development of a process for microbial sulfate reduction
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Environmental Pollution 252 (2019) 281e288
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Environmental Pollution
journal homepage: www.elsevier .com/locate/envpol
Development of a process for microbial sulfate reduction in coldmining waters e Cold acclimation of bacterial consortia from anArctic mining district☆
Hanna Virpiranta*, Sanna Taskila, Tiina Leivisk€a, Jaakko R€am€o, Juha TanskanenUniversity of Oulu, Chemical Process Engineering, PO Box 4300, 90014, Oulu, Finland
a r t i c l e i n f o
Article history:Received 21 March 2019Received in revised form10 May 2019Accepted 16 May 2019Available online 22 May 2019
Keywords:Cold acclimationDesulfobulbusMixed culturePostgate mediumSulfate-reducing bacteria
☆ This paper has been recommended for acceptanc* Corresponding author.
E-mail addresses: [email protected] (H. Virp(S. Taskila), [email protected] (T. Leivisk€a), [email protected] (J. Tanskanen).
https://doi.org/10.1016/j.envpol.2019.05.0870269-7491/© 2019 The Authors. Published by Elsevie
a b s t r a c t
Biological sulfate removal is challenging in cold climates due to the slower metabolism of mesophilicbacteria; however, cold conditions also offer the possibility to isolate bacteria that have adapted to lowtemperatures. The present research focused on the cold acclimation and characterization of sulfate-reducing bacterial (SRB) consortia enriched from an Arctic sediment sample from northern Finland.Based on 16S rDNA analysis, the most common sulfate-reducing bacterium in all enriched consortia wasDesulfobulbus, which belongs to the d-Proteobacteria. The majority of the cultivated consortia were ableto reduce sulfate at temperatures as low as 6 �C with succinic acid as a carbon source. The sulfatereduction rates at 6 �C varied from 13 to 42mg/L/d. The cultivation medium used in this research was aPostgate medium supplemented with lactate, ethanol or succinic acid. The obtained consortia were ableto grow with lactate and succinic acid but surprisingly not with ethanol. Enriched SRB consortia areuseful for the biological treatment of sulfate-containing industrial wastewaters in cold conditions.© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Sulfate and metals are the most common pollutants in miningand metallurgical wastewaters (Huisman et al., 2006). Sulfate-containing wastewaters are also produced by the pulp and paper,food, and petroleum industries (Wolicka, 2008; Lens et al., 2003).Sulfate can be removed from water by membrane or ion exchangeprocesses, or by chemical precipitation as gypsum for instance.These methods are efficient but have some disadvantages such asfouling of membranes, the need for pre-precipitation or down-stream treatment, high costs in the case of membrane processes,and production of sludge in large volumes in the case of chemicalprecipitation (Silva et al., 2012). Gypsum precipitation is suitablefor sulfate concentrations of several grams per liter and the ach-ieved residual concentrations are around 1500e2000mg/L(Geldenhuys et al., 2003), whereas limits for sulfate concentrationsin mining effluent discharge in Finland range from 1000 to
e by Joerg Rinklebe.
iranta), [email protected]@oulu.fi (J. R€am€o), juha.
r Ltd. This is an open access article
4000mg/L (AVI, 2013; AVI, 2015; AVI, 2016; AVI, 2017).Sulfate-reducing bacteria (SRB) are anaerobic microbes that use
sulfate as an electron acceptor for the oxidation of organic com-pounds, which results in the production of sulfide (de Matos et al.,2018). Some SRB are able to oxidize organic compounds completelyresulting in the production of CO2; however, some of the bacteriacan perform only partial oxidation of organic compounds whichleads to the production of acetate (Gibson, 1990). According toSulaiman et al. (2008), the most effective biological sulfate reduc-tion by SRB is achieved when the initial sulfate concentration is2500mg/L, but bacterial growth inhibition is clearly detected whenthe sulfate concentration reaches 4000mg/L. Biological sulfatereduction can also be used for much higher sulfate concentrations,but the method is economically more viable with moderate sulfateconcentrations. The achieved residual sulfate concentrations can beeven lower than 100mg/L (Runtti et al., 2018). In the bioremedia-tion process by SRB, the removal of sulfate and organic substancesand the precipitation of base metals occur simultaneously, whichimproves the cost-effectiveness of the method.
