The Effect of Decreasing Alkalinity on Microbial Community Dynamics in a Sulfate-Reducing Bioreactor As Analyzed by PCR-SSCP

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370 Science in China Series C: Life Sciences 2006 Vol.49 No.4 370—378

DOI: 10.1007/s11427-006-2004-3

The effect of decreasing alkalinity on microbial community dynamics in a sulfate-reducing bioreactor as analyzed by PCR-SSCP

REN Nanqi1, ZHAO Yangguo1, WANG Aijie1, GAO Chongyang2, SHANG Huaixiang1, LIU Yiwei1 & WAN Chunli1

1. School of Environmental and Municipal Engineering, Harbin Institute of Technology, Harbin 150090, China; 2. Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China Correspondence should be addressed to Ren Nanqi (email: [email protected])

Received August 2, 2005; accepted September 2, 2005

Abstract PCR-single-strand conformation polymorphism (SSCP) and Southern blotting techniques were adopted to investigate microbial community dynamics in a sulfate-reducing bioreactor caused by decreasing influent alkalinity. Experimental results indicated that the sulfate-removal rate approached 87% in 25 d under the conditions of influent alkalinity of 4000 mg/L (as CaCO3) and sulfate-loading rate of 4.8 g/(L·d), which indicated that the bioreactor started up successfully. The analysis of microbial community structure in this stage showed that Lactococcus sp., Anaerofilum sp. and Kluyvera sp. were dominant populations. It was found that when influent alkalinity reduced to 1000 mg/L, sulfate-removal rate decreased rapidly to 35% in 3 d. Then influent alkalinity was increased to 3000 mg/L, the sulfate-removal rate rose to 55%. Under these conditions, the populations of Dysgo- nomonas sp., Sporobacte sp., Obesumbacterium sp. and Clostridium sp. got to rich, which predomi- nated in the community together with Lactococcus sp., Anaerofilum sp. and Kluyvera sp. However, when the alkalinity was decreased to 1500 mg/L, the sulfate-removal rate rose to and kept stable at 70% and populations of Dysgonomonas sp., Sporobacter sp. and Obesumbacterium sp. died out, while some strains of Desulfovibrio sp. and Clostridium sp. increased in concentration. In order to determine the minimum alkalinity value that the system could tolerate, the influent alkalinity was decreased from 1500 to 400 mg/L secondly. This resulted in the sulfate-removal rate, pH value and effluent alkalinity dropping quickly. The amount of Petrotoga sp., Prevotella sp., Kluyvera sp. and Neisseria sp. reduced obviously. The result data from Southern blotting indicated that the amount of sulfate-reducing bacteria (SRBs) decreased with influent alkalinity dropping. Analysis of the microbial community structure and diversity showed that the SRBs populations were very abundant in the in- oculated activated sludge and the alkalinity decrease caused the reduction of the populations noted. Most of resident populations in the bioreactor were fermentative acidogenic bacteria (FABs), among which the phylum Firmicute was in the majority, but SRBs were very few. This community structure demonstrates the cooperation between SRBs and FABs, which sustains the system’s high sulfate-removal and operation stability.

Keywords: alkalinity, community dynamics, PCR-single-strand conformation polymorphism (SSCP), www.scichina.com www.springerlink.com 转载中国科技论文在线 http://www.paper.edu.cn

Effect of decreasing alkalinity on microbial community dynamics in a SR bioreactor as analyzed by PCR-SSCP 371

Southern blotting, sulfate-reducing bacterium (SRB), fermentative acidogenic bacterium (FAB).

