Focused-Pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester
Introduction
Methanogenesis is a mature process for the stabilization of municipal and industrial sludge (Rittmann, 2008, Speece, 2008), which is a practical means to capture energy from residual biomass and reduce waste sludge disposal. A drawback today is that the methane yield often is low in anaerobic digesters treating waste activated sludge (WAS). Currently, anaerobic digestion captures about 50% of the incoming organic matter to CH4 on the basis of COD equivalents (Tchobanoglous et al., 2003). One limiting factor for high methane yield is that the organic matter is mostly solid and not readily bioavailable to the methane-producing microbial community (Li and Noike, 1992, Rittmann, 2008, Weemaes and Verstraete, 1998). Various sludge pre-treatment methods – thermal, chemical, biological, ultrasonic, and mechanical – have been attempted for pre-treating biosolids before entering the anaerobic digester (Li and Noike, 1992, Weemaes and Verstraete, 1998). Although many of these methods are effective to make the complex organic matter more bioavailable to microorganisms, none has been put into widespread commercial use due to problems such as high energy cost, high chemical usage, odors, corrosion, and toxicity (Rittmann et al., 2008, Stuckey and McCarty, 1984).
Recently, Focused-Pulsed (FP) sludge pre-treatment was adapted from the technology of pulsed electric fields (PEF) commonly used for disinfecting foods (Rittmann et al., 2008, Salerno et al., 2009, Topfl, 2006). In FP pre-treatment, polar groups, including phospholipids in the periplasmic membrane and peptidoglycan in the cell wall, are exposed to a strong electrical field that is pulsed on and off, creating pore openings in the membranes. When the pores are opened large enough and held open long enough, osmotic pressure can provide enough force to lyse the cell, often destroying the integrity of the cells. In particular, FP treatment makes the cell membrane permeable for releasing soluble components of the cytoplasm and breaks the biomass into small colloids; both effects cause the biomass to be more bioavailable. More detailed descriptions of FP and PEF technology can be found in elsewhere (Rittmann et al., 2008, Topfl, 2006).
The rapid development of molecular techniques to study the structure of microbial communities is making it possible to understand the complex interactions occurring in methanogenic systems and find and test ways to improve the design and operation of engineered systems, such as anaerobic digesters (Chouari et al., 2005, Godon et al., 1997, Raskin et al., 1994, Sekiguchi et al., 1998). However, little is known about how pre-treatment to make input sludge more bioavailable alters microbial community structure in methanogenic digesters. Furthermore, the evaluation of microbial diversity has been limited by the number of sequences that can be obtained by the traditional methods of clone library and Sanger sequencing. The two most comprehensive microbial diversity surveys for anaerobic treatment systems have each sampled no more than 1000 sequences (Chouari et al., 2005, Godon et al., 1997), which were used to infer the diversity for the 109 prokaryotes in 1 g of wet waste sludge (Curtis et al., 2002, Rittmann and McCarty, 2001). These studies reported 133–206 Bacteria and 6–20 Archaea phylotypes, respectively, based on a sequence similarity of 97% as a proxy for a species identity. Assuming a log-normal species distribution, Curtis and co-workers extrapolated experimental clone-abundance data and predicted as many as 9000 Bacteria phylotypes in anaerobic digesters (Curtis et al., 2002). The disparity of the predicted and observed phylotypes suggests that the bacterial community in anaerobic digesters is even more diverse than we previously thought. Rare members that may collectively perform important functions likely have thus escaped detection by Sanger sequencing.