SRB can be exploited in biological sulfate removal either as purecultures or as mixed consortia with other bacteria unable to utilizesulfate as an electron acceptor. The use of diverse bacterial con-sortia in the microbial sulfate reduction process has some
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H. Virpiranta et al. / Environmental Pollution 252 (2019) 281e288282
advantages compared to the use of pure cultures, such as a lowerrisk of contamination by other organisms and enhanced adaptationto changes in conditions (Boothman et al., 2006; White et al., 1998).In addition, in mixed consortia the utilization of substrates is morecomprehensive than in single strain cultures, for instance the ace-tate produced by SRB in the partial oxidation of organic compoundscan act as a carbon and energy source for other microorganisms. Ahigh diversity of bacteria also allows for the degradation of a largervariety of organic contaminants (Boothman et al., 2006; White andGadd, 2000).
In boreal areas, the cold climate causes problems in biologicalwastewater treatment related to the slower metabolism andnutrient uptake of bacteria. Microbes are found in a wide varietyof environmental conditions and are able to adapt rather quicklyto environmental changes. The relative content of various bac-terial genera is strongly dependent on the origin of the soil andthe climate. Bacterial consortia used for the treatment of coldwastewaters can be screened from samples collected from Arcticareas where the microbial populations have already adapted tothe cold climate. For example, Knoblauch et al. (1999) have re-ported that the SRB isolated from Arctic marine sediments hadsignificantly higher metabolic rates than the comparable meso-philic strains at low temperatures. SRB are able to tolerate tem-peratures from �5 �C to 75 �C and they adapt easily to changes intemperature (Cocos et al., 2002). However, for the majority ofSRB, the optimal growth temperature is from 28 to 30 �C (Hao,2003) and the metabolic rates of the bacteria are highlyaffected by temperature. It is known that rather than beingactually psychrophilic, the majority of cold-tolerant bacteria aremesophilic strains with the ability to grow also in cold temper-atures, referred to as psychrotolerant bacteria or psychrotrophs(Morita, 1975; Russell, 1990).
The purpose of the present research was to enrich bacterialconsortia with the ability to reduce sulfate efficiently also at coldtemperatures, as the performance of biological sulfate reductioncould be a major concern in boreal areas with a cold climate. Sinceonly limited information on psychrophilic and psychrotolerantnon-marine SRB is available, the aim was also to identify theenriched bacteria and the most important factors affecting theirperformance at cold temperatures. Even though some SRB con-sortia have been successfully cultivated at temperatures as low as4e6 �C (Nielsen et al., 2018; Tsukamoto et al., 2004), in this study,cold acclimation occurred more quickly. In addition, this studyexamined the effect of different carbon sources and temperature onthe composition of microbial communities. Thus, the present studyprovides new and significant information about low-temperatureexperiments and the cold acclimation of SRB consortia.
A sediment sample from wetlands near a mine site operatingin an Arctic environment was used as the SRB source. The soiltype of the sampling site indicates a possibly high content ofhumic substances, which can serve as carbon and nitrogensources for the soil microbiota. The capability of the enriched SRBconsortia to utilize different carbon sources was investigated.Succinic acid and ethanol were chosen as alternative carbonsources since they are economically more viable than sodiumlactate, which is the most commonly utilized carbon source inSRB cultivation. Succinic acid is also one of the main degradationproducts of soil humic acids (Tsutsuki and Kuwatsuka, 1979) andthereby closely related to the likely nutrient sources of the mi-crobial community in the sediment sample. In addition, ethanol iswidely used in commercial applications (Bomberg et al., 2017)and, furthermore, there is a lack of knowledge on succinic acid asa carbon source in SRB cultivations. To the best of our knowledge,this is the first study to test succinic acid in the cultivation of SRBat low temperatures.
2. Material and methods
2.1. Enrichment of sulfate-reducing bacteria
A Postgate medium is commonly used for SRB cultivation(Barbosa et al., 2014; Ismail et al., 2014; de Matos et al., 2018). Themedium used in this study was modified from DSMZ Desulfovibrio(Postgate) Medium (Leibniz-Institut DSMZ GmbH, 2017). A moredetailed composition and preparation of the medium is presentedin the supplementary material.
The mixed cultures used in this study were enriched from asediment sample which was collected in September 2017 from aditch near amine located in the Lapland region of northern Finland.At the time, the average temperature in the area was around 7 �C,while the average annual temperature variation is from �15 to15 �C. Approximately 50mL of the sediment sample was suspendedin 450mL of 0.9% w/v saline solution, which was first sparged withN2 gas to exclude oxygen. When the suspension was settled, ali-quots of 3mL were transferred into six 40mL glass vials containing30mL of the modified Postgate medium with an initial sulfateconcentration of approximately 1.7 g/L. The vials were then sealedwith screw caps with septa and incubated at three different tem-peratures: 6 �C, 16 �C and RT (approximately 22 �C).
