Research On The Microbial Diversity Of Granular Sluge Based On PCR-DGGE

As an excellent biological treatment process which can realize the resourcezation ofwastewater, the anaerobic-aerobic biological treatment process with anaerobic reactorprocessing as the core technique, has received common concern of the domestic andforeign researchers recent years. Because of its high treatment efficiency, and thesubsequent production of biological energy methane, this process has become an importantdevelopment trend in future wastewater treatment field.Within anaerobic reactor represented by UASB, EGSB, IC, whether the anaerobicdigestion can be processed smoothly depends largely on the activity of granular sludge andmicrobial diversity characteristics. Granular sludge was formed through theself-immobilization of different species of microbes; these microbes synergistically carryout the metabolic digestion process of the organic matter in wastewater. A significant shiftin the microbial community would occur when the anaerobic granular sludge weresubjected to different substrate of wastewater in the treating process, which impact thetreating efficiency subsequently.Therefore, dynamic change characteristics of the organisms of granular sludge in thereactor at different operation stages and treating different wastewater substrate was studiedin this paper by PCR-DGGE technique with four same volume UASB reactor as mainsubject. The main research contents and conclusions are as follows:(1) Start-up and stable operation of the reactors: Optimal operating conditions of fourreactors were explored, different industrial wastewaters were processed according to acertain proportion (artificial glucose wastewater to R1and R2, papermaking wastewater toR3, dyeing wastewater to the4th reactor) and reactor operation parameters and sludgeproperties were monitored in this part. The results indicated that: when COD concentrationwas2000mg·L-1, hydraulic retention time22.8h, and inflow was3.16L·d-1, four reactorscan run stably. At the end of start-up, the average volume load of four reactors reached2.5kgCOD·m-3·d-1, with stable COD removal rate of more than95%; when treating practicalindustrial wastewater, no significant change happened to treating efficiency and granularsludge in R3treating papermaking wastewater, the COD removal rate was above90%with0.37gCOD·gVSS-1·d-1of the specific methanogenic activity; while owing to the toxicity ofdyeing wastewater, COD removal rate of R4was only89%in the end, and the specificmethanogenic activity was just less than0.30gCOD·gVSS-1·d-1;(2) Optimization of experimental conditions by PCR-DGGE in studying microbialdiversity of anaerobic granules. Contrast experiments indicated that DNA template concentration, annealing temperature and extension time had significant effects on theresult of PCR. For nest-PCR, PCR template of the second round should be the product ofthe first round after being diluted by50times; the second round amplification was at58°Cfor Archaeal anneal,40s for extension using nest-PCR; no obvious effect was observed bytwo different polymerases; when denaturant gradient of Archaea were40~60%andBacteria30~45%respectively and both were electrophoresed at80V for16h, theidentification of a major number of genera can be gained; from three staining methods,modified silver-nitrate staining was preferred.(3) Under optimal condition, microbial diversity of the anaerobic granular sludge fromthree reactors treating different wastewater was studied by use of PCR-DGGE technique atdifferent stage. The results showed that: there were two group of Bacteria in granularsludge, one group, regulatory bacteria, was responsible for adjusting water quality changeand environment fluctuation; the other group was thought as metabolic function bacteria,which would be enriched and gradually outstood to be advantage strains at stable operationstage of the reactor, assuming the degradation of organic matter and the production ofhydrogen and acetic acid. A complex shift of the microbial structure of Archaea occurred inthe operation process, but no big change of the advantage strains was observed. Thefluctuation of water quality can facilitate the breed of some Archaea species, but after aperiod of time to adapt the Archaea structure in the reactor would gradually restore to itsoriginal levels, namely the structure state of methanogen as advantage strains, thus ensuringa smooth accomplishment of the methane-producing process, the last step of anaerobicdigestion.