Alkalinity refers to the substance that can react with reactor (CSTR) was introduced to study treatment ef- acid in an aqueous system, which measures its capac- ficiency using simulated wastewater under different ity to neutralize hydrogen iron (H+). Alkalinity influ- influent alkalinity concentrations. Simulated waste- ences greatly the stability and treatment ability of an- water contained waste sugar beet molasses and sodium aerobic bioreactor. It has been well accepted that an sulfate (COD/sulfate ratio is 5), as well as a small acidogenic phase reactor of two-phase anaerobic amount of nitrogen phosphorus fertilizer, with the final treatment process has advantages for sulfate-laden ratio of C, N and P being 200:5:1. During bioreactor wastewater, and several types of acidogenic sulfate- start-up, the alkalinity was kept around 4000 mg/L by ― reducing processes have been developed earlier[1 3]. adding sodium bicarbonate and the loading rates of Zuo[4] and Wang et al.[5,6] have investigated quantifi- 2− COD and sulfate (SO4 ) were maintained at 24 and 4.8 cation and control strategy of key ecological factors, g/(L·d) respectively, and influent pH was about 6.7. i.e. sulfate to chemical oxygen demand (COD) ratio, When the bioreactor ran stably and reached maximum pH value, oxidation-reduction potential (ORP) and sulfate-removal, the influent alkalinity was decreased alkalinity in acidogenic sulfate-reducing treatments, to 3000 mg/L and other parameters, such as the load- presented control of alkalinity during sulfate reduction, 2− ing rates of COD and SO4 , influent pH value, and so and analyzed the components and conversion of alka- on, were invariable. After the bioreactor ran stably, the linity theoretically. Zhao et al.[7] found that fluctuation influent alkalinity was decreased from 3000 to 1500 of alkalinity remarkably affects the activity of sul- mg/L and other parameters were still invariable. fate-reducing bacteria (SRBs) and sulfate-removal. Until now few reports related to the influence of alka- 1.2 Sampling and extraction of sludge DNA [5 ― 7] linity on sulfate reduction have appeared , and 1.0 mL aliquots of activated sludge was withdrawn none of them covered the influence of alkalinity on with a sterile pipette from the bioreactor at different microbial community structure and function. times of operation, and put into a sterile 1.5 mL cen- Recently, as a culture-independent genetic finger- trifuge tube on ice. The samples were used to extract printing technique, the single-strand conformation total DNA or stored in a −80℃ refrigerator. 0.5 mL of polymorphism (SSCP) has been widely used to ana- activated sludge was used to extract total DNA with a lyze the microbial diversity and community succession bacterial genomic mini extraction kit (Huashun, [8―10] [11,12] in natural ecosystems and in bioreactors . The Shanghai China) according to the manufacturer’s advantage of this technique is that the DNA in the manual. Finally the total DNA was suspended in 50

modified PCR-SSCP profiles is single-strand, which μL of ddH2O containing 2 mmol/L Tris-HCl (pH 8.0). makes later Southern blotting easier. This study fo- And the DNA extraction procedure was repeated once. cuses on investigating the influence of decreasing alkalinity on microbial dynamics and structure in a con- 1.3 SSCP analysis tinuous-flow acidogenic sulfate-reducing bioreactor For SSCP analysis, partial 16S ribosomal RNA using PCR-SSCP and Southern blotting techniques to gene (16S rDNA) fragments were amplified by using provide guidance in its operation. primer SRV3-1: 5′-CGG(C/T)CCAGACTCCTACGG- G-3′, and primer SRV3-2: 5′-TTACCGCGGCTG CT- [8] 1 Materials and methods GGCA-3′, the latter was phosphorylated at the 5′ end . The primers set was used to amplify 16S rDNA from 1.1 Operation of sulfate-reducing bioreactor nucleotide 330 to nucleotide 533 (E. coli numbering), In order to investigate the effect of decreasing alka- including one highly variable region (region V3). Each linity on sulfate-reduction, continuously stirred tank PCR was done by using a total volume of 50 μL in a