Sulfate concentrations were determined using Hach Lange Sul-fate cuvette tests LCK 353 or LCK 153 and a UV/Vis Spectropho-tometer DR 2800. The principle of the cuvette tests is that sulfateions react with barium chloride and form poorly soluble bariumsulfate thereby causing turbidity, which can be measured photo-metrically at 450 nm.
2.2. Sulfate reduction using alternative carbon sources and coldacclimation of the enriched consortium
After one month of incubation of the enrichment cultures at 16and 22 �C, 1mL aliquots were inoculated into 35mL of fresh culturemedia. Four new cultures were inoculated from each vial (6 sam-ples from enrichment), making a total of 24 cultures. The mediawere prepared as described above, but sodium lactate was replacedwith succinic acid or ethanol, both at a concentration of 30mM.Incubation was continued at the same temperature for 10 days.After the 10-day incubation, 1mL aliquots from the succinic acidcultivations were again inoculated into 35mL of fresh culture me-dia and the incubation temperatures were decreased first from 16to 14 �C and from 22 to 20 �C (¼ initial temperatures in cold accli-mation tests). Two new cultures were inoculated from every vialwhere growth was clearly detected, i.e. from all 12 succinic acidcultivations. Samples were collected periodically through the septawith a syringe and needle. When sulfate reduction was observed,the temperatures were again decreased a few degrees at a time,until a temperature of 6 �C was reached for all the cultures. Freshmediumwas fed with a syringe and needle to the culture vials twotimes during the cold acclimation. The points at which the mediumwas added are shown in Fig. 4.
2.3. Identification of the enriched cultures by 16S rDNA sequencingand data analysis
The enriched cultures, with the exception of the cultivationswith ethanol and the cultivations at 6 �C without acclimation, werecharacterized by 16S rDNA sequencing at the Biocenter OuluSequencing Center. All 32 samples for 16S rDNA sequencing anal-ysis were taken at the end of all the parallel cultivations: enrich-ment with lactic acid e 6 samples, cultivation with succinic acid e
12 samples, and cold acclimation e 14 samples from the consortiathat grew at 6 �C. The genomic DNA of these strains was isolated
Fig. 2. Sulfate consumption during 28 days of incubation in Postgate medium withlactate as a carbon source. Symbols: incubation at 16 �C (dot), incubation at 22 �C(square). Averaged values of three parallel cultivations with standard deviations arepresented in both data series.
H. Virpiranta et al. / Environmental Pollution 252 (2019) 281e288 283
from the cell pellets using the standard protocol (QIAGEN, 2017). Aportion of the 16S small sub-unit ribosomal gene was amplifiedwith standard primers F519 (5-CAGCMGCCGCGGTAATWC-3) andR926 (5-CCGTCAATTCCTTTRAGTTT-3) modified by the addition ofbarcodes, namely: the F519 primer contained an Ion Torrentadapter sequence A, a 9-bp unique barcode sequence, and onenucleotide linker whereas the R926 primer contained an IonTorrent adapter trP1 sequence. Polymerase chain reaction (PCR)assays were performed in 25 mL reactions in three replicates, eachcontaining 1� Phusion GC master mix (Thermo Scientific, Espoo,Finland), 0.4 mM of forward and reverse primers and 20 ng ofgenomic DNA as the template. After an initial 3-min denaturation at98 �C, the following cycling conditions were used: 20 cycles of98 �C,10 s; 64 �C, 20 s; 72 �C, 20 s. After PCR amplification reactions,the samples were purified using the AMPure XP reagent (AgencourtBioscience, CA, USA). The amplicon concentration of the purifiedsamples was measured on a Bioanalyzer DNA-1000 chip (AgilentTechnologies, CA, USA) and individual samples were pooled inequivalent amounts. The pooled sample was further purified withAmpure XP and sequencing performed with Ion Torrent PGM on a316 chip using Ion View chemistry (ThermoFisher Scientific, USA).The obtained sequences were compared with those present inGenBank using a BLAST tool (National Center for BiotechnologyInformation, U.S. National Library of Medicine, USA).
3. Results
3.1. Culture enrichment
In the first step of the enrichment, after twoweeks of incubationat 16 and 22 �C, the growth of SRBwas observed by the formation ofblack iron sulfide precipitate in the vials (Fig. 1). However, a cleardecrease in sulfate concentration was detected only after threeweeks of incubation (Fig. 2). After 28 days, the sulfate haddecreased by approximately 500mg/L (30% removal). There was nosignificant difference in sulfate removal between samples incu-bated at 16 and 22 �C. At 6 �C without cold acclimation, sulfateremoval of 500mg/L was attained only after 84 days of incubation(Fig. S1).
