Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

The effect of biomass density on cellulose solubilisation rates

The aim of this work was to compare the impact of inoculation density on the rate of cellulose hydrolysis by a rumen derived culture with that of a microbial enrichment from an organic waste anaerobic digester. The results showed a linear relationship between the mass of biomass at the start of the first order degradation phase (Xo) and the first order hydrolysis rate (r) for both rumen inoculated and leachate inoculated cellulose digestions and that the slopes of these relationships were not distinguishable. This suggested that differences in the microbial community, media and other environmental factors had a lesser impact on the hydrolysis rate compared to the effect of the number of cells in the system. This could be of great importance to industrial applications of anaerobic digestion technologies as it suggested that if cells densities in the waste treatment digesters could be boosted to match those seen in the rumen, then the rates of the cellulose hydrolysis would rise.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

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

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.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Production of H2 from cellulose by rumen microorganisms: effects of inocula pre-treatment and enzymatic hydrolysis

H₂ production from cellulose, using rumen fluid as the inoculum, has been investigated in batch experiments. Methanogenic archaea were inhibited by acid pre-treatment, which also inhibited cellulolytic microorganisms, and in consequence, the conversion of cellulose to H₂. Positive results were observed only with the addition of cellulase. H₂ yields were 18.5 and 9.6 mmol H₂ g cellulose^?1 for reactors with 2 and 4 g cellulose l^?1 and cellulase, respectively. H₂ was primarily generated by the butyric acid pathway and this was followed by formation of acetic acid, ethanol and n-butanol. In reactors using 4 g cellulose l^?1 and cellulase, the accumulation of alcohols negatively affected the H₂ yield, which changed the fermentation pathways to solventogenesis. PCR–DGGE analysis showed changes in the microbial communities. The phylogenetic affiliations of the bands of DGGE were 99 % similar to Clostridium sp.     

Effect of feeding yeast culture (Yea-sacc1026) on rumen fermentation in vitro and production performance in crossbred dairy cows

The effect of supplementing diet with yeast culture (Yea-sacc¹⁰²⁶) on dairy cattle was studied by rumen in vitro studies and a feeding trial. Using rumen inoculum from a cow, yeast adapted (YA) and yeast unadapted (YU), incubations were carried out with finger millet (Eleusine coracana) straw (FMS) and a commercial cattle feed (CCF). The 24 h cumulative gas production and digestibility of neutral detergent fibre and acid detergent fibre were not different with YU and YA rumen inoculum for both FMS and CCF. Microbial nitrogen synthesis (mg N ml⁻¹ gas produced) with rumen inoculum from YA feeding regime was higher (P < 0.01) than YU with starch (0.1068 vs. 0.1008) and cellulose (0.0900 vs. 0.0859).A feeding trial was conducted using twelve multiparous cows in midlactation in a switch over design. The cows were divided into two groups of six cows in each group. The duration of the trial was fourteen weeks, each period lasting for 7 weeks. There were no differences in dry matter intake, body weight gain and milk yield. Milk composition for the two groups were also similar.     

Autohydrolysis pretreatment of Arundo donax: a comparison between microwave-assisted batch and fast heating rate flow-through reaction systems

Background

Autohydrolysis of lignocellulosic biomass in liquid hot water has been widely studied owing to its high efficiency and relatively low cost. In the perspective of industrial applications, continuous or semi-continuous processes are more interesting than batch systems. Moreover, microwave heating of pretreatment systems has been proposed to intensify the kinetics of the process. In this study, the autohydrolysis of Arundo donax was performed in pure liquid hot water using a microwave-heated batch reactor and a semi-continuous flow-through reaction system with fast heating rate at the same operating conditions with the aim of performing a systematic comparison between the two different experimental apparatuses.

