Christopher J. Rivard

Anaerobic digestion of lignocellulosic biomass and wastes : cellulases and related enzymes

Anaerobic digestion represents one of several commercially viable processes to convert woody biomass, agricultural wastes, and municipal solid wastes to methane gas, a useful energy source. This process occurs in the absence of oxygen, and is substantially less energy intensive than aerobic biological processes designed for disposal purposes. The anaerobic conversion process is a result of the synergistic effects of various microorganisms, which serve as a consortium. The rate-limiting step of this conversion process has been identified as the hydrolysis of cellulose, the major polymeric component of most biomass and waste feedstocks. Improvements in process economics therefore rely on improving the kinetic and physicochemical characteristics of cellulose degrading enzymes. The most thoroughly studied cellulase enzymes are produced by aerobic fungi, namelyTrichoderma reesei. However, the pH and temperature optima of fungal cellulases make them incompatible for use in anaerobic digestion systems, and the major populations of microorganisms involved in cellulase enzyme production under anaerobic digestion conditions are various bacterial producers. The current state of understanding of the major groups of bacterial cellulase producers is reviewed in this paper. Also addressed in this review are recently developed methods for the assessment of actual cellulase activity levels, reflective of the digester “hydrolytic potential,” using a series of detergent extractive procedures.

Effects of natural polymer acetylation on the anaerobic bioconversion to methane and carbon dioxide

The successful production of novel biodegradable plastic copolymers incorporating both synthetic plastic formulations, such as polystyrene, and naturally occurring biodegradable polymer components, such as cellulose, starch, or xylan, requires stable chemical bonding between these polymers. Modification of the natural polymers through acetylation of the available hydroxyl groups permits the formation of appropriate film-forming plastic copolymers. However, modification of natural polymers has been demonstrated to result in decreased attack by microbial catalysts. For this study, the abundant natural polymers cellulose, starch, and xylan were substituted with acetate to various degrees, and the effect of this modification on the anaerobic biodegradation was assessed using the biochemical methane potential (BMP) protocol. Significant reduction in anaerobic biodegradability resulted with all polymers at substitution levels of between 1.2-1.7. For the xylan acetate series, the trends for anaerobic biodegradation were in good agreement with reduced enzymatic hydyolysis using commercially available xylanase preparations.

Degradation of furfural (2-furaldehyde) to methane and carbon dioxide by an anaerobic consortium.

Furfural, a byproduct formed during the thermal/chemical pre-treatment of hemicellulosic biomass, was degraded to methane and carbon dioxide under anaerobic conditions. The consortium of anaerobic microbes responsible for the degradation was enriched using small continuously stirred tank reactor (CSTR) systems with daily batch feeding of biomass pretreatment liquor and continuous addition of furfural. Although the continuous infusion of furfural was initially inhibitory to the anaerobic CSTR system, adaptation of the consortium occurred rapidly with high rates of furfural addition. Addition rates of 7.35 mg furfural/700-mL reactor/d resulted in biogas productions of 375%, of that produced in control CSTR systems, fed the biomass pretreatment liquor only. The anaerobic CSTR system fed high levels of furfural was stable, with a sludge pH of 7.1 and methane gas composition of 69%, compared to the control CSTR, which had a pH of 7.2 and 77% methane. CSTR systems in which furfural was continuously added resulted in 80% of the theoretically expected biogas. Intermediates in the anaerobic biodegradation of furfural were determined by spike additions in serum-bottle assays using the enriched consortium from the CSTR systems. Furfural was converted to several intermediates, including furfuryl alcohol, furoic acid, and acetic acid, before final conversion to methane and carbon dioxide.