3.2. Effect of alternative carbon sources on sulfate reduction
After one week of incubation with alternative, economicallymore viable carbon sources (ethanol and succinic acid), a cleardifference in sulfate reduction rates was detected (Fig. 3). At 16 �C,sulfate concentrations had decreased by approximately 10% with
Fig. 1. Growth of sulfate-reducing bacteria was observed by the formation of a black iron susource. Vials after inoculation in the left photo, and vials after two weeks of incubation at
ethanol as a carbon source and 40% with succinic acid as a carbonsource. At 22 �C, sulfate removals were 20% with ethanol and 65%with succinic acid. The average sulfate reduction rates (SRR) werealso higher with succinic acid (98mg/L/d at 16 �C and 169mg/L/d at22 �C) than in the enrichment step with lactic acid (70mg/L/d at16 �C and 46mg/L/d at 22 �C) as a carbon source at both tempera-tures. Therefore, the cultivations were continuedwith succinic acid.
3.3. Cold acclimation of the enriched sulfate-reducing cultures
Fig. 4 shows the sulfate consumption during cold acclimationwith succinic acid as a carbon source. The sulfate reduction rateswere more repeatable in the cultures with an initial temperature of14 �C (Fig. 4b) than in the cultures with an initial temperature of20 �C, which had more diversity (Fig. 4a). Both test series withdifferent initial temperatures followed a similar trend at thebeginning. The sulfate decreased to a level of about 600mg/L but,after the second substrate addition, the sulfate concentrationincreased in the cultures with an initial temperature of 20 �C due tothe low temperature and slower kinetics. Since the cultures alsocontained other bacteria than SRB, the other reason for the slight
lfide precipitate in the liquid Postgate medium supplemented with lactate as a carbon22 �C in the right photo. Vials 7 to 9 are replicates.
Fig. 3. Sulfate consumption during nine days of incubation with ethanol or succinicacid as a carbon source. Symbols: ethanol at 16 �C (dot), ethanol at 22 �C (square),succinic acid at 16 �C (diamond), succinic acid at 22 �C (triangle). Averaged values of sixparallel cultivations with standard deviations are presented in each data series.
H. Virpiranta et al. / Environmental Pollution 252 (2019) 281e288284
increase in sulfate concentration could be bioleaching of sulfatesfrom the precipitated iron sulfide. However, the sulfate concen-tration continued to decrease with a delay in reaching the previ-ously obtained sulfate level for both test series. The cultures with alower initial temperature reached a somewhat lower sulfate level.At 6 �C, the average SRRs were 22mg/L/d and 31mg/L/d for initialtemperatures of 20 �C and 14 �C, respectively.
Fig. 4. Sulfate consumption during cold acclimation with succinic acid as a carbonsource, a) with an initial temperature of 20 �C, b) with an initial temperature of 14 �C.Averaged values of the parallel cultivations with standard deviations are presented.The series shown in Figure (a) contains the data on eight parallel cultivations andFigure (b) shows data on six parallel cultivations.
3.4. Identification of the enriched cultures
Most of the bacteria in all the experiments belonged to the phylaBacteroidetes, Proteobacteria and Firmicutes (Fig. 5). Some differ-ences in bacterial distribution were detected after the change ofsubstrate and the cold acclimation. At both temperatures (22 and16 �C) after sodium lactate was replaced with succinic acid, theamount of Firmicutes increased and the amount of Proteobacteriaand Bacteroidetes decreased, but during the cold acclimation theamount of Firmicutes decreased again and the amount of Bacter-oidetes increased. Statistically significant changes (according to t-test, p-value< 0.05) were the increase in Bacteroidetes during thecold acclimation from 16 to 6 �C, and the decrease in Proteobacteriaafter the substrate replacement at 22 �C, as well as changes in theamount of Firmicutes except during the cold acclimation from 22 to6 �C.