Results

The effect of process temperature and time, biomass to water mass to volume ratio and water flow rate on the concentration and yield of hydrolysis products was investigated. The flow-through set-up allowed us to reach biomass solubilization up to 44.5 wt% on dry basis, while the batch system stopped at 34.5 wt% suggesting that the mass transfer could be the rate-determining step in the solubilization of the constituting biopolymers. For example, in the flow-through layout, using a flow rate of 3.5 mL/min at 200 °C with 20 min of processing time, quantitative recovery of hemicellulose was obtained with limited formation of degradation products. Interestingly, higher cellulose/hemicellulose extraction ratios were found using the microwave-assisted batch reactor. FTIR analyses of the solid residues recovered after the pretreatment offered independent information on the fractions of liquefied biopolymers complementary to those derived from HPLC and UV–Vis spectroscopy.

Conclusions

Collected experimental results indicated that the flow-through system can be adopted to obtain complete solubilization of the hemicellulose fraction of Arundo donax addressing the product distribution in soluble compounds towards fermentable sugars with limited formation of sugar degradation products and with limited penalty in terms of dilution of the hydrolysate solution. It was also found that microwaves can promote cellulose depolymerization and solubilization, thus allowing a more comprehensive utilization of the biomass and that infrared spectroscopy can be a useful technique to estimate the effect of the pretreatment.

     

Non-linear dynamics of carbon and hydrogen isotopic signatures based on a biological kinetic model of nitrite-dependent methane oxidation by “Candidatus Methylomirabilis oxyfera”

System dynamics of nitrite-dependent anaerobic methane oxidation (N-DAMO) in a “Candidatus Methylomirabilis oxyfera” culture are described using a mathematical model based on chemical kinetics, microbial growth dynamics and equations for ¹³C and ²H isotopic fractionation. Experimental data for the N-DAMO model were taken from Rasigraf et al. (2012), who studied N-DAMO in a batch culture of “Ca. M. oxyfera” started at two different conditions with varying methane, nitrite and biomass concentrations. In the model, instead of using concentrations of each isotopologue (¹²C and ¹³C, ¹H and ²H), total concentrations and respective isotope ratios were considered as variables. The empirical Monod equations, which included methane and nitrite as two rate-limiting substrates, a threshold methane concentration CH _4min below which there was no biomass growth, and the same kinetic coefficients for the separate batch experiments, fitted the experimental data much better than apparent first-order kinetics that required rather different kinetic coefficients for the two experiments. Non-linear dynamics of ¹³C and ²H isotopic signatures were obtained based on the N-DAMO model. It was shown that rate limitation by methane or nitrite concentrations significantly affected the dynamics of carbon and hydrogen isotopic signatures. Fractionation rate increased at higher initial biomass concentration. The non-linear N-DAMO model satisfactorily described experimental data presented in the two-dimensional plot of hydrogen versus carbon stable isotopic signatures.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis

Population dynamics was studied in a 52-l biotrickling filter (BTF) operated for 182 days and used to clean air contaminated with styrene vapors. In the BTF, biomass grew either as free-floating (planktonic) or attached (sessile) microorganisms. PCR-amplified 16S rDNA fragments from planktonic and sessile cells within the bioreactor were analyzed using denaturing gradient gel electrophoresis (DGGE). The results indicated that the complexity of biofilm community was always more pronounced than the complexity of the planktonic cell community. Notably, Rhodococcus erythropolis was identified, based on DNA sequence analysis, as one of the biofilm-specific strains. It was also shown that the inoculum, even when enriched with styrene-degrading bacteria, was not adapted to the growth conditions imposed by the BTF. After a 35-day microbial acclimation period, the DGGE analysis also showed less variation in the banding pattern representing the microbial complexity of the biofilm. In addition, the phylogenic fingerprinting method used demonstrated similar banding profiles in the biofilm along the filter bed. Electronic Publication     

The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover,Cellulase Structure,and Adsorption

