkoxysilanes,fattyacid,oxalic

acid,catecho

l

Preventsulfid

eoxidatio

nby

inorganica

ndorganicc

oatin

g

Long

-term

effectiv

enessisstillin

questio

n,organicc

oatin

gexpensive

Electro

chem

icalcover

Tailing

/wasterock

Con

ductives

teelmesh,cathod

e,metalanod

eRe

ducing

DOby

electrochem

ical

process

Highcapitalcosto

fano

des,no

inform

ationavailableo

nlarge

scalea

pplication


Table1:Con

tinued.

Syste

mtype

Applicability

Supp

ortm

aterials

Mechanism

sLimitatio

nPh

ysica

l

Dry

cover

Sulfide

bearingwastesrockpiles

Fine-grained

soil,organic

materials,

synthetic

material

(plasticliners),vegetation

Minim

izeo

xidatio

nby

physical

barrier,neutralization

precipitates

Shortterm

effectiv

eness

Wetcover

Sulfide

wastes

Und

erwater

Disp

osingwasteun

derw

ater

anoxiccond

ition

sRe

quire

rigorou

seng

ineerin

gdesig

n,high

maintenance

Gas

redo

xanddisplacement

syste

m(G

aRDS)

Und

ergrou

ndmines

CO2andCH4gas

Gas

mixturesp

hysic

allydisplace

O2

Itson

lyfeasiblewhere

partialor

completefl

ooding

isno

tfeasib

le


MetalSul�des(ZnS)

Air

Excess

Sulfur

Puri�ede�uent

Recycle

Neu

troph

ilic

SRB

Bior

eact

or o

ne

Bior

eact

or tw

oSu

l�de

oxid

ising

bac

teriaWaste water or

process water(３／4

2− and metals,such as ：Ｈ2+)

（2-rich gas

＃／2

（３−

(３Ｉ)

３／42−

+ 4（2 + （+→ （３

−+ 4（2／

：Ｈ2+

+ （３−→ ：Ｈ３ + （

+

2（３−+ ／2 + 2（

+→ 2３

Ｉ+ 2（2／

(a)

Bior

eact

orsta

ge I

Stag

e II

Ana

erob

ic ag

itate

dco

ntac

tor

SulfurReagents

Feed water(AMD)

sulfu

r red

ucin

g ba

cter

ia

Clari�erTreated water

Filter

Metal sul�de (ZnS)to smelter

（2３

Gen

erat

ion

by

（2３

(b)

Figure 2: Schematic overview of the Thiopaq (a) and Biosulfide (b) processes (adapted from Adams et al. [12], Muyzer and Stams [13]).

simpler engineering designs and reduce operational costs byusing single on-line reactor vessels that could be used to bothgenerate sulfide and selectively precipitate target metal(s).Precipitation and removal of many soluble transition metals,often present in AMD emanating from metal mines, maybe achieved by ready biomineralization as their sulfides. Theproduced metal sulfides have different solubilities; thereforemetals can be precipitated together or selectively by con-trolling concentrations of the key reactant S2−, which maybe achieved by controlling pH (S2− + H+ ↔ HS−). Coppersulfide, for example, is far less soluble than ferrous sulfide(respective log Ksp values of −35.9 and −18.8) and thereforeCuS precipitates at pH 2, whereas FeS needs much higher pHto precipitate. Diez-Ercilla et al. [31] have also demonstratedthat selective precipitation of metal sulfides occurs naturallyin Cueva de la Mora pit lake (SW Spain) and the geochemicalcalculations match perfectly with the results of chemicaland mineralogical composition. Ňancucheo and Johnson[3] showed that it was possible to selectively precipitate

stable metal sulfides in inline reactor vessel testing twosynthetic AMDs in acidic conditions (pH 2.2–4.8). In the firstbioreactor, with a composition of feeding similar to AMD atthe abandoned Cwm Rheidol lead-zinc mine in mid-Wales,zinc was efficiently precipitated (>99%) as sulfide inside thereactor while both aluminum and ferrous iron remain insolution (>99%) and were washed out of the reactor vessel.The second sulfidogenic bioreactor was challenged with asynthetic AMD based on that from Mynydd Parys, NorthWales. Throughout the test period, all the copper presentin the feed liquor was precipitated (confirmed as coppersulfide) within the bioreactor, but none of the ferrous ironwas present in the solids. Although the initial pH at whichthe bioreactor was operated (from pH 3.6 to 2.5) causedsome coprecipitation of zinc with the copper, by progressivelylowering the bioreactor pH and the concentration of theelectron donor in the influent liquor, it was possible toprecipitate >99% of the copper within the bioreactor as CuSand to maintain >99% of the zinc, iron, and aluminum in


solution. Glycerol was used as energy and carbon source(electron donor) and the generalized reaction is [1]