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PCR tube. Reaction mixtures contained 1× PCR buffer loading amount. Finally, PCR-SSCP electrophoreto- with Mg2+, deoxynucleoside triphosphate solution gram was collected by a UMAX scanner (model Pow- (200 μmol/L each), primers SRV3-1 and SRV3-2 (0.6 erlook 1000, TX USA). μmol/L each), and 0.125 U of EX Taq DNA poly- 1.4 Southern blotting merase (Takara, Dalian China). The total amount of genomic DNA added to PCR mixtures was approxi- According to a previous study, SRBs group made [15] mately 5 ng. Thermocycling was carried out on 9700 up less than 1% of a bacterial community in the PCR system (ABI) and started with an initial denatu- acidogenic sulfate-reducing bioreactor, which cannot ration for 5 min at 94℃. A total of 30 cycles, each in- be observed by the usual molecular fingerprinting techniques, such as SSCP and DGGE[8,16]. So the cluding 40 s at 94℃, 30 s at 50℃, and 40 s at 72℃, 32P-labeled SRBs-specific oligonucleotide probe was followed by a final primer extension step of 10 SRB385 (5′-CGGCGTCGCTGCGTCAGG-3′[17], cor- min at 72℃. The purity and amount of PCR products responding to positions 385―402 bp in the 16S rRNA were determined by running 3 μL of the reaction mix- gene sequence of E. coli) was used to hybridize with ture on 1% agarose gel and comparing their brightness the SSCP profiles and demonstrate the real status of with the quantitative marker DL2000 (Takara, Dalian SRBs in whole community. China). The polyacrylamide gel was separated from the Lambda-exonuclease can specifically digest the glass plates and the single-stranded DNA in SSCP gel 5′-terminal phosphorylated strand of DNA molecule was transferred onto positively charged nylon mem- and not react with the non-phosphorylated strand[13]. branes (TotalBLOT+, Amresco, OH USA) in an elec- In order to obtain single-stranded DNA from PCR tric field for 10 h with 2 mA/cm2 using the electroblot- products and simplify the SSCP profile, the phos- ting unit (Liuyi, Beijing China) and 1 × TBE buffer. phorylated strand was removed by lambda-exonuc- The membrane was air-dried at room temperature and lease digestion. For the digestion of the phosphory- the DNA was crosslinked to the membranes with UV lated strand, 30 U of lambda-exonuclease (New Eng- light at 266 nm for 5 min. The membrane was prehy- land Biolabs, MA USA) was mixed with 30 μL of the bridized for 1 h at 50℃ with 20 mL of solution con- PCR product in a total volume of 60 μL. The reaction taining 0.5% (w/v) sodium dodecyl sulfate (SDS), 6× mixtures were incubated at 37℃ for 4h. Protein was SSC (1× SSC is 150 mmol/L NaCl plus 15 mmol/L removed by phenol-chloroform extraction, and finally sodium citrate) and 50× Denhardt using Hybaid Shake single-stranded DNA was resuspended in 50 μL of ‘n’ Stack Hybridization Ovens (Thermo Electron, MA 32 ddH2O. USA). 30 pmol of P-labelled oligonucleotide probe The samples were electrophoresed in 12% poly- SRB385 was added into the prehybridization solution acarylamide gel (Sigma, MO USA) plus 10% glycerol and incubated for 12 h at 50℃. After hybridization, (Amresco, OH USA) with 1 × TBE buffer. Gel was the membrane was washed for 30 min in each of the run at 250 V for 11 h in a refrigerator with electropho- following solutions in turn at 50℃: 2×SSC-0.5% (w/v) resis apparatus PowerPac 1000 (Bio-Rad, CA USA). 5 SDS; 2×SSC-0.1% (w/v) SDS; and 1×SSC-0.1% (w/v) μL of denaturing loading buffer (95% formamide, 10 SDS. Subsequently, the membrane was air-dried and mmol/L NaOH, 0.25% bromophenol blue, 0.25% xy- incubated with Kodak film at −80℃ for 7 d. Finally, lene cyanol)[8] was added to 10 μL of sample before the film was developed and then scanned by a UMAX electrophoretic analysis. Then the samples were incu- scanner. bated at 95℃ for 5 min and immediately cooled on ice before being loaded onto the gel. The gel was run 1.5 Diversity analysis of microbial community in according to the above mentioned conditions and then bioreactor silver stained in accordance with the description of Recovery of DNA molecules in the high intensity Bassam et al.[14]. Other repeated parallel samples were and variation of bands was based on the method de- electrophoresed according to the same condition and scribed by Schmalenberger and Tebbe[9]. Reapplica-

