Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions—II: microbial population dynamics
Introduction
The feasibility of codigestion of two or more organic waste streams (e.g., organic fraction of municipal solid waste (OFMSW), sewage sludge or biosolids, animal waste, agricultural solid waste) has been demonstrated at both the laboratory-scale (Poggi-Varaldo and Oleszkiewicz, 1992; Rivard et al., 1990; Saunders et al., 1990; Stenstrom et al., 1983) and the full-scale level (Ahring, 1994; Cecchi et al., 1988). A variety of startup strategies, operating conditions, and reactor configurations were evaluated, but microbial community structure generally was not linked to digester performance. Until recently, such analyses were difficult due to a lack of adequate tools to study microbes directly in their natural habitats. Culture-based methods have been especially difficult to use in anaerobic systems because syntrophic interactions, low growth rates, unknown growth requirements, and obligate anaerobiosis make anaerobic microorganisms difficult to isolate and identify. Molecular tools based on sequence comparison of small-subunit (SSU) ribosomal RNA (rRNA) molecules have made it possible to study complex microbial communities without the need to culture microorganisms, thereby reducing the widely acknowledged biases associated with culturing (Ward et al., 1992). Oligonucleotide probes targeting SSU rRNAs of phylogenetically defined groups of microbes (methanogens, sulfate-reducing bacteria (SRB), fiber digesting microbes) already have been used for the quantification of population abundance in a variety of anaerobic environments (e.g., gastrointestinal environments (Lin et al., 1997; Stahl et al., 1988), sediments (Devereux et al., 1992; MacGregor et al., 1997), and bioreactors (Hansen et al., 1999; Harmsen et al., 1996; Raskin et al., 1995)). We previously used oligonucleotide probe hybridizations to evaluate methanogen population dynamics in anaerobic codigesters (Griffin et al., 1998), and demonstrated how this technology can be used to link microbial community structure and digester performance.
The performance of an anaerobic codigestion system is tied closely to the structure of its microbial community. Stable anaerobic digestion is carried out by representatives of four major metabolic groups: hydrolytic-fermentative bacteria, proton-reducing acetogenic bacteria, hydrogenotrophic methanogens, and aceticlastic methanogens (Zinder et al., 1984). During balanced carbohydrate fermentation, the majority of electrons are channeled through acetate, hydrogen, and formate (Schink, 1992). If these intermediates build up, more reduced organic intermediates (e.g., propionate, butyrate, lactate, ethanol) accumulate, often resulting in a drop in pH. The accumulation of volatile fatty acids (VFA) during anaerobic digester overload is well documented (e.g., Ahring et al., 1995; Harper and Pohland, 1986; Hickey and Switzenbaum, 1991; McCarty et al., 1963; McCarty and Mosey, 1991) and it has been demonstrated that VFA toxicity effects are exacerbated at low pH values (Barredo and Evison, 1991; Fukuzaki et al., 1990). Without adequate levels of populations that can remove hydrogen and other intermediates, VFA continue to accumulate, inhibiting methanogenesis and causing further imbalance. Since the microorganisms responsible for VFA consumption (i.e., proton-reducing acetogens) are very sensitive to the accumulation of their own metabolites (hydrogen, formate, and acetate) (Stams, 1994), the inhibition of methanogenesis by these products causes further VFA build up (Schink, 1997). Therefore, the rapid acidification of an overloaded digester can bring VFA oxidation and methanogenesis to a complete halt, preventing digester recovery (Kaspar and Wuhrmann, 1978; Zinder, 1993). Ironically, recovery can be difficult for digesters with a history of very stable operation, since propionate-degrading organisms are thought to be virtually absent from such systems, due to a lack of steady substrate (propionate) supply (McCarty and Mosey, 1991). A successful recovery from overload requires adequate levels of VFA-degrading microbes to metabolize the surplus intermediates, sufficient quantities of methanogens to consume the hydrogen and acetate produced during VFA oxidation, and environmental conditions which encourage their close association.
The recent phylogenetic characterization of several syntrophic propionate-oxidizing bacteria (SPOB) and saturated fatty acid-β-oxidizing syntrophs (SFAS) resulted in the development of SSU rRNA-based oligonucleotide probes for these organisms (Hansen et al., 1999; Harmsen et al., 1995; McMahon et al., in preparation; Stams, 1994; Zhao et al., 1993). Herein, we present the application of these probes and previously designed probes for methanogens (Raskin et al., 1994; Zheng and Raskin, 2000) to study the population dynamics in mesophilic anaerobic digesters treating OFMSW, primary sludge, and waste activated sludge (WAS). Operating conditions and detailed digester performance data are presented in the accompanying paper (Stroot et al., 2001), in which we report that propionate and acetate accumulated to high levels in continuously mixed digesters that were subjected to aggressive startups and overloading. While acetate was eventually consumed, propionate persisted throughout system operation. When the mixing level was reduced, propionate was degraded and digester operation was stabilized, indicating that adjustment of the mixing level could potentially serve as an effective operational tool for stabilizing unstable digesters. To help explain these observations, we here emphasize the importance of linking digester performance results to microbial population dynamics, with a focus on syntrophic bacteria and their methanogenic partners.
Section snippets
Digester operation and chemical analyses
In Experiment 1, four laboratory-scale digesters were operated in a semi-continuous mode (daily feeding and wasting) at mesophilic conditions (37°C) with an initial target retention time of 20 days, as described in detail by Stroot et al. (2000). One digester was started without an exogenous inoculum (Digester 1), while the three other digesters were seeded with cattle manure and anaerobic digester sludge (Digester 2), cattle manure only (Digester 3), and anaerobic digester sludge only
Microbial community structure in inocula
Table 2 presents the hybridization results for the two inoculum sources used in Experiment 1 (cattle manure and anaerobic digester sludge) and for the anaerobic digester sludge inoculum used in Experiment 2. Anaerobic sludge for Experiments 1 and 2 was obtained from the same sewage sludge digester, but on separate occasions. Methanogens (Archaea) were significantly more abundant in anaerobic sludge than in manure, though their levels in the sludge for Experiment 1 were lower than those in the
Discussion
The methanogenic population dynamics observed in this study support previous hypotheses about the importance of these organisms in anaerobic digestion processes (Griffin et al. , 1998; Novaes, 1986; Raskin et al., 1994; Zinder, 1984). Methanogen (archaeal) abundance increased dramatically as VFA were consumed during digester stabilization following a reduction in the mixing level. The most dramatic increases were observed during propionate turnover in Digesters 2 (Fig. 1E) and 7-MM (Fig. 2C).
Acknowledgements
We are thankful to Fons Stams and David Boone for providing syntrophs; to Kaare Hansen for cloning SSU rDNA from Syntrophomonas wolfeii LYB; to Jim Danalewich, José Barrios-Perez, and David Schumacher for help with digester maintenance and analyses; to Bryan White for access to laboratories; and to Donna Hilton for VFA analyses. This research was supported by the Office of Solid Waste Research (Project no. OSWR-12-013), University of Illinois. K. D. McMahon was supported by a US National
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