After the cold acclimation, the majority of the Proteobacteriapresent in the cultures were sulfate reducers. All the SRB found inthe cultivations were members of d-Proteobacteria. The mostcommon sulfate reducer in all cultivations was Desulfobulbus(Fig. 6). Also other members of the family Desulfobulbaceae werepresent. Other genera detected were Desulfovibrio and Desulfo-tignum. The highest amount of sulfate reducers, 19% of all bacteriapresent in the consortium, was detected after cold acclimationwithan initial incubation temperature of 16 �C, which signifies that alower temperature is more favourable to SRB than to the otherbacteria present in the consortia. In particular, the amount ofDesulfobulbus spp. grew during cold acclimation and the increasewas also statistically significant (p-values< 0.05). When comparingthe Desulfobulbus spp. amounts between 22 and 16 �C, it can bestated that the amounts show distinct homogeneity (p-
value> 0.05). With succinic acid as a carbon source, the amount ofDesulfovibrio spp. was significantly higher (p-value< 0.05) at 16 �Cthan at 22 �C as well as after cold acclimation with the initial in-cubation temperature of 16 �C rather than 22 �C as the initialtemperature.
4. Discussion
In the present research, SRB were enriched from an environ-mental sample using a liquid Postgate medium, which is selectivetowards Desulfovibrio species. In addition to lactate, the perfor-mance of succinic acid and of ethanol as carbon sources was alsoinvestigated. The detected diversity of SRB was low in all cultiva-tions and SRB accounted for 6e19% of the bacterial consortia. Theresults are in agreement with Boothman et al. (2006) who detectedonly three different SRB species in their consortium, constituting21% of the consortium. In addition, Bomberg et al. (2017) detected
Fig. 5. Bacterial phylum distribution in the original sediment sample and cultivationswith lactic acid and succinic acid at different temperatures. Averaged values of theparallel cultivations with standard deviations are presented: LA e 3 cultivations atboth temperatures, SA e 6 cultivations at both temperatures, acclimation from 16 to6 �C e 6 cultivations, acclimation from 22 to 6 �C e 8 cultivations. Symbols: LA e lacticacid, SA e succinic acid.
Fig. 6. SRB distribution in the original sediment sample and cultivations with lacticacid and succinic acid at different temperatures. Averaged values of the parallel cul-tivations with standard deviations are presented: LA e 3 cultivations at both tem-peratures, SA e 6 cultivations at both temperatures, acclimation from 16 to 6 �C e 6cultivations, acclimation from 22 to 6 �C e 8 cultivations. Symbols: (F) e bacterialfamily, (G) e bacterial genus, LA e lactic acid, SA e succinic acid.
H. Virpiranta et al. / Environmental Pollution 252 (2019) 281e288 285
four different d-proteobacterial SRB genera in their bioreactor cul-tivations and 1e15% of all bacteria in the consortia were sulfatereducers. Zeng et al. (2018) reported that less than 10% of theirenrichment culture constituted of SRB, and the only SRB genusdetected was Desulfovibrio.
In the present study, the dominating SRB genus was Desulfo-bulbus and the secondmost abundant genus was Desulfovibrio, bothof which are gram-negative mesophiles (Barbosa et al., 2014).Desulfobulbus propionicus and various Desulfovibrio spp. have also
been found to be microaerophilic (Dannenberg et al., 1992).Desulfotignum spp. were detected only in the sediment sample;however, some bacteria belonging to the same Desulfobacteraceaefamily were detected in cultivations conducted at 22 �C. Desulfo-tignum spp. are strictly anaerobic, growing in temperatures rangingfrom 10 to 42 �C (Kuever et al., 2005). Desulfobulbus spp. prevailedover Desulfovibrio spp. probably because they were already presentin larger amounts in the original sediment sample. The enrichmentof Desulfobulbus spp. has also been detected in other studies onsediment samples collected from an urban pond in Brazil (Barbosaet al., 2014) and an area impacted by drainagewater discharge fromtailings in northern Siberia (Karnachuk et al., 2005).
For most SRB the optimal growth temperature is from 28 to32 �C. Optima of 24e28 �C have been observed with some Desul-fobacterium andDesulfobacter strains (Hao, 2003). In addition, somepsychrophilic strains with optima below 20 �C have been foundfrom Arctic marine sediments (Knoblauch et al., 1999). ManyDesulfobulbus strains have an optimal growth temperature ofaround 30 �C, but this study confirmed that they are able to grow attemperatures as low as 6 �C. In comparison, D. aggregans (Kharratet al., 2017), D. mediterraneus (Sass et al., 2002), D. propionicus(Widdel and Pfennig, 1982) and D. rhabdoformis (Lien et al., 1998)are able to grow at a minimum temperature of 10 �C.