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Effect of decreasing alkalinity on microbial community dynamics in a SR bioreactor as analyzed by PCR-SSCP 373

tion of the eluted DNA was performed using the same sulfate-removal and pH. It is obvious that the start-up primers set SRV3-1/SRV3-2 and conditions as previ- of bioreactor lasts about 25 d until the sulfate-removal ously detailed. After being purified using an agarose rate rises steadily to 87%. On d 44, the influent alka- gel recovery kit (Huashun, Shanghai China), the PCR linity was adjusted by mistake from 4000 mg/L to less products were cloned into T-vector (Takara, Dalian than 1000 mg/L (It should have been 3000 mg/L), China). Positive clones were screened out by which resulted in great changes in the sulfate-removal blue-white selection and PCR methods. 5 clones in rate, effluent alkalinity and pH. The sulfate-removal one band were sequenced using M13 universal primers rate dropped from 85% to 35%, the effluent alkalinity by model 3730 sequencer (ABI, CA USA). declined from 6000 to 3200 mg/L, and pH from 7.0 to Sequence classifying online was carried out using less than 6.0 within 3 d. sequence match software in RDP[18]. Then the similar In order to amend the operating error, the influent sequences (>95%) were regarded as one Operating alkalinity was corrected to 3000 mg/L on d 47. Under Taxonomy Unit (OTU), which wasP compared with that this condition, the sulfate-removal rate increased to in GenBank by BLAST software[19]. 55% and effluent alkalinity and pH also increased to 4000 mg/L and 6.2. On d 56, influent alkalinity was 2 Results and discussions secondly decreased to 1500 mg/L. Effluent alkalinity dropped quickly to 3000 mg/L, but the effluent pH 2.1 Effect of decreasing alkalinity on sulfate reduc- was still about 6.2. The sulfate-removal rate fluctuated tion downwards initially, but then increased slowly and Fig. 1 shows the effect of alkalinity adjustment on stabilized at 70%. Fig. 1 also indicates that microbes

Fig. 1. Variations of sulfate-removal rate and pH value subjected to decreasing influent alkalinity. The symbols ▲ under X show the sampling times.

A1―A5 on the top of the figure indicate the five stages with different concentrations of influent alkalinity.

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will accustom themselves to different concentrations nity of the bioreactor was sampled at various times (d of alkalinity within their tolerant threshold, which 0, 25, 44, 55, 65, 75, 85, 91) (Fig. 1) and the results makes the rate of sulfate-removal increase to 70%. are shown in Fig. 2. Then Southern blotting using Decreasing the alkalinity to less than 1000 mg/L SRBs-specific 16S rRNA probe SRB385 with Fig. 2 is had a very obvious effect on sulfate-removal. To de- shown in Fig. 3. As seen in Figs. 2 and 3, decreasing termine its extent, the alkalinity was reduced to 400 alkalinity during sulfate reduction resulted in measur- mg/L on d 87. This resulted in the percent of sulfate able microbial community successions. removed dropping quickly to 45%, the effluent alka- When the microbial community lane on d 25 was linity and pH declined to 2000 mg/L and 6.0 respec- compared with that on d 0, it was found that the later tively within 3 d. time was lower than that of the initial stage and the Fig. 1 also shows that 1000 mg/L of influent alka- community structure was remarkably altered. The ini- linity perhaps is the minimum value of threshold that tial population of organisms disappeared and was re- sustains a high sulfate-removal rate (>50%), which placed by strains that must have been present at levels can be proved by the effect of manually decreasing below detectability initially. Specifically, the popula- alkalinity to less than 1000 mg/L on sulfate-removal tion band u1 dies out, while population bands b1, b2, rate on d 87. At the same time, the decrease in alkalin- u2, u3, u4 and b11 are enriched. The enriched popula- ity leads to a weakening of the buffering capacity of tions are perhaps related to sulfate reduction because system and lowering of the pH. there is a positive correlation between their presence and a high sulfate-reducing rate. However, comparing 2.2 Microbial community dynamics activated by the Fig. 2 and 3, it appears that the enriched populations adjustment of alkalinity might not be SRBs, but fermentative acidogenic bac- The PCR-SSCP analysis of the microbial commu- teria (FABs), which help sulfate metabolism.

