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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
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Bioremediation: An Overview on CurrentPractices, Advances, and New Perspectivesin Environmental Pollution Treatment
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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
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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.
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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
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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
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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
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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].
HindawiBioMed Research InternationalVolume 2017, Article ID 7256582, 17 pageshttps://doi.org/10.1155/2017/7256582
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2 BioMed Research International
(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]).
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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
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4 BioMed Research International
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
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BioMed Research International 5
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
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6 BioMed Research International
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
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BioMed Research International 7
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
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8 BioMed Research International
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
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BioMed Research International 9
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
Sulfate,thiosulfate,
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]
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10 BioMed Research International
Table2:Con
tinued.
Microorganism
Temperature
(∘ C)
pHa
Carbon
andele
ctron
source
Electro
nacceptor
Source
Reference
Thermodesulfobacterium
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]
Thermodesulfobacterium
thermophilum
nd6.0–
8.0(7.0)
H2,C
O2,pyruvate,
lactate
Sulfate,thiosulfate
nd[55]
Thermodesulfobacterium
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]
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BioMed Research International 11
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
Sulfate,thiosulfate,
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]
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12 BioMed Research International
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]
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BioMed Research International 13
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.
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14 BioMed Research International
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
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BioMed Research International 15
(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
HindawiBioMed Research InternationalVolume 2017, Article ID 6571671, 7 pageshttps://doi.org/10.1155/2017/6571671
https://doi.org/10.1155/2017/6571671
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2 BioMed Research International
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
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BioMed Research International 3
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
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4 BioMed Research International
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