4C3H8O3+ 10H+ + 7SO

4

2−+ Cu2+ + Zn2+ + Fe2+

→ 12CO2+ 5H2S + CuS + ZnS + Fe2+ + 16H

2O

(1)

This low sulfidogenic bioreactor system was also demon-strated to be effective at processing complex acidic waterdraining from the Mauriden mine in Sweden [18]. Through-out the test period, zincwas removed from the syntheticminewater as ZnS, from which the metal could be recovered, asin the case at the Budel zinc refinery in The Netherlands[24]. Recently, Falagán et al. [32] have operated this sulfi-dogenic reactor to mediate the precipitation of aluminumin acidic mine waters as hydroxysulfate minerals. Besides,this bioreactor was tested to demonstrate the recovery ofover 99% of the copper present in a synthetic mine waterdrained from a copper mine in Carajás in the State of Pará,Brazil [33]. The sulfidogenic system was also operated underdifferent temperatures. Although there were large variationsin rates of sulfate reduction measured at each temperature,the bioreactor operated effectively over a wide temperaturerange (30–45∘C) which can have major advantages in somesituations where temperatures are relatively high for examplein mine sites located in northern Brazil and in other regionswhere high temperatures are observed. Therefore, therewould be no requirements to have temperature control (heat-ing or cooling) to preserve the integrity of the acidophilic SRBreactor [33]. The perceived advantages of this system are thatthere are simple engineering and relatively low operationalcost. The system can be configured to optimize mine waterremediation and metal recovery according to the nature ofthe mine water, which are the constraining factors in usingactive biological technologies to mitigate AMD.

Metalloids such as arsenic are a common constituent ofmine waters. Battaglia-Brunet and colleagues [34] demon-strated that As (III) can be removed by precipitation as asulfide.The results demonstrated the feasibility of continuoustreatment of an acidic solution (pH 2.75–5) containing up to100mgAs (V).Under this approach,As (V)was reduced toAs(III) directly or indirectly (via H

2S) by the SRB and orpiment

(As2S3) generated within the bioreactor. In addition, this

process was also observed to occur naturally in an acidic pitlake [31].

Recently, Florentino and colleagues [35] studied themicrobiological suitability of using acidophilic sulfur reduc-ing bacteria for metal recovery. These authors demonstratedthat the Desulfurella strain TR1 was able to perform sulfurreduction to precipitate and recover metals such as copperfrom acidic waste water and mining water, without the needto neutralize the water before treatment. One drawback onthe of use sulfur reducing microorganisms is that a suitableelectron donor needs to be added for sulfate reduction. Eventhough sulfate is present in AMD, the additional cost ofelectron donors (such as glycerol) for sulfate reduction ishigher than the cost of the combined addition of elementalsulfur and electron donors. Subsequently elemental sulfur asan electron acceptor can be more economically attractive for

the application of biogenic sulfide technologies. On the otherhand, cheaper electron donor such organic waste materialmay be used but their variable composition makes it lesssuitable for controlled high rate technologies. Besides, deadalgal biomass can release organic products suitable to sustainthe growth of SRB. Therefore, Diez-Ercilla et al. [31] haveproposed that under controlled eutrophication it could bepossible to decrease the metal concentrations in acidic minepit lakes.

3. Microbiology in Remediating AcidicMine Waters

Based on 16S rRNA sequence analysis, microorganisms thatcatalyze the dissimilatory reduction of sulfate to sulfideinclude representatives of five phylogenetic lineages ofbacteria (Deltaproteobacteria, Clostridia, Nitrospirae, Ther-modesulfobiaceae, and Thermodesulfobacteria) and twomajor subgroups (Crenarchaeota and Euryarchaeota) of theArchaea domain (Table 2 shows a summary of sulfidogenicmicroorganisms used for their main characteristics). SRB arehighly diverse in terms of the range of organic compoundsused as a carbon source and energy, though polymericorganic materials generally are not utilized directly by SRB[13]. In addition, some SRB can grow autotrophically usinghydrogen as electron donor and fixing carbon dioxide,though others have requirement for organic carbon such asacetate, when growing on hydrogen. Besides, many SRB canalso use electron acceptors other than sulfate for growth,such as sulfur, sulfite, thiosulfate, nitrate, arsenate, iron, orfumarate [78].