Fig. 2. SSCP electrophoretogram of community samples during start-up and alteration of alkalinity in a sulfate-removal bioreactor. M is the simulated community containing 4 strains: A, Bacillus.subtilis (GenBank Accession No. AF548498); B, W-1 (AY434721), isolating from biohydrogen reactor[20]; C, Desulfovibrio desulfuricans (DQ092636); and D, Escherichia coli (AF076037). b1―b12 indicate the bands cloned and sequenced, while u1―u7 are the bands not cloned.

Fig. 3. Southern blotting of the isotope 32P-labeled probe SRB385 hybridizing with SSCP profiles (Fig. 2). M shows the biomarker; C is Desulfovi- brio desulfuricans (GenBank Accession No. DQ092636).

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Effect of decreasing alkalinity on microbial community dynamics in a SR bioreactor as analyzed by PCR-SSCP 375

The sulfate-removal rate had approached 87% when that were recovered. Five transformants were selected bioreactor came into the stable status. However, at the from each band to determine their sequences. Totally same time the microbial community structure is shift- 60 of 200 bp-long clone sequences were obtained. The ing as seen by comparing the community lane on d 44 sequences were classified by sequence match software with that on d 25. On d 44, population band u3 dies in RDP which indicated these sequences belong to 5 out and b8 appears and is enriched. phyla, i.e. Firmicutes, 58.33%; Actinobacteria, 8.33%; The microbial community lane on d 55 demon- Proteobacteria, 16.67%; Bacteroidetes, 15.00%; strates a community structure, in which the alkalinity Thermotogae, 1.67%. drops to less than 1000 mg/L and then recovers to 29 different OTUs were obtained by comparing 60 3000 mg/L, and the sulfate-removal rate reaches 55%. clones with sequencher 5.0 software, in which the In this community lane, population bands b3, b5 and minimum match percentage was established by the b10 are enriched and predominate, but the quantity of software at 95% and a minimum overlap was 100 bp. population band b12 is declining. Hybridization indi- The clones’ diversity can be substituted for the micro- cated that the quantity of SRBs in this stage is too bial community structure because almost all the main small to quantitate. In addition, the sulfate-removal PCR-SSCP bands have been sequenced. One band rate is at its lowest (only 54%) at all sampling points. sometimes contains more than one OTU, which is So, there is an obvious positive correlation between perhaps caused by similar conformations of different the quantity of SRBs and sulfate-removal. sequences in non-denaturing gel[9]. Muyzur has re- The community lane on d 65 is a reflection of the ported that this phenomenon cannot be avoided using alkalinity that decreased to 1500 mg/L with the biore- the DGGE technique[21]. actor operating stably. The population bands b5 and The result of comparing the 29 OTUs with that in b10 die out and u5, u6, b6 and b7 are enriched. Under GenBank is shown in Table 1. According to Fig. 2 and the same alkalinity concentration, two other commu- Table 1, most population bands (b1, b2, b3, b8, b9, nity SSCP profiles are obtained, i.e. the communities b11 and b12), which have only a small quantitative on d 75 and 85. The community lane on d 75 has al- change when the bioreactor starts-up and alkalinity is most no difference with that on d 65, but on d 85, altered, are resident populations. These resident popu- population bands b4, b6, u3 and b8 gradually become lations are similar to some species of the following dominant. genera: Lactococcus sp., Bifidobacterium sp., Obe- On d 91 the decrease in alkalinity has an effect on sumbacterium sp., Prevotella sp., Anaerofilum sp., microbial community biomass. As shown the intensity Clostridium sp., Ruminococcus sp., Aquaspirillum sp., of population bands b4, b11 and b12 declines and the Citrobacter sp., Kluyvera sp., Streptococcus sp. and hybridization indicates the SRBs biomass also drops at Neisseria sp. All of these genera are anaerobes and can this time. live in many different anaerobic environments. And Results from Figs. 1 and 2 also indicate that some some of them can ferment many substrates to produce populations are almost not influenced by alkalinity [22,23] alterations, such as the population bands b1, b2, b3, b8, acid . b9, b11 and b12. These populations are present from There is also Desulfovibrio sp. except for Clostrid- start-up (d 25) successful to its operational end (d 91) ium sp. in the population band b6. Desulfovibrio sp. is and so are called resident populations. Hybridization the genus directly correlated with sulfate metabolism. demonstrates that these populations are not SRBs, but From the hybridization as shown in Fig. 3, there is they are required for sulfate metabolism in this system. only this group of SRBs. And the SRBs abundance is Furthermore, the pH they can stand is very wide, and very high on d 0; simplification of SRBs is caused by their quantities fluctuate little when the pH drops from provision of simple substrates. Figs. 1 and 3 indicate 7.0 to 6.0. that the quantity and activity of this group are the final determining factor for high sulfate-removal. When 2.3 Microbial community diversity analysis sulfate-removal is low on d 55 and 91, the corre- Fig. 2 shows partial PCR-SSCP bands (b1―b12) sponding SRBs bands are quantitatively too small to