Development of a novel laboratory scale high solids reactor for anaerobic digestion of processed municipal solid wastes for the production of methane

Economic evaluations of the capital costs for anaerobic digestion systems for gas production show that the reactor is a significant cost component. The successful application of high solids digestion of processed MSW (e.g., greater than 10% solids within the digester) would allow a decrease in reactor volume with maintenance of relatively high gas production rates. However, high solids slurries do not mix well in conventional stirred tank reactors. A horizontal shaft, hydraulically driven reactor was designed and fabricated to test the anaerobic digestion of high solids concentrations. Digester performance was evaluated as a function of experimental parameters such as nutrient requirements, feeding rates, pH control, and agitator design/ rotation speed; horsepower of mixing was also evaluated for the reactor. Several startup protocols were examined to obtain a biologically stable anaerobic fermentation at high solids levels.

Measurement of the inhibitory potential and detoxification of biomass pretreatment hydrolysate for ethanol production

The Microtox assay represents a rapid, accurate, and reproducible method for determining general microbial toxicity. This assay was used to evaluate the relative toxicity of a variety of hydrolysate samples derived from dilute-acid and alkaline biomass pretreatment. Toxicity is elicited from biomass degradation products, such as furfural, hydroxymethyl furfural, and acetic acid, generated during pretreatment. Microtox results indicate that the pretreatment samples examined ranged from 9 to 71 toxicity units (TU). Correlations of TU and sample absorbance at several wavelengths were evaluated for all sample series. Sample TU values best agreed with absorbance at 230 nm, but the unsatisfactory fit suggests that absorbance should not be used as an absolute measure of sample toxicity.Microtox data for pretreatment hydrolysate samples were correlated with the inhibition experienced by the ethanologenic yeast,Saccharomyces cerevisiae strain D5A, during the simultaneous saccharification and fermentation (SSF) process of pretreated biomass. None of the alkaline pretreatment conditions produced inhibition during SSF (data not shown). However, the acid pretreatment conditions did produce a wide range of inhibitory and noninhibitory hydrolysates. In general, fermentation was inhibited for acid-pretreated hydrolysate samples with values exceeding 45 TU. Preliminary studies that focused on reducing hydrolysate sample toxicity (detoxification) indicate that adding perlite and zeolite had little effect. However, the use of charcoal, a universal flocculent, or ion-exchange resins significantly reduced sample toxicity, holding promise for the efficient bioconversion of pretreated biomass to ethanol. Moreover, the developed toxicity measurement assay can quickly monitor the quality of the pretreatment process. In this way, biomass conversion operation processes can be reliably controlled at the pilot and commercial scales.

Anaerobic digestion of municipal solid waste : utility of process residues as a soil amendment

Tuna processing wastes (sludges high in fat, oil, and grease [FOG]) and municipal solid waste (MSW) generated on Tutuila Island, American Samoa, represent an ongoing disposal challenge. The biological conversion of the organic fraction of these wastes to useful products, including methane and fertilizer-grade residue, through anaerobic high-solids digestion is currently in scale-up development. The suitability of the anaerobic digestion residues as a soil amendment was evaluated through extensive chemical analysis and greenhouse studies using corn as an indicator crop. Additionally, native Samoan soil was used to evaluate the specific application rates for the compost. Experiments established that anaerobic residues increase crop yields in direct proportion to increases in the application rate. Additionally, nutrient saturation was not demonstrated within the range of application rates evaluated for the Samoan soil. Beyond nutrient supplementation, organic residue amendment to Samoan soil imparts enhanced water- and nutrient-binding capacities.

Pretreatment technology for the beneficial biological reuse of municipal sewage sludges