Most species of SRB that have been isolated from acidicmine waste such asDesulfosarcina,Desulfococcus,Desulfovib-rio, and Desulfomonile are neutrophiles and are active atneutral pH [14, 25]. Besides, for a long time the accepted viewwas that sulfate reducing activity was limited to slightly acidicto near neutral pH explained by the existence of micronichesof elevated pH around the bacteria [21, 31]. Attempts to isolateacidophilic or acid-tolerant strains of SRB (aSRB) havemostlybeen unsuccessful, until recently [79]. One of the reasons forthe failure to isolate aSRB has been the use of organic acidssuch as lactate (carbon and energy source) which are toxicto many acidophiles. In acidic media, these compounds existpredominantly as nondissociated lipophilicmolecules and, assuch can transverse bacterial membranes, where they disso-ciate in the circumneutral internal cell cytoplasm, causing adisequilibrium and the influx of further undissociated acids,and acidification of the cytosol [80]. In contrast, glycerolcan be used as carbon and energy source as it is unchargedat low pH. In addition, many SRB are incomplete substrateoxidizers, producing acetic acid as a product, enough to limitthe growth of aSRB even at micromolar concentration. Tocircumvent this problem and for isolating aSRB, overlay platecan be used to remove acetic acid. This technique uses adouble layer where the lower layer is inoculatedwith an activeculture of Acidocella (Ac.) aromatica while the upper layer isnot. Therefore, the heterotrophic acidophiles metabolize thesmall molecular weight compounds (such as acetic acid) thatderive from acid hydrolysis of commonly used gelling agents


Table2:Isolated

sulfido

genicm

icroorganism

sand

theirm

aincharacteris

tics.

Microorganism

Temperature

(∘ C)

pHa

Carbon

andele

ctron

source

Electro

nacceptor

Source

Reference

Thermocladium

modestiu

s45–82

(75)

2.6–

5.9(4.0)

Glycogen,

starch,

proteins

Sulfu

r,thiosulfate,

L-cyste

ine

Hot

sprin

gs(w

ater,

mud

),Japan

[43–45]

Caldivirg

amaquilin

gensis

70–9

02.3–6.4(3.7–4

.2)

Glycogen,

beefextract

pepton

e,tryptone,yeast

extract

Sulfu

r,thiosulfate,

L-cyste

ine

Hot

sprin

gs(w

ater,

solfataric

soilmud

),Mt

Maquilin

g,Ph

ilipp

ines

[46]

Archaeoglobu

slithotro

phicu

snd

6.0

Acetate

Sulfate,L-cysteine

nd[47,48]

Archaeoglobu

sveneficus

nd6.9

H2,acetate,formate,

pyruvate,yeastextract,

citrate,lactate,sta

rch,

pepton

e

Sulfite,thiosulfate

Wallsof

activ

eblack

smoker

atmiddle

AtlanticRidge

[44]

Archaeoglobu

sprofund

usnd

4.5–7.5

H2,acetate,pyruvate,

yeastextract,lactate,

meatextract,peptone,

crud

eoilwith

acetate

Sulfate,thiosulfate,

sulfite

Deepseah

ydrothermal

syste

moff

Guaym

as,

Mexico

[49,50]

Archaeoglobu

sfulgidu

s60–75

(70)

5.5–7.5

(6,0)

H2,C

O2,formate,

form

amide,D(−)-and

L(+)-lactate,glucose,

starch,calam

inea

cids,

pepton

e,gelatin,casein,

meatextract,yeast

extract


sulfite

Marineh

ydrothermal

syste

m,N

eron

e,Ita

ly[49,51]

Thermodesulfatatorind

icus

55–80

(70)

6.0–

6.7(6.25)

H2,C

O2;stim

ulated

bymethano

l,mon

omethylamine,

glutam

ate,pepton

e,fumarate,tryptone,

isobu

tyrate,3-C

H3

butyrate,ethanol,

prop

anolandlow

amou

ntso

facetate.

Sulfate

Marineh

ydrothermal

syste

m,C

entralIndian

Ridge

[52]

Thermodesulfobacterium

hydrogeniphilum

50–80

(75)

6.3–6.8(6.5)

H2,C

O2;stim

ulated

byacetate,fumarate,

3-methylbutyrate,

glutam

ate,yeastextract,

pepton

eortrypton

e

Sulfate

Marineh

ydrothermal

syste

m,G

uaym

asBa

sin[53]


Table2:Con

tinued.