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be measured. sugar beet molasses is used as a carbon resource[3]. Population bands b5 and b10 are similar to genera FABs contain many different metabolic types of bac- Dysgonomonas sp., Citrobacter sp. and Clostridium teria which are responsible for some steps when mo- sp., Dysgonomonas sp. is an anaerobic gram-negative lasses is broken down. In this system, all populations coccobacillus and can ferment many kinds of carbo- except for population band b6 belong to FABs. FABs hydrate. When metabolizing glucose, they produce are in charge of metabolizing and converting starch, only acid, but no carbon dioxide and hydrogen gas[24]. sucrose, glucose, and fructose into acetic acid, ethanol, These two population bands appear when the sul- methanol, formic acid and hydrogen, which can be fate-removal rate is lowest, so they perhaps have no metabolized by SRBs easily. They play a very impor- direct relation with sulfate metabolism. tant role in providing substrate for SRBs, buffering The microbes in this bioreactor can be divided into acid accumulation and sustaining predominant me- two groups according to their function during sulfate tabolism conditions[3]. The proportion of SRBs is very reduction, i.e. FABs and SRBs. The two groups estab- small. However, they can transfer the electrons to lish chain-like metabolic relationship when the waste 2− 2− SO4 and reduce SO4 to sulfide.

Table 1 Retrieve results of 29 OTUs by BLAST OTU Band No. Accession No. Most similar sequence GenBank No. Similarity Most similar Genus 1 b1-1,2,3,4,5 DQ088176 Uncultured bacterium clone 3 AY652853 99% Lactococcus 2 b2-2,3,5,6 DQ088177 Uncultured bacterium clone 3 AY652853 99% Lactococcus 3 b2-4 DQ088178 Uncultured bacterium clone 3 AY652853 99% Lactococcus 4 b3-1 DQ088179 Bifidobacterium psychroaerophilum AY174108 99% Bifidobacterium 5 b3-2 DQ088180 Obesumbacterium proteus AY077753 95% Obesumbacterium 6 b3-3 DQ088181 Uncultured bacterium clone nc-12c AY368603 98% Prevotella 7 b3-4,6 DQ088182 Uncultured bacterium clone 3 AY652853 99% Lactococcus 8 b4-1 DQ088183 Petrotoga olearia AJ311703 86% Petrotoga 9 b4-2,4,5,6 DQ088184 Prevotella sp. oral clone AA020 Y005057 95% Prevotella Uncultured Bacteroidetes bacterium clone 10 b5-1,3,4,5 DQ088185 AY582893 99% Dysgonomonas GMA-H46 11 b5-2 DQ088186 Uncultured bacterium clone 7-2 AY883107 98% Sporobacter 12 b6-1 DQ088187 Desulfovibrio desulfuricans AF098671 98% Desulfovibrio 13 b6-2,3,4,5 DQ088188 Clostridium sp. 44a-T5zd AY082483 99% Clostridium 14 b7-1,2,3,4,5 DQ088189 Uncultured bacterium clone B3-6-3 AY675996 99% Anaerofilum 15 b8-1,5 DQ088190 Clostridium sp. 44a-T5zd AY082483 99% Clostridium 16 b8-2 DQ088191 Ruminococcus sp. 25F7 AJ515917 95% Ruminococcus 17 b8-3 DQ088192 Bifidobacterium psychroaerophilum AY174108 99% Bifidobacterium 18 b8-4 DQ088193 Uncultured bacterium clone 24 AY652859 98% Bifidobacterium 19 b9-2,5,6 DQ088194 Bifidobacterium psychroaerophilum AY174108 99% Bifidobacterium 20 b9-7 DQ088195 Aquaspirillum peregrinum subsp. Integrum AB074521 97% Aquaspirillum 21 b9-8 DQ088196 Citrobacter sp. HPC784 AY838373 97% Citrobacter 22 b10-2,5 DQ088197 Uncultured bacterium clone 7-2 AY883107 99% Sporobacter. 23 b10-3 DQ088198 Obesumbacterium proteus AY077753 96% Obesumbacterium 24 b10-4,6 DQ088199 Clostridium longisporum X76164 99% Clostridium 25 b11-1,4,5,6 DQ088200 Kluyvera sp. Tf 250 AY461697 99% Kluyvera 26 b11-7 DQ088201 Uncultured bacterium clone 5-1 AY883103 99% Anaerofilum 27 b12-1,3 DQ088202 uncultured bacterium AJ576427 100% Streptococcus 28 b12-2 DQ088203 Bacterium N40 AB182202 92% Neisseria 29 b12-6,7 DQ088204 Uncultured bacterium clone DONOR_90 AY343317 97% Anaerofilum

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Effect of decreasing alkalinity on microbial community dynamics in a SR bioreactor as analyzed by PCR-SSCP 377

