Identification, Detection, and Spatial Resolution of Clostridium Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor

Abstract

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.Landfilling is still one of the most common forms of disposal of organic solid waste worldwide. However, it is becoming increasingly obvious that current waste disposal practices are not sustainable. The development of anaerobic digestion technologies to efficiently convert organic solid wastes, like municipal solid waste (MSW) and agricultural waste, to methane is driven by the need for alternative sources of fuels and the need to mitigate the environmental impacts of landfills, such as uncontrolled greenhouse gas emissions and leachate production (9, 36).The composition of MSW tends to vary depending on climatic, seasonal, and cultural factors, but it is commonly rich in biodegradable material. In general, MSW contains between 40 and 70% cellulosic waste, depending on the factors mentioned above and the level of processing to which the waste is subjected (9, 19, 25). The conversion of cellulosic material to methane is mediated by four microbial populations, including cellulolytic microbes, noncellulolytic saccharolytic microbes, syntrophic hydrogen-producing bacteria, and methanogenic Archaea (4, 9).It is generally accepted that hydrolysis is the slowest and therefore the rate-limiting step in biomethanogenesis of cellulosic material (4,19,25). Therefore, an increase in the rate...

Isolation and Characterization of a New Clostridium sp. That Performs Effective Cellulosic Waste Digestion in a Thermophilic Methanogenic Bioreactor

“…Two primer sets were used for the analysis; one was designed for the general detection of eubacteria, and the other was designed for the specific detection of group III clostridia. Evidence has indicated that group III contains effective cellulose-digesting strains (3,10,17,21). The result revealed a marked transition of the bacterial population during the fermentation period.…”

“…In fact, electron microscopic observation demonstrated that the cells of this organism are frequently distributed inside the cellulose fiber structure. Burrell et al (3) recently reported that the strains of Clostridium group III (strain EBR45 belongs to this group) are often found to be associated with cellulose fibers, while those belonging to Clostridium group XIVa are distributed in the planktonic phase. A successive inoculation method would be effective in enriching the strains belonging to the former group of clostridia.…”

Evidence of cellulose metabolism by the giant panda gut microbiome

“…In Clostridia, we detected 13 OTUs closely related to Clostridium group I (10 OTUs, 1,457 sequences, 26.4% of the total sequences) and XIVa (3 OTUs, 185 sequences, 3.3% of the total sequences) (SI Appendix, Fig. S3), groups that contain organisms known to digest cellulose (15)(16)(17)(18). Moreover, we found that 7 of these 13 OTUs were unique to giant pandas compared with other mammals, including Artiodactyla, Perissodactyla, Rodentia, Diprotodontia, and primates (2, 19-21) (SI Appendix, Table S2).…”

Novel Clostridium populations involved in the anaerobic degradation of Microcystis blooms

“…Furthermore, microorganisms affiliated with Clostridium have been found to be major players in the fermentation of organic substances. The absolute dominance of this genus was detected during the anaerobic fermentation of cellulose in a landfill leachate bioreactor (Burrell et al, 2004) and the Bacterial succession during degradation of Microcystis P Xing et al production of hydrogen from heat-treated activated sludge (Wang X et al, 2007). It is interesting that so abundant Clostridium spp.…”