Microorganism

Temperature

(∘ C)

pHa

Carbon

andele

ctron

source

Electro

nacceptor

Source

Reference


commun

e41–83

6.0–

8.0(7.0)

H2,C

O2,pyruvate,

lactate

Sulfate,thiosulfate

Hot

sprin

gs(w

ater,

sedimentand

mats)

Yellowsto

neNational

Park,U

SA[54,55]


thermophilum

nd6.0–

8.0(7.0)

H2,C

O2,pyruvate,

lactate

Sulfate,thiosulfate

nd[55]


hveragerdense

754.5–7.0

(7.0)

H2,pyruvate,lactate

Sulfate,sulfite

Hot

sprin

gs(m

icrobial

mats),Iceland

[56]

Thermodesulfobium

narugense

694.0–

6.0(5.5–6

.0)

H2,C

O2

Sulfate,n

itrate,

thiosulfate

Hot

sprin

gs(m

icrobial

mats),Japan

[57]

Desulfotomaculum

spp.(30

species)

nd2.3–5.5

H2,C

O2,formate,some

(organicacids;lip

ids;or

mon

oaromatic

hydrocarbo

ns)

Sulfide,sulfur,

thiosulfate,A

cetate,

some(Fe

(III),Mn(IV),

U(V

I)or

Cr(V

I))

Subsurface

environm

ents,

rice

fields,mines,oilspills

[58–62]

Desulfosporosinus

meridiei

10–37

6.1–7.5

H2,C

O2,acetate,som

e(la

ctate,pyruvate,

ethano

l)Sulfate,som

e(nitrate)

Groun

dwater

contam

inated

with

polycyclica

romatic

hydrocarbo

ns,inSw

anCoastalPlain,

Australia

[63]

Desulfosporosinus

youn

gii

8–39

(32–35)

5.7–8.2(7.0–

7.3)

Beefextract,yeast

extract,form

ate,

succinate,lactate,

pyruvate,ethanoland

toluene

Fumarate,sulfate,sulfite,

thiosulfate

Artificialwetland

(sedim

ent)

[64]


Table2:Con

tinued.

Microorganism

Temperature

(∘ C)

pHa

Carbon

andele

ctron

source

Electro

nacceptor

Source

Reference

Desulfosporosinus

orien

tis37–4

86.0–

6.5

H2,C

O2,formate,

lactate,pyruvate,m

alate,

fumarate,succinate,

methano

l,ethano

l,prop

anol,butanol,

butyrate,valerate,

palm

itate

Sulfate,sulfite,

thiosulfate,sulfur

nd[65]

Desulfosporom

usapolytro

pa4–

376.1–8.0

H2,C

O2,formate,

lactate,bu

tyrate,several

alcoho

ls,organica

cids,

carboh

ydrates,some

aminoacids,choline,

betaine

Sulfate,Fe(OH) 3

Oligotroph

iclake

(sedim

ent),

German

[66]

Thermodesulfovibrio

yellowstonii

41–83

6.0–

8.0(7.0)

H2,C

O2,acetate,

form

ate,lactate,

pyruvate


sulfite

Hot

sprin

gs(w

ater,

sedimentand

mats)

Yellowsto

neNational

Park,U

SA

[67]

Thermodesulfovibrio

islandicus

554.5–7.0

(7.0)

H2,pyruvate,lactate,

form

ate

Sulfate,n

itrate

Bioreactor

inoculated

with

hotsprings

(microbialmats)sample,

Iceland

[56]

Desulfohalobium

spp.(6

species)

nd5.5–8.0(6.5–7.0)

H2,lactate,ethanol,

acetate

Sulfite

hypersaline

environm

ents

[68,69]

Desulfocaldus

terraneus

58nd

H2,C

O2,aminoacids,

proteinaceou

ssub

strates

andorganica

cids,

prod

ucingethano

l,acetate,prop

ionate,

isovalerate/2-

methylbutyrate,

Cystine,sulfu

r,sulfate

Seao

ilfacilities,Alaksa

[70]


Table2:Con

tinued.

Microorganism

Temperature

(∘ C)

pHa

Carbon

andele

ctron

source

Electro

nacceptor

Source

Reference

Desulfomicrobium

spp.(4

species)

25–30

ndH2,lactate,pyruvate,

Ethano

l,form

ate

Sulfate,sulfoxyanions

Anaerob

icsediments

(Freshwater,brackish

,marine),anaerob

icstrata

oroverlyingwater,and

insaturatedmineralor

organicd

eposits.

[54,69]

Desulfonatro

novibrio

hydrogenovoran

s37–4

09.0

–10.2(9.0–9.7)

H2,formate

Sulfate,sulfite,

thiosulfate

Alkalines

odalakes

(anaerob

ic)

[71]

Desulfonatro

num

spp.(3

species)

20–4

5(37–45)

8.0–

10.0(9.0)

H2,form

ate,Yeast

extract,ethano

l,lactate

Sulfate,sulfite,

thiosulfate

Alkalines

odalakes

(anaerob

ic)

[72]

Desulfovibriospp.(47

species)

25–4

4(25–35)

ndH2,C

O2,acetate,lactate,

carboh

ydrates,

Sulfate,n

itrate

nd[73]