Sulfate reductions rates at different temperatures are summa-rized in Table 1. Biological sulfate reduction has been investigatedon reactor scale at a temperature of 9 �C with H2 as the electrondonor (Nevatalo et al., 2010), at 8 �C with ethanol as the electrondonor (Sahinkaya et al., 2007), at 6 �C with ethanol or methanolcombined with spent manure as the electron donor (Tsukamotoet al., 2004) and at 4e8 �C with molasses as carbon source(Nielsen et al., 2018). Low temperature batch experiments havebeen conducted at 15 �C with acetate as an electron donor (Qianet al., 2019a) and at 5 �C with methanol or ethyl glycol as carbonsources (Nielsen et al., 2019). Tsukamoto et al. (2004) acclimatedthe SRB to cold conditions by lowering the temperature from 22 to6 �C for 266 days, whereas in the present research cold acclimationfrom 22 to 6 �C was conducted in 61 days using succinic acid as acarbon source. In addition, in the study made by Nielsen et al.(2019), adaptation to the temperature of 5 �C lasted over 80 daysbefore any significant sulfate reduction occurred. The result wasquite similar compared to the cultivation at 6 �C without coldacclimation in the present study (Fig. S1).
Consequently, sulfate reduction rates also depend on tempera-ture. Sulfate reduction may increase over 3-fold with a 10 �C in-crease in temperature (Nielsen, 1987). The present researchachieved quite similar results, i.e. much lower reduction at lowertemperature, which signifies that the SRB obtained are not psy-chrophilic but more likely psychrotolerant. As a result, there is anoticeable difference in the duration of the lag phase even between16 and 22 �C with succinic acid as a carbon source. At 22 �C sulfatereduction starts faster but the difference in reduction rates seems todecrease in the long term (Figs. 3 and 4). Even though psychrophilicSRB could be more efficient sulfate reducers at low temperatures,there may still be some advantages to the use of psychrotolerantcultures. In boreal areas, the seasonal temperature variation is wideand in summer the metabolism of psychrophilic bacteria might beaffected once again when the temperature increases above 20 �C.The results obtained in cultivations under cold temperaturescannot be directly comparedwith those achieved in other studies athigher temperatures and in reactor experiments (Table 1). In thereported low temperature bioreactor experiments, substrates werefed to the reactors continuously (Nevatalo et al., 2010; Tsukamotoet al., 2004; Sahinkaya et al., 2007; Nielsen et al., 2018), so thatthe SRRs were unaffected by any nutrient limitations in contrast tothe small-scale batch experiments in this study. SRB adapt rather
Table 1Sulfate reduction rates reported for various temperatures and electron donors.
SRB source T (�C) Electron source SRR (mg/L/d) System Reference
Enriched from sediment near mining district 22 Succinic acid, yeast extract 169 Batch This studyEnriched from sediment near mining district 16 Succinic acid, yeast extract 98 Batch This studyEnriched from sediment near mining district 6 Succinic acid, yeast extract 13e42 Batch This studySewage sludge 15 Acetate, glucose, yeast extract 1300 Batch Qian et al. (2019a)Sewage sludge NA Acetate, glucose, yeast extract 2487 SRUSB Qian et al. (2019b)Creek sediment near mining district 5 Methanol 26.7a Batch Nielsen et al. (2019)Creek sediment near mining district 5 Ethylene glycol 4.1a Batch Nielsen et al. (2019)Mine impacted waters 4e8 Molasses 0e22 PBR Nielsen et al. (2018)Psychrotolerant enrichment culture obtained
from cold temperature mining areas9 H2 451e663 MBR Nevatalo et al. (2010)
Psychrotolerant enrichment culture obtainedfrom cold temperature mining areas
8 Ethanol 250b FBR Sahinkaya et al. (2007)
Spent manure 6 Ethanol, spent manure 961e1345 AF Tsukamoto et al. (2004)Spent manure 6 Methanol, spent manure 1057e1441 AF Tsukamoto et al. (2004)AMD affected creek sediment 22 Wood chips, leaf compost,
poultry manure103.7a Batch Cocos et al. (2002)
Creek sediment 21 Compost ND Batch Gibert et al. (2004)Creek sediment 21 Oak leaf 24 Batch Gibert et al. (2004)Creek sediment 21 Sheep manure 24 Batch Gibert et al. (2004)Creek sediment 21 Poultry manure 7 Batch Gibert et al. (2004)Sewage sludge 35 Sewage sludge 184 CSTR Ristow et al. (2018)Isolated from soil and wastewater containing
petroleum residues30 Dairy wastewater 74 Batch Wolicka (2008)
Enriched from soil polluted by oil-derived products 25 Whey, yeast extract 88 Batch Wolicka and Borkowski (2009)Enriched from soil polluted by oil-derived products 25 Whey 40 Batch Wolicka and Borkowski (2009)
AF e anaerobic filter, AMD e acid mine drainage, CSTR e continuous stirred-tank reactor, FBR e fluidized-bed reactor, MBR e (sub-merged) membrane bioreactor, NA e notavailable, ND e not detectable, PBR e packed bed reactor, SRB e sulfate-reducing bacteria, SRR e sulfate reduction rate, SRUSB e sulfate-reducing up-flow sludge bed.
a Average SRR without the lag phase and sulfate-limited phase.b Maximum SRR.
