Assessing Biohydrogen Production Capabilities in Secondary Clarifier and Anaerobic Digester Sludges Using Molecular Techniques

Introduction: Although anaerobic digestion processes are currently optimized for methane (CH4) gas production at most wastewater facilities, hydrogen (H2) gas represents a desirable alternative energy source. Hydrogen-based fuel cells yield high efficiencies of conversion and low pollution emissions [Benneman, 2004]. Since wastewater typically contains nutrients capable of promoting abundant microbial growth, it represents a potential renewable energy source for hydrogen production by bacteria and other microbes present in the domestic waste sludges. To date, only a few studies have examined natural systems such as wastewater at the biochemical and molecular level for the presence of hydrogen-producing microorganisms. To assess the feasibility of biohydrogen production during the acid phase of anaerobic digestion, we quantified major gas evolution, the occurrence and the mRNA transcription of specific bacterial genes involved in H2 gas production in secondary clarifier and anaerobic digester sludges after sugar supplementation.The hydrogenase-4 system (hyfB and hyfG genes) and the Fdh-H system (fdhF gene) associated with Escherichia coli were chosen for examination because this organism is abundant in municipal wastewater and its hydrogen pathways are well described. Additionally, to supplement our understanding of this complex environment, the hydA gene (encoding a hydrogenase) associated with Clostridium acetobutyricum was also quantified via qPCR reactions, and methanogens were identified using 16S rDNA techniques. E. coli, a facultative microorganism, can generate energy by acid fermentation using various glycolytic carbon sources. Formate is produced [Kessler and Knappe, 1996] and can be either excreted or further metabolized to H2 and CO2 by a membrane-associated formate hydrogenlyase (Fhl-1) system composed of formate dehydrogenase H (Fdh-H) and the hydrogenase-3 (Hyc), or by the formate hydrogenlyase (Fhl-2) system that consists of formate dehydrogenase H (Fdh-H) and the hydrogenase-4 complex (Hyf) [Sawers, 1994; Böck and Sawers, 1996; and Gennis and Stewart, 1996].Sludge samples, either secondary clarifier sludge (solid loading rate ∼0.3 lb/ft2·hr, pH 6.3) or anaerobic digester sludge (solid loading rate ∼ 0.08 lb VSS/day/ft3, pH 7.2) were blended, aliquoted into subsamples that received no further pretreatment ("as is" sludge) or were heat treated (boiled or autoclaved). Batch experiments were performed at 30° C in agitated sealed 125-ml serum bottles containing sludge, MES buffer, nutrients, 10 g/L glucose or lactose and N2 gas (anaerobic). Each experiment consisted of the 3 different types of samples prepared in triplicate for each sampling time. Sludge-containing bottles were sampled periodically for: 1) production of total biogas, H2, CO2, and CH4 [Owen et al., 1979], 2) gene copy number of specific E. coli or C. acetobutyricum genes involved in biohydrogen production, and 3) number of viable total bacteria and E. coli. Gas samples were analyzed by GC (TCD detector). H2 gas production was calculated from serum bottle headspace measurements and the total volume of biogas produced for each time interval using the mass balance equation [Logan et al., 2002]. Total nucleic acids (DNA and RNA) were extracted [Yu et al., 1999] and prepared for PCR and qPCR analyses. RNA was prepared by DNAase and RNAase inhibitor treatments and cDNA produced by reverse transcription using the GeneAmp PCR Kit (Applied Biosystems) with recommended reaction mixtures. Microorganisms were enumerated in triplicate on LB agar (total counts), mTEC selective media agar (E. coli counts) plates using serial dilutions of blended sludge samples after anaerobic incubation. Anaerobic digester sludges were serially diluted in thioglycollate medium, incubated and enumerated.Hydrogen production correlated with increases in the number of hydrogen(ly)ase genes, mRNA transcripts of the hyfB, hyfG, and fdhF genes and cell counts (CFU/mL) for "as is" samples in all experiments. Glucose addition produced about 5 times more hydrogen than unsupplemented sludge samples (data not shown). Methane production showed seasonal variance, as reflected by changes in the types of methanogens (data not shown), and increased with the addition of lactose for the unheated samples. Summer or spring samples typically produced more hydrogen than winter sludges. hyfB and hyfG copy numbers reached an asymptote at 12 hr and maximum biohydrogen production was between 12-24 hr. Maximum hydrogen production in anaerobic sludges containing glucose occurred between 24-36 hr and correlated with the hydA gene (hydrogenase operon) copy number of C. acetobutyricum. Net hydrogen production decreased as methane production increased, confirming interspecies hydrogen transfer among sludge microbes. The H2 gas yield was approximately 1.5 L H2 per L of sludge (wet wt). These experiments suggest that anaerobic digestion could be expanded to include an initial acid phase reactor designed to produce hydrogen. Molecular monitoring of pathway genes important in hydrogen or methane production could be used to optimize gas production in reactors. cDNA transcripts of mRNA of hyfG, hyfB and fdhF clearly showed whether E. coli was involved in biohydrogen production.