Desulfomonile

spp.(2

species)

30–30

(37)

6.5–7.8

(6.8–7.0)

H2,C

O2,benzoate,

pyruvate,organic

carbon

,halogens

Sulfate,sulfite,

thiosulfate,sulfur,Fe

(III),Nitrate,U(V

I)Slud

ge[74]

Syntroph

obacteraceae

(8genera)

31–6

07.0

–7.5

H2,C

O2,acetate,

form

ate,lactate,

pyruvate,

Sulfate,sulfite,

thiosulfate

Sewages

ludge,

freshwater,brackish

,marines

edim

ent,

marineh

ydrothermal

vents,ho

tspring

sediments

[73,75]

Desulfobacterium

anilini

306.9–

7.5H2,C

O2,butyrate,

high

erfatty

acids,other

organica

cids,alcoh

ols

Sulfate,sulfite,

thiosulfate

Freshw

ater,B

rackish

water,M

arine,and

Haloalkalineh

abitats

[76]


Table2:Con

tinued.

Microorganism

Temperature

(∘ C)

pHa

Carbon

andele

ctron

source

Electro

nacceptor

Source

Reference

Desulfarculus

baarsii

35–39

7.3H2,C

O2,butyrate,

high

erfatty

acids,other

organica

cids,alcoh

ols

Sulfate,sulfite,

thiosulfate

Freshw

ater,B

rackish

water,M

arine,and

Haloalkalineh

abitats

[76]

Desulfobacteraceae(12

genera)

10–4

0nd

H2,C

O2,L

ong-chain

fatty

acids,Alcoh

ols,

Polara

romatic

compo

unds,and

insome

casese

venAlip

hatic

,arom

atichydrocarbo

ns

Sulfate,sulfite,

thiosulfate

Freshw

ater,B

rackish

water,M

arine,and

Haloalkalineh

abitats

[77]

Desulfosporosinus

acidophilus

25–4

03.6–

5.2(5.2)

H2,lactate,pyruvate,

glycerol,glucose

and

fructose

Sulfate

Sedimentfrom

anacid

effluent

pond

[26]

Desulfosporosinus

acididuran

s15–4

03.8–7.0

(5.5)

H2,formate,lactate,

butyrate,fum

arate,

malate,pyruvate,

glycerol,m

ethano

l,ethano

l,yeastextract,

xylose,glucose,fructose

Ferriciro

n,nitrate,

sulfate,elem

entalsulfur,

thiosulfate

Whiteriv

erdraining

from

theS

oufriere

hills

inMon

serrat(pH3.2)

[78]

a Valuesc

losedby

parenthesis

arec

onsid

ered

optim

alpH

;nd:no

tinformed

byconsultedreference.


such agar.The advantage ofAc. aromatica is its use of a limitedrange of organic donors and that it does not grow on yeastextract, glucose, glycerol, or many other small molecularweight organic compounds that are commonly metabolizedby acidophilic heterotrophic microorganisms. Overlay platesare considered to be more versatile and efficient, particularlyfor isolating acidophilic sulfidogens from environmentalsamples, given that these microorganisms cannot completelymetabolize the substrate [20]. Using this technique, aSRB andnonsulfidogens have been isolated from acidic sulfidogenicbioreactors. Two acidophilic sulfidogens (Desulfosporosinus(D.) acididurans and Peptococcaceae strain CEB3) and strainIR2 were all isolated from a low pH sulfidogenic bioreactorat different stages of operation, previously inoculated withan undefined microbial mat found at abandoned coppermine in Spain [3]. Although not yet fully characterized,Peptococcaceae CEB3 appears to be a more thermotolerantand acidophilic SRB that can oxidize glycerol to CO

2[33].

In addition, D. acididurans grew successfully togetherwith Ac. aromatica in a pH controlled bioreactor, showingan example of microbial syntrophy where this heterotrophicbacterium converted acetic acid into CO

2and H

2[17].

D. acididurans tolerates relatively high concentrations ofaluminum and ferrous iron and can grow in a pH range of3.8–7, with and optimum pH at 5.5. The temperature rangefor growth was 15–40∘C with (optimum pH at 30∘C), andit can use ferric iron nitrate, sulfate, elemental sulfur, andthiosulfate as electron acceptors [78]. D. acidophilus, thesecond acidophilic SRB validly described [26] isolated froma sediment sample collected in a decantation pond receivingacidmine effluent (pH ∼ 3.0), showed high tolerance to NaCl.SRB belonging to the genus Desulfosporosinus are known tothrive in low pH environments together with members ofthe closely related genus Desulfitobacterium which have alsobeen detected in reactors operating at low pH. Interestingly,Desulfitobacterium is a genus with members that can usesulfite as electron acceptor, but not sulfate. Some bacteria,phylogenetically related to sulfur reducers, have been alsodetected in AMD bioreactors as well in natural acidic con-ditions [29].