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easily to changes in temperature (Cocos et al., 2002), which wasalso observed in the present study in the cold acclimation step.Bacterial growth was detected each time within a few days of thetemperature changes and, interestingly, all parallel cultures alsoreduced sulfate at a remarkably similar rate even during coldacclimation (Figs. 2e4). This finding shows that cold acclimation isan effective method to adapt SRB to low temperatures.
Ethanol is widely used as a carbon and electron source incommercial applications of biological sulfate reduction and it is arelatively economical option (Bomberg et al., 2017). However, it iseven more economical to use ethanol-containing wastes (Costaet al., 2009; Martins et al., 2009). In addition to ethanol, othereconomically viable substrates suitable for the growth of SRB havebeen investigated, such as acetate (Qian et al., 2019a; Qian et al.,2019b), animal manure and cellulosic materials (Gibert et al.,2004; Cocos et al., 2002), sewage sludge (Ristow et al., 2018), andwhey or dairy wastewater (Wolicka, 2008; Wolicka and Borkowski,2009) (Table 1). Despite all these studies, efficient and cheap carbonsources still require further examination especially for cold-adapted bacteria, since the suitability of the carbon sources formicrobes also depends on the cultivation temperature.
Desulfobulbus is an incomplete oxidizer, which means that it canperform only partial oxidation of organic compounds, resulting inthe production of acetate instead of CO2. According to Sahinkayaet al. (2007), acetate oxidation is usually the limiting factor inoxidation of ethanol by psychrotrophic SRB. This results in theaccumulation of acetate, which also lowers the pH value. As can beobserved from equations (1)e(4), lactate oxidation (Eq. (1)) andsuccinate oxidation (Eq. (3)) produce bicarbonate alkalinity alreadyin the first oxidation step, unlike ethanol oxidation (Eq. (2)). Afterethanol oxidation, acetate should also be oxidized to produce bi-carbonate alkalinity (Eq. (4)).
2CH3CHOHCOO� þ SO2�
4 /2CH3COO� þ 2HCO�
3 þ H2S (1)
2CH3CH2OHþ SO2�4 /2CH3COO
� þ H2Sþ 2H2O (2)
4ðCH2Þ2ðCOO�Þ2 þ3SO2�4 þ 6Hþ/4CH3COO
� þ 4HCO�3 þ 3H2S
þ 4CO2
(3)
CH3COO� þ SO2�
4 þ Hþ/2HCO�3 þ H2S (4)
Higher bicarbonate alkalinity production may be one reasonwhy better sulfate reduction rates were observed in cultivationswith succinic acid as a carbon source thanwith ethanol as a carbonsource (Fig. 3), even though ethanol is known to be suitable forvarious SRB species. Moreover, succinic acid contains more organiccarbon compared to ethanol. In addition, Karnachuk et al. (2005)reported that, in their experiments, Desulfobulbus spp. were themost dominant SRB at 4 �C and pH 7.2 with lactate as a carbonsource as well as at 28 �C and pH 7.2 with ethanol as a carbonsource. However, at pH 7.2, Desulfobulbus cells were not detected at28 �C with lactate or at 4 �C with ethanol as a carbon source.Moreover, at acidic pH values, Desulfobulbus spp. were not detectedat 4 �C with lactate as a carbon source. In the present study, themost likely cause of the enrichment of Desulfobulbus spp. is pro-duction of propionate, which is known to be readily utilized byDesulfobulbus spp. In the cultivation medium lactate, succinate andamino acids provided by yeast extract can be fermented to propi-onate by the other bacteria present (Stams et al., 1998).
All the cultivations in the present study were dominated by thephyla Bacteroidetes, Proteobacteria and Firmicutes (Fig. 5). This isunsurprising since Proteobacteria, Firmicutes, Bacteroidetes, Acti-nobacteria and Acidobacteria are usually widespread in soil and arealmost always present in any type of soil (Lauber et al., 2009). Thelack of Actinobacteria in the enriched cultures is self-evident,considering that most of the bacteria belonging to this phyloge-netic group are aerobic. In addition, the majority of Acidobacteria
H. Virpiranta et al. / Environmental Pollution 252 (2019) 281e288 287
are acidophilic and cultivations in this study were conducted atneutral or slightly alkaline pH.