Modern municipal sewage waste treatment plants use conventional mechanical and biological processes to reclaim waste waters. This process has the overall effect of converting a water pollution problem into a solid waste disposal problem (sludges). The costs for conventional disposal of sewage sludges have risen dramatically because of increased environmental mandates, which restrict their disposal, as well as a dwindling number of landfills. Previously, we determined that secondary bioprocessing (specifically anaerobic digestion) was not effective in reducing the organic content and bulk of the sludge waste (1). Therefore, we have examined the potential of a variety of pretreatment technologies designed to disrupt the macrostructure of the sludge and thereby enhance its subsequent biodegradation. Two thermal/mechanical pretreatments tested were found to have a dramatic effect on the subsequent bioconversion of the microbial sludges. Both technologies evaluated, sonication and shear, were found to be affected by sludge solids levels, duration of treatment, and treatment temperature. Optimum sonication pretreatment occurred with sludge solids of 1% and treatment times of 4–8 min. The most effectivee treatment temperature tested was 55°C. The optimum enhancement in bioconversion potential for the sonication pretreatment was 80–83% of the materials carbon oxygen demand (COD) content. The optimum shear pretreatment occurred with sludge solids of 1–2% and treatment times of 6–10 min. The most effective treatment temperature tested was 87°C. The optimum enhancement in bioconversion potential for the shear pretreatment was 88–90% of the material’s COD content. These data were the basis for US patent no. 5,380,445, granted January 10, 1995.

Horsepower requirements for high-solids anaerobic digestion

Improved organic loading rates for anaerobic bioconversion of cellulosic feedstocks are possible through high-solids processing. Additionally, the reduction in process water for such a system further improves the economics by reducing the overall size of the digestion system. However, mixing of high-solids materials is often viewed as an energy-intensive part of the process. Although the energy demand for high-solids mixing may be minimized by improving the agitator configuration and reducing the mixing speed, relatively little information is available for the actual horsepower requirements of a mechanically mixed high-solids digester system.The effect of sludge total solids content and digester fill level on mixing power requirements was evaluated using a novel NREL laboratory-scale high-solids digester. Trends in horsepower requirements are shown that establish the optimum parameters for minimizing mixing energy requirements, while maintaining adequate solids blending for biological activity. The comparative relationship between laboratory-scale mixing energy estimates and those required for scale-up systems is also established.

Anaerobic bioconversion of municipal solid wastes using a novel high-solids reactor design. Maximum organic loading rate and comparison with low-solids reactor systems.

Novel, laboratory-scale, high-solids reactors operated under mesophilic conditions were used to study the anaerobic fermentation of processed municipal solid waste (MSW) to methane. Product gas rate data were determined for organic loading rates ranging from 2.99–18.46 g of volatile solids (VS) per liter (L) per day (d). The data represent the anaerobic fermentation at high-solids levels within the reactor of 21–32%, while feeding a refuse-derived fuel (RDF)/MSW feedstock supplemented with a vitamin/mineral/nutrient solution. The average biogas yield was 0.59 L biogas/g VS added to the reactor system/d. The average methane composition of the biogas produced was 57.2%. The data indicate a linear relationship of increasing total biogas production with increasing organic loading rate to the process. The maximum organic loading rate obtainable with high-solids anaerobic digestion is in the range of 18–20 g VS/L·d to obtain 80% or greater bioconversion for the RDF/MSW feedstock. This loading rate is approximately four to six times greater than that which can be obtained with comparable low-solids anaerobic bioreactor technology.

Efficacy of hydrolytic enzyme augmentation and thermochemical pretreatments for increased secondary anaerobic digestion of treated municipal sewage sludges

Rising costs for landfill disposal of municipal sewage residues have prompted evaluation of alternative methods for reducing the bulk of the final waste. Representative samples of municipal sewage sludge residues were obtained from three major treatment plants in the United States, including Los Angeles (Hyperion), Denver (North Metro), and Chicago (Stickney). The majority of the treated, dewatered sewage sludge solids was found to be volatile (50–60%) and, presumably, biodegradable. Additionally, much of the volatile content was solubilized by both acid detergent fiber and neutral detergent fiber treatments, and was presumed to be proteineous microbial biomass in nature. Both low- and high-solids anaerobic digester systems, as well as the standard biochemical methane potential (BMP) assay, were utilized to evaluate the anaerobic digestibility of these sewage sludge residues. The low methane yields and, thus, the poor organic waste conversion indicated the need for treatment prior to bioconversion. The effectivenesss of various pretreatments based on assessment of increased soluble protein or organics and anaerobic digestibility as determined by the BMP assay was evaluated.