4. Natural Attenuation for the Design of AMDRemediation Strategies

Natural remediation of metal pollutants generally involvesthe catalytic action of microbial activities that can acceler-ate the precipitation reaction of soluble toxic compoundsresulting in their accumulation in precipitates [81]. Suchinformation fromnatural systems can be useful for the designof engineered systems. Natural attenuation of transitionmetals in AMD has been described, for example, at theCarnoulès mine in France [81] and the Iberian Pyrite Belt(IPB) in Spain [10]. Rowe and colleagues [82] described indetail such process at a small site at the abandonedCantarerascopper mine, which is located in theTharsis, mine district inthe IPB.They reported that SRB other thanDesulfosporosinusspp. were responsible for precipitating copper (as CuS) ina microbial mat found at the bottom layer and dissolvedorganic carbon (DOC) originated from photosynthetic and

chemosynthetic primary producers serving as substrates forthe aSRB. The pH of AMD obtained from this bottomlayer was extremely acidic (pH < 3), and the dark greycoloration was due to the accumulation of copper sulfide,presumably as a result of biosulfidogenesis. No iron sulfides(e.g., hydrotroilite; FeS⋅nH

2O)were detected, presumably due

to the low pH of the mine water even at depth. Because thesolubility product of CuS (log Ksp at 25∘C is −35.9) is muchlower than that of FeS (−18.8), this sulfidemineral precipitatesin acidic waters whereas FeS does not.

Furthermore, Sánchez-Andrea and colleagues [83] describedin detail the importance of sulfidogenic bacteria of the TintoRiver sediments (Spain) and their role in attenuating acidmine drainage as an example of performing natural biore-mediation. The results showed that, for attenuation in layerswhere sulfate reducing genera such as Desulfosporosinusand Desulfurella were abundant, pH was higher and redoxpotential and levels of dissolved metals and iron were lower.They suggested that sulfate reducers and the consequentprecipitation of metals as sulfides biologically drive theattenuation of acid rock drainage. Lastly, the isolation andfurther understanding of anaerobic acidophiles in naturalenvironments such as Cantareras and Rio Tinto have ledto the proposal of new approaches to selectively precipitatetoxic metals from AMD, turning a pollution problem into apotential source of metals [3, 83].

5. Concluding Remarks

Mining companies are increasing the extraction of mineralresources guided by a higher market demand, and also sup-ported by productivity improvement resultant from advanceson prospection and extraction technologies. Increased pro-duction consequently results in a higher generation ofresidues that is a global concern. The mining process hasbeen significantly developed; however, pollution is still one ofthe main challenges of the mining industry and will requireinnovative management tools.

Given the fact that protecting aquatic and terrestrialecosystems from pollutants generated from mine wastes isa major concern, new strategies must be employed such asthe application of robust and empirically design bioreactorsas part of an integrated system for remediation of acidicmine water and metal recovery. Using novel acidophilic andacid-tolerant sulfidogenic microorganisms that are the keycomponents for bioremediation and knowledge about themicrobial interactions that occur in extremely acidic, metal-rich environments will help in the development of newmethods for bioremediation purposes.

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper.

Acknowledgments

The authors acknowledge the financial support by Con-selho Nacional de Desenvolvimento Cient́ıfico e Tecnológico


(CNPq) to José O. Siqueira and Guilherme Oliveira and Valeand the sponsorship of SENAI/SESI Innovation Call. IvanNancucheo is supported by Fondecyt, Chile (no. 11150170).

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Research ArticleEffect of Free Ammonia, Free Nitrous Acid, and Alkalinityon the Partial Nitrification of Pretreated Pig Slurry, Using anAlternating Oxic/Anoxic SBR

Marisol Belmonte,1,2,3 Chia-Fang Hsieh,1 José Luis Campos,4 Lorna Guerrero,5

RamónMéndez,6 Anuska Mosquera-Corral,6 and Gladys Vidal1

1Engineering and Environmental Biotechnology Group, Environmental Science Faculty & Center EULA-Chile,University of Concepción, P.O. Box 160-C, Concepción, Chile2School of Biochemical Engineering, Pontificia Universidad Católica de Valparaı́so, 2362803 Valparaı́so, Chile3Laboratory of Biotechnology, Environment and Engineering, Faculty of Engineering, University of Playa Ancha,2340000 Valparaı́so, Chile4Facultad de Ingenieŕıa y Ciencias, Universidad Adolfo Ibáñez, 2503500 Viña del Mar, Chile5Department of Chemical and Environmental Engineering, University Federico Santa Maŕıa, 2390123 Valparaı́so, Chile6Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela,15782 Santiago de Compostela, Spain