The majority of the Firmicutes found in sulfate-reducing con-sortia belonged to the class Clostridia e commonly knownfermentation organisms. The most dominant genus within Clos-tridia was Acidaminococcus, which are anaerobic cocci that utilizeamino acids, especially glutamic acid (Rogosa, 1969). Their amountincreased significantly after the change of substrate from lactate tosuccinic acid (Fig. 5). The Postgate mediumwas supplemented with1 g/L yeast extract, which contains glutamic acid, and has probablyenhanced the growth of the Acidaminococcus spp. The genus is alsoable to produce propionate as a fermentation product (Shimizuet al., 2018). Another common family within Clostridia was Pep-tostreptococcaceae, including the genus Proteocatella. For example,P. sphenisci was found to be a psychrotolerant organism able toferment yeast extract (Pikuta et al., 2009). Most of the Bacteroidetespresent in the enriched cultures belonged to the family Porphyr-omonadaceae, and one common genus detected within this familywas Paludibacter. Propionate is one of their major fermentationend-products (Sakamoto, 2014) and most likely further utilized bythe Desulfobulbus spp. Oxidation of ethanol to propionate by Acid-aminococcus spp. or Porphyromonadaceae is very unlikely, whichwould consequently explain why the sulfate reduction was insig-nificant in the case of ethanol as carbon source. Since the enrichedconsortia included rather high amounts of different fermentingbacteria, utilization of more complex substrates by the consortiawould be very likely and will be examined in further studies.
The most common genus within b-Proteobacteria was Dechlor-omonas. According to the literature, D. agitata utilizes acetate,lactate and succinate while reducing chlorate, otherwise sulfideand ferrous iron can act as alternative electron donors for D. agitataand be oxidized to elemental sulfur and ferric iron oxide, respec-tively (Achenbach et al., 2001). Consequently, the change of sub-strate did not have an effect on the amount of Dechloromonas spp.,but the genus was more abundant at 22 �C and the amount reducedsignificantly after cold acclimation. In addition to sulfate reducers,one common genus within d-Proteobacteria was Geobacter. UsuallyGeobacter spp. reduce iron and other metals, even uranium(Anderson et al., 2003), and oxidize organic compounds and con-taminants (Childers et al., 2002). For instance, G. sulfurreducens isknown to oxidize acetate and reduce sulfur and iron (Caccavo et al.,1994). However, Geobacter spp. died out in the cultivations almostcompletely after the substrate change. Interesting genera were alsofound within the phylum Spirochaetes, including Sphaerochaetaspp., which are able to reduce ferrous iron (Ritalahti et al., 2012).They were found first only at 22 �C although the amount increasedafter the substrate change and was still detectable after coldacclimation (Fig. 5). Consequently, the consortia contained a highdiversity of different anaerobic bacteria with specific functions inoxidizing and reducing the compounds of the cultivation media.
5. Conclusions
The enriched sulfate-reducing cultures were able to grow andreduce sulfate at as low as 6 �C with rates varying from 13 to 42mg/L/d. As can be expected, the sulfate reduction rates were not as highas the values reported in continuously operated bioreactor exper-iments at low temperatures, but they give a promising start forfurther investigation. Interestingly, the cultures were not able togrow with ethanol as a carbon source. As an alternative, succinicacid was found to be an effective substrate for the cultivated con-sortia. The dominant SRB genus in all the cultivations was Desul-fobulbus, which, as an incomplete oxidizer, produces acetate as anoxidation product. Acetate can be further utilized by other micro-organisms in the consortia. Further research is required to find
suitable low-cost carbon sources for the sulfate-reducing bacterialconsortia enriched in this research. Use of a low growth tempera-ture, an easily available low-cost carbon source and comprehensiveutilization of substrate by the consortium make it possible todevelop a cost-effective biological method for sulfate reduction.With further development, the enriched consortia could be appli-cable for sulfate management in cold mining waters.
Compliance with ethical standards
Declarations of interest
None.
Ethical approval
This article did not entail any studies using human participantsor animals by any of the authors.
Availability of data and material
The datasets generated and/or analysed during the currentstudy are available from the corresponding author upon reasonablerequest.
Acknowledgements
The study was conducted as part of the Comprehensive SulfateManagement in the Cold Mining Waters (COSUMA) project (grantID 295050), funded by the Academy of Finland. Marko Suokas,M.Sc., at the Biocenter Oulu Sequencing Center is acknowledged forthe 16S rDNA sequencing.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.envpol.2019.05.087.
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