http://www.paper.edu.cn 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 tech- niques 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 sul- fate-loading rate of 4.8 g/(L·d), which indicated that the bioreactor started up successfully. The analy- sis 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 de- creased from 1500 to 400 mg/L secondly. This resulted in the sulfate-removal rate, pH value and ef- fluent 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 sul- fate-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 al- kalinity 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 中国科技论文在线 http://www.paper.edu.cn 372 Science in China Series C: Life Sciences 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.