Correspondence should be addressed to Marisol Belmonte; [email protected]

Received 18 May 2017; Accepted 1 August 2017; Published 6 September 2017

Academic Editor: Giuseppe Olivieri

Copyright © 2017 Marisol Belmonte et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The effect of free ammonia (NH3or FA), free nitrous acid (HNO

2or FNA), and total alkalinity (TA) on the performance of a partial

nitrification (PN) sequencing batch reactor (SBR) treating anaerobically pretreated pig slurry was studied. The SBR was operatedunder alternating oxic/anoxic (O/A) conditions and was fed during anoxic phases. This strategy allowed using organic matter topartially remove nitrite (NO

2

−) and nitrate (NO3

−) generated during oxic phases.The desiredNH4

+ toNO2

− ratio of 1.3 gN/gNwasobtained when an Ammonium Loading Rate (ALR) of 0.09 g NH

4

+-N/L⋅dwas applied.The systemwas operated at a solid retentiontime (SRT) of 15–20 d and dissolved oxygen (DO) levels higher than 3mg O

2/L during the whole operational period. PN mainly

occurred caused by the inhibitory effect of FNA on nitrite oxidizing bacteria (NOB). Once HNO2concentration was negligible,

NH4

+ was fully oxidized to NO3

− in spite of the presence of FA.The use of biomass acclimated to ammonium as inoculum avoideda possible effect of FA on NOB activity.

1. Introduction

The intensive swine production is creating scenarios wheregenerated waste is not correctly disposed, exceeding theassimilation capability of the soil-water-plant ecosystem ofthe crop lands [1]. The anaerobic digestion is the most usedtechnology to treat this kind of wastes [2]. In this pro-cess, high removal efficiencies of carbonaceous compoundscontained in the wastewater are achieved while nitrogenremoval is scarce, only due to biomass growth. Since theeffluent from the anaerobic digester has a low C/N ratio, to

perform nitrogen removal by the combination of nitrification(sequential ammonium (NH

4

+) oxidation to nitrite (NO2

−)and nitrate (NO

3

−)) and denitrification (nitrate or nitritereduction to nitrogen gas (N

2)) processes is not economically

feasible due to the requirements of organic matter. Theapplication of the combined partial nitrification (oxidationof ammonium to nitrite with around 50% efficiency) andanammox (combination of previously generated nitrite andammonium to produce nitrogen gas) processes could avoidthis drawback. However, some studies reflected problematicsituations for nitrogen removal in thisway due to the presence


https://doi.org/10.1155/2017/6571671


Aeration

Stirring

Feeding

Settling

Withdrawal

Time (min)

105 5 55 105 5 55 105 5 55 105 5 55 45 15

Figure 1: Distribution of the operational cycle.

of relevant concentrations of residual organic matter in thetreated effluent [3]. In this sense, Wett et al. [4] proposedto treat a municipal wastewater, with a low C/N ratio, ina partial nitrification unit operated in alternated oxic andanoxic periods in order to promote the use of the organicmatter present for denitrification.This strategy together withthe control of the solid retention time (SRT) also allowedsuppressing the growth of nitrite oxidizers when the unitwas operated at low temperature and low ammonium con-centrations and, therefore, improving the stability of partialnitrification. Moreover, as the organic matter was removedby denitrification, alkalinity was generated which partiallycompensated for the alkalinity consumption due to partialnitrification.

During the treatment of wastewater with high ammo-nium concentrations, as the effluent of pig slurry comingfrom the anaerobic digestion, the presence of free ammonia(NH3or FA) and/or free nitrous acid (HNO

2or FNA) can

affect the performance of the partial nitrification process.These compounds can cause inhibition of nitrifying anddenitrifying bacteria and provoke the nitrite accumulationin the system [5, 6]. The nitrifying bacteria are inhibitedat concentrations of FA and FNA within 0.1–150mg NH

3-

N/L and 0.2–2.8mg HNO2-N/L, respectively [5], while the

effect of FNA on denitrifying bacteria was observed within0.01–0.20mgHNO

2-N/L [7]. Another factor to be considered

is the inlet total alkalinity/ammonium ratio (TA/NH4

+-N)since it will determine the pH value inside the reactor and,therefore, the concentrations of FNA and/or FA [8–10].

In the present research the effect of FA, FNA, andtotal alkalinity/ammonium ratio on the performance of apartial nitrification sequencing batch reactor (SBR) operatedunder alternating oxic/anoxic conditions was studied. Ananaerobically pretreated pig slurry and acclimated biomassto high ammonium concentrations were used as feedingand inoculum, respectively. The operational conditions wereadjusted to achieve the desired nitrite to ammonium ratioin the effluent and promote the consumption of the presentorganic matter by means of the denitrification process.