A new technology of sequencing by synthesis, called pyrosequencing, can achieve the much higher number of sequences needed to reveal the diversity of the microbial community at a lower cost than the Sanger method (Sogin et al., 2006). One run of the 454-Roche Genomic Sequencer FLX system can produce 400,000 sequences that are 100–400 bp in length, compared with 192 sequences of 700 bp by one-run of the most up-to-date capillary Sanger sequencer. The cost of pyrosequencing, on a per-base basis, is 30 times less than that of Sanger sequencing (Hugenholtz and Tyson, 2008). Pyrosequencing of PCR amplicons from variable regions of the 16S rRNA gene has recently enabled deep bacterial diversity surveys (1000–18,000 phylotypes) in human intestines (Zhang et al., 2009) and oceans (Sogin et al., 2006). Given its success in these other ecological systems, pyrosequencing will likely produce new insights on the microbial community structure in anaerobic treatment and microbial energy systems.
For the full-scale digesters reported by Rittmann et al. (2008), an average of 53% of the influent sludge was pre-treated by FP in 2008, and this resulted in increased methane production by an average of 31% and decreased biosolids required for disposal of 17%. Thus, FP pre-treatment improved the breakdown of complex organic materials carried out by the microbial community in the anaerobic digester. For a plant treating 380 m3-sludge/day, full FP treatment of 100% of the influent sludge should generate an annual economic benefit of approximately $540,000 net of electricity and other operating and maintenance costs (Rittmann et al., 2008). For the present study, we conducted an in-depth molecular ecological survey on microbial communities in the same full-scale anaerobic digester (Rittmann et al., 2008) whose input sludge was subjected to FP pre-treatment to make the organic solids more bioavailable. Our goals were to reveal the true microbial diversity in the anaerobic digester and to determine if and how FP pre-treatment changed community compositions. We used three complementary methods. For Bacteria and Archaea, we built 16S rRNA gene clone libraries using the traditional Sanger-sequencing method so that we could perform phylogenetic analysis for both domains. We also explored the Bacteria diversity in much greater depth by analyzing 36,797 sequences produced by pyrosequencing. For Archaea and based on our Archaea clone libraries, we also performed quantitative real-time PCR (QPCR) to quantitatively assess the importance of acetoclastic methanogens within the total Archaea.
Section snippets
Methanogenic digester operation
The two full-scale anaerobic digesters at the Northwest Water Reclamation Plant (NWWRP) at the City of Mesa, Arizona, were operated in parallel at an average hydraulic retention time (HRT) of 30–35 days and a temperature of 35–38 °C. Because the digesters were completely mixed and there was no recycle back to the digester, the solids retention time (SRT) was equal to the HRT. The input biosolids were a mixture of primary and waste activated sludge at a ratio of approximately 60:40 by volume. An
Enhanced digester performance by FP treatment
At the time of sampling for the sequencing and the first QPCR analysis after FP startup, FP pre-treatment (defined as a 3-month moving average of the fraction of sludge volume treated) of 39% of the digester incoming sludge resulted in an increase of methane production (over the pre-FP baseline) of about 15% and a decrease of biosolids removed for disposal of about 2%. At the time of the last sample taken for QPCR analysis, FP pre-treatment was 63% of the digester incoming sludge, the methane
Discussion
As a major and sustained perturbation to the digester community, FP pre-treatment made the complex organic matter more bioavailable and increased methane production in the digesters studied here (Rittmann et al., 2008). Parallel laboratory-based tests with waste activated sludge from the same wastewater treatment plant showed that FP treatment could increase methane production by 2-fold (Salerno et al., 2009). The present study provides further confirmation that the addition of more
Conclusions
Prolonged sludge pre-treatment by FP increased methane production and changed the composition of the microbial community in a full-scale anaerobic digester. At the time of our last sampling, methane production increased by 30% with FP pre-treatment of 63% of the input sludge. In parallel, acetoclastic methanogens affiliated with Methanosaetaceae steadily became dominant in the Archaea community. Methanosaeta are known for being excellent scavengers of acetate, and the acetate concentration in
Acknowledgements
We thank Ronald Lopez from the Northwest Water Reclamation Plant at the City of Mesa for assistance in digester effluent analysis, and Elizabeth Edwards at University of Toronto for providing plasmids used for real-time PCR.
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