Microbial community composition and dynamics in a semi-industrial-scale facility operating under the MixAlco™ bioconversion platform.

AIMS: To monitor microbial community dynamics in a semi-industrial-scale lignocellulosic biofuel reactor system and to improve our understanding of the microbial communities involved in the MixAlco™ biomass conversion process. METHODS AND RESULTS: Reactor microbial communities were characterized at six time points over the course of an 80-day, mesophilic, semi-industrial-scale fermentation using community qPCR and 16S rRNA tag-pyrosequencing. We found the communities to be dynamic, bacterially dominated consortia capable of changing quickly in response to reactor conditions. Clostridia- and Bacteroidetes-like organisms dominated the reactor communities, but ultimately the communities established consortia containing complementary functional capacities for the degradation of lignocellulosic materials. Eighteen operational taxonomic units were found to share strong correlations with reactor acid concentration and may represent taxa integral to fermentor performance. CONCLUSIONS: The results of this study indicate that the emergence of complementary functional classes within the fermentor consortia may be a trait that is consistent across scales, and they suggest that there may be flexibility with respect to the specific identities of the organisms involved in the fermentor's degradation and fermentation processes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information regarding the composition, dynamics and potential flexibility of the microbial communities associated with the MixAlco™ process and is likely to inform the improvement of this and other applications that employ mixed microbial communities.

Development of highly digestible animal feed from lignocellulosic biomass Part 2: Oxidative lime pretreatment (OLP) and shock treatment of corn stover.

Oxidative lime pretreatment (OLP) increases lignocellulose digestibility by removing lignin and hemicellulose acetyl content. Digestibility is improved further by adding mechanical shock treatment, which subjects aqueous slurry of biomass to an explosive pressure pulse. Shock treatment mechanically disrupts the microscopic structure while maintaining the macroscopic integrity of the biomass particle. This study determined the effectiveness of these pretreatments to enhance the ruminant digestibility of corn stover. In terms of compositional changes, OLP and shock treatment should negatively affect the feed value of corn stover; however, digestibility analysis provides a significantly different conclusion. With corn stover, shock + OLP improved the 48-h neutral detergent fiber digestibility (NDFD) to 79.0 g neutral detergent fiber (NDF) digested/100 g NDF fed, compared to 49.3 for raw corn stover. The 48-h in vitro total digestible nutrients (TDNom, g nutrients digested/100 g OM) was 51.9 (raw), 59.7 (OLP), and 72.6 (shock + OLP). Adding extracted corn stover solubles