Enzymatic hydrolysis of lignocellulosic biomass is limited by rapid cellulase deactivation, consequently requiring large amounts of enzyme to maintain acceptable biomass conversion. In this study, a new approach to improve lignocellulose hydrolysis was investigated. Performing enzymatic hydrolysis of corn stover (CS) in the presence of polymeric–surfactant micelles (PMs) was demonstrated to improve hydrolysis yield to a greater extent than using only surfactant micelles. Application of 2 % (w/w) of polyethylene glycol (PEG 6000) with casein, Tween-20, and Triton X-100 at levels above the critical micelle concentrations increased the hydrolysis yield of CS containing high-bound lignin (extrusion-pretreated) by up to 87.8, 11.7, and 7.5 %, respectively. These PMs were not effective during enzymatic hydrolysis of biomass lacking lignin (Avicel) or alkali-pretreated CS (7.2 % lignin). The main reasons for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween, and PEG-Triton were enhanced cellulase solubilization; reformation of α-helix substructure; and combination of induced cellulase solubilization, α-helix reformation, and chemical changes in the microstructure of biomass, respectively. Deformation of the cellulase substructure during hydrolysis of biomass and its subsequent reformation in the presence of surfactants were shown in this study for the first time. Chemical changes in the microstructure of biomass (e.g., lignin side changes, C–O bonds, and amorphous cellulose) were found to be another potential reason for the effectiveness of surfactants when they are incubated at above 6 g/L for 72 h with biomass.     

Efficacy of a quaternary ammonium compound against planktonic and sessile populations of differentLegionella pneumophila strains

Efficacy of Gemacide PN-50^TM (a quaternary ammonium compound) as a commercial formulation recommended for disinfecting heat exchangers was determined for both planktonic and sessile populations of variousLegionella pneumophila strains. The quaternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. PlanktonicL. pneumophila strains were suspended in tap water while sessile ones were grown on stainless steel that is used in construction of the cooling towers, then both group of strains were exposed to the biocide. The sensitivity of both planktonic and sessile populations ofL. pneumophila strains to the biocide was different. The biocide was found effective below recommended dosages (1000–2000 mg/L) against planktonic populations ofL. pneumophila, whereas it was determined that higher than the recommended dosages were required for sessile populations. The environmental isolates were more resistant to the biocide than the ATCC isolate was. The results indicated that studying only the planktonic populations ofL. pneumophila for biocide tests might not be sufficient to provide the optimum dosage and contact time information for field trials. Therefore, biocidal activity of a water treatment chemical must be evaluated in terms of dosage and contact times on both planktonic and sessile bacteria.     

Cellulose hydrolysis by a methanogenic culture enriched from landfill waste in a semi-continuous reactor

This study investigates the hydrolysis of cellulose by a mixed culture enriched from landfill waste in a continuous reactor operated under prolonged residence times to accommodate methanogenic conditions. Chemostat studies of hydrolysis under balance methanogenic conditions are rarely reported, despite the importance of hydrolysis under these conditions in waste management and renewable energy industries. Continuous digestion was studied in a 1.25l digester, fed with a 1% (w/v) slurry of 50mum cellulose in sterilized leachate drawn from a 220l digester operated on a feedstock of mixed municipal solid waste. Unsterilized leachate was used as the inoculum. Stable and rapid hydrolytic conditions were established at residence time of 2.5, 3.5 and 5d with a 1st order hydrolysis rate 0.45+/-0.07d(-1) and high methane yields ranging from 57% to 62% of solubilised cellulose on a COD basis. Biomass yields were between 32% and 35% of solubilised cellulose on a COD basis, over three times that observed with fermentative cultures. This is attributed to the diversity of the microbial population which fully converted solubilised COD to methane, as evident by VFA yields of less than 8% on a COD basis.     

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor

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.     