2. Materials and Methods

2.1. Reactor SBR Description and Operational Conditions. Alaboratory scale SBR with a working volume of 1.5 L and

a total volume of 2.5 L was used. Dimensions of the unitwere height of 540mm (𝐻), inner diameter of 77mm (𝐷),and the 𝐻/𝐷 ratio of 7. Oxygen was supplied by means ofa ceramic air diffuser located at the bottom of the reactorconnected to an air pump. The system was equipped witha mechanical stirrer operated at 80 RPM. The reactor wasmaintained in a thermostated chamber at 33 ± 2∘C. ThepH was not controlled and ranged between 6.2 and 8.5. Aprogrammable logic controller (PLC) was used to control thecycle.

The reactor was operated in cycles of 12 h distributed asshown in Figure 1. The volume exchange ratio was fixed at8.3% and the hydraulic retention time (HRT) was of 6 days.The DO was supplied only during the oxic period and itsconcentration was kept higher than 3mg O

2/L. In the anoxic

phase the mixture inside the reactor was achieved throughmechanical stirring.

The reactor was fed with the effluent coming from ananaerobic digester treating diluted pig slurry [2], whosetotal alkalinity/NH

4

+ ratio ranged from 4.0 to 9.4 g/g. Thereactor was operated during 270 days divided into threestages according to the inlet ammonium concentrations of350, 550, and 880mg NH

4

+-N/L, which corresponded toapplying Ammonium Loading Rates (ALRs) of 0.06, 0.09,and 0.15 g NH

4

+-N/L⋅d, respectively (Table 1). The SRT wasnot controlled and ranged from 15 to 20 d during the wholeoperational period.

2.2. Activity Assays. Periodical samples of biomass werecollected from the reactor during the operational stagesto evaluate their specific ammonium and nitrite oxidizingactivities (AOB and NOB, resp.) and specific denitrifyingactivity (SDA). The specific nitrifying activity (ammoniumand nitrite oxidizing) of the biomass was determined byrespirometric assays, applying the methodology described byLópez-Fiuza et al. [11], while themaximum SDA of the sludgewas determined according to the methodology proposed byBuys et al. [12].

2.3. Inoculum. The SBR was inoculated with 5 g volatilesuspended solids (VSS)/L of activated sludge collected froman aerobic reactor, used to remove both organic matterand nitrogen from pig slurry, located in the Region of theLibertador Bernardo O’Higgins, Chile. The initial specific


Table 1: Characterization of the different operational stages of the SBR reactor.

Parameter UnitStage

I II IIIInfluent Effluent Influent Effluent Influent Effluent

Operation time d 0–75 76–190 191–270ALRs g NH

4

+-N/L⋅d 0.06 0.09 0.15Total alkalinity/NH

4

+-N g/g 9.4 ± 0.0 — 7.5 ± 0.0 — 4.1 ± 0.0∗ —pH 7.5 ± 0.1 7.4 ± 1.3 7.5 ± 0.1 6.8 ± 0.9 7.5 ± 0.1 7.2 ± 1.3CODS mg/L 734 ± 85 415 ± 28 801 ± 100 363 ± 189 1907 ± 319 293 ± 58NH4

+-N mg/L 350 ± 26 82 ± 25 550 ± 67 128 ± 77 880 ± 100 102 ± 60NO2

−-N mg/L


Nitr

ogen

conc

entr

atio

n (m

g/L)

Stage I Stage II Stage III

50 100 150 200 250 3000Time (d)

0

200

400

600

800

1000

1200

(a)

Stages I and IIStage III

0

3

6

9

12

15

．（

3-N

(mg/

L)

20 40 60 80 1000

NAR (%)

0.0

0.1

0.2

0.3

0.4

0.5

Stage ）） → ）））

2-N

(mg/

L)H

NO

(b)

Stages I and IIStage III

Stage ）） → ）））

9.4 g/Ａ → 7.57.5 g/Ａ → 4.1 g/gTA/．（4+-N inf: 4.1 g/g

0

2000

4000

6000

8000

10000

Tota

l alk

alin

ity (m

g Ca

C／

3/L

)

20 40 60 80 1000NAR (%)

(c)

Figure 2: Evolution of nitrogen compounds. (a) Behavior of nitrogen concentration inside the reactor: NH4

+-N influent (diamond), NH4

+-Neffluent (square), NO

2

−-N effluent (triangle), and NO3

−-N effluent (circle). (b) Nitrite accumulation ratios (NAR) as percentages obtained atdifferent HNO

2-N (asteris

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