3 Conclusions 2 Mizuno O, Noike T. The behavior of sulfate-reducing bacteria in acid-producing phase of anaerobic digestion. Water Res, 1998, Alkalinity is a key parameter in sulfate-laden 32(5): 1626―1634 wastewater treatment bioreactor. It could affect the 3 Wang A J, Ren N Q, Liu W, Huang Z, Zhen W D. The role of sul- microbial activity markedly and further impact the fate reducing bacteria population in acidogenic-desulfate bioreac- sulfate-removal rate. Sulfate-removal rate is lower tor. China Environ Sci (in Chinese), 2001, 21(2): 119―123 than 50% when influent alkalinity is less than 1000 4 Zuo J E. Study on new processes of high sulfate organic wastewater biotreatment (in Chinese). Dissertation for the Doctoral mg/L. The authors presume that there exists a mini- Degree. Beijing: Tsinghua University, 1996 mum threshold value of alkalinity in acidogenic sul- 5 Wang A J, Ren N Q, Zhen W D, Zhang Y. Study on the quantify- fate-reducing bioreactor. ing of restrictive ecological factors in acidogenic desulfate biore- Microbial community analysis by the PCR-SSCP actor. Acta Sci Circumst (in Chinese), 2002, 22(2): 177―182 technique demonstrates that microbial community 6 Wang A J, Ren N Q, Lin M, Du D Z. Stabilization and control of succession takes place in the acidogenic sulfate-re- alkalinity during sulfate-reduction. J Harbin Inst Technol (in Chi- nese), 2003, 35(6): 651―654 ducing bioreactor along with decreasing alkalinity. 7 Zhao Y H, Ye Y F, Liu X D. Sulfate-reducing bacteria and their Dysgonomonas sp., Sporobacte sp., Obesumbacterium impacted factors. Environ Pollut Contr (in Chinese), 1997, 19(5): sp. and Clostridium sp. increase in amount when alka- 1041―1043 linity is decreased from 4000 to 3000 mg/L. When 8 Lee D H, Zo Y G, Kim S J. Nonradioactive method to study ge- alkalinity was decreased to 1500 mg/L, Dysgonomo- netic profiles of natural bacterial communities by PCR-single- strand-conformation polymorphism. Appl Environ Microbiol, nas sp., Sporobacte sp., Obesumbacterium sp. die out, 1996, 62(9): 3112―3120 some strains of Desulfovibrio sp. and Clostridium sp. 9 Schmalenberger A, Tebbe C C. Bacterial diversity in maize appear and are enriched. During the course of alkalin- rhizospheres: Conclusions on the use of genetic profiles based on ity decrease, the amount of resident populations of PCR-amplified partial small subunit rRNA genes in ecological Lactococcus sp., Bifidobacterium sp., Obesumbacte- studies. Mol Ecol, 2003, 12(1): 251―262 rium sp., Prevotella sp., Anaerofilum sp., Clostridium 10 Sliwinski M K, Goodman R M. Spatial heterogeneity of crenar- chaeal assemblages within mesophilic soil ecosystems as revealed sp., Ruminococcus sp., Aquaspirillum sp., Citrobacter by PCR-single-stranded conformation polymorphism profiling. sp., Kluyvera sp., Streptococcus sp. and Neisseria sp. Appl Environ Microbiol, 2004, 70(3): 1811―1820 has no remarkable shift. 11 Delbès C, Moletta R, Godon J-J. Bacterial and archaeal 16S The 60 sequences originating from SSCP main rDNA and 16S rRNA dynamics during an acetate crisis in an an- bands belong to 5 different phyla, i.e. Firmicutes, aerobic digestor ecosystem. FEMS Microbiol Ecol, 2001, 35(1): 16―26 58.33%; Actinobacteria, 8.33%; Proteobacteria, 12 Zumstein E, Moletta R, Godon J-J. Examination of two years of 16.67%; Bacteroidetes, 15.00%; Thermotogae, 1.67%. community dynamics in an anaerobic bioreactor using fluores- And 29 different OTUs have been obtained by com- cence polymerase chain reaction (PCR) single-strand conforma- paring the sequences. Only one OTU correlates di- tion polymorphism analysis. Environ Microbiol, 2000, 2(1): 69― rectly with sulfate reduction and others all correlate 78 with FABs. SRBs populations are very abundant in 13 Subramanian K, Rutvisuttinunt W, Scott W, Myers R S. The en- zymatic basis of processivity in λ exonuclease. Nucleic Acids Res, seed sludge, but their diversity tends to decrease 2003,31(6): 1585―1596 caused by provision of simple substrates. 14 Bassam B J, Caetano-Anolles G, Gresshoff P M. Fast and sensi- tive silver staining of DNA in polyacrylamide gels. Anal Biochem, Acknowledgements This work was supported by the Na- 1991, 196(1): 80―83 tional Natural Science Foundation of China (Grant No. 15 Zhao Y G, Ren N Q, Wang A J Liu G M, Zhao S Q, Shang X X. 50208006), and National 863 Plan of the Ministry of Sci- Microbial community structure in sulfate-reducing reactor ana- ence and Technology of China (Grant No. 2002AA001036). lyzed by single-strand conformation polymorphism technique. 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