A pore‐hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass

Until now, most efforts to improve monosaccharide production from biomass through pretreatment and enzymatic hydrolysis have used empirical optimization rather than employing a rational design process guided by a theory‐based modeling framework. For such an approach to be successful a modeling framework that captures the key mechanisms governing the relationship between pretreatment and enzymatic hydrolysis must be developed. In this study, we propose a pore‐hindered diffusion and kinetic model for enzymatic hydrolysis of biomass. When compared to data available in the literature, this model accurately predicts the well‐known dependence of initial cellulose hydrolysis rates on surface area available to a cellulase‐size molecule. Modeling results suggest that, for particles smaller than 5 × 10^?3 cm, a key rate‐limiting step is the exposure of previously unexposed cellulose occurring after cellulose on the surface has hydrolyzed, rather than binding or diffusion. However, for larger particles, according to the model, diffusion plays a more significant role. Therefore, the proposed model can be used to design experiments that produce results that are either affected or unaffected by diffusion. Finally, by using pore size distribution data to predict the biomass fraction that is accessible to degradation, this model can be used to predict cellulose hydrolysis with time using only pore size distribution and initial composition data. Biotechnol. Bioeng. 2013; 110: 127–136. © 2012 Wiley Periodicals, Inc.     

Catalysis with CuII(bpy) improves alkaline hydrogen peroxide pretreatment

Copper(II) 2,2′‐bipyridine (Cu^II(bpy))‐catalyzed alkaline hydrogen peroxide (AHP) pretreatment was performed on three biomass feedstocks including alkali pre‐extracted switchgrass, silver birch, and a hybrid poplar cultivar. This catalytic approach was found to improve the subsequent enzymatic hydrolysis of plant cell wall polysaccharides to monosaccharides for all biomass types at alkaline pH relative to uncatalyzed pretreatment. The hybrid poplar exhibited the most significant improvement in enzymatic hydrolysis with monomeric sugar release and conversions more than doubling from 30% to 61% glucan conversion, while lignin solubilization was increased from 36.6% to 50.2% and hemicellulose solubilization was increased from 14.9% to 32.7%. It was found that Cu^II(bpy)‐catalyzed AHP pretreatment of cellulose resulted in significantly more depolymerization than uncatalyzed AHP pretreatment (78.4% vs. 49.4% decrease in estimated degree of polymerization) and that carboxyl content the cellulose was significantly increased as well (fivefold increase vs. twofold increase). Together, these results indicate that Cu^II(bpy)‐catalyzed AHP pretreatment represents a promising route to biomass deconstruction for bioenergy applications. Biotechnol. Bioeng. 2013; 110: 1078–1086. © 2012 Wiley Periodicals, Inc.     

Functional and Structural Responses of a Degradative Microbial Community to Substrates with Varying Degrees of Complexity in Chemical Structure

Abstract The objective of the present study was to determine whether cultivation of a degradative community on substrates with varying degrees of chlorination and complexity in chemical structure, as well as cultivation in batch and flow cell culture, would alter the community's functional capability. The community was isolated from oil-contaminated soil and maintained in the laboratory on 2,4,6-trichlorobenzoic acid for 5 months before its ability to grow on 15 different chemicals as sole carbon source was evaluated in batch and flow cell systems. While the community could grow and develop biofilms in flow cells on all the substrates, only 11 of the 15 substrates could support growth in batch culture. Although biofilm development was less extensive on chemicals such as pentachlorophenol (2.09% average area covered by biofilm; average biofilm depth = 3 μm) than on 2,4,6-trichlorobenzoic acid (50.84% area covered; biofilm depth = 6.4 μm), no correlation was observed between the degree of chlorination, or number of rings, and the number of planktonic cells or biofilm biomass. In contrast, physicochemical characteristics such as the octanol/water partition coefficient had a significant effect on the development of biofilm biomass. In the case of planktonic communities, the degree of chlorination and ring number also had no effect on the BIOLOG carbon utilization profiles of the resulting communities. Although the sessile communities generally clustered separately from their planktonic counterparts, principal component analysis of carbon utilization profiles of the sessile communities showed different grouping between growth on chlorinated and nonchlorinated substrates. Analysis of the degradative community maintained on 2,4,6-trichlorobenzoic acid over an extended period further showed that adaptation to a new chemical environment is a rather slow process, since the substrate utilization profiles did not stabilize even after 12 months. These results demonstrate the flexibility in metabolic ability and community structure found in microbial communities. Received: 30 November 1998; Accepted: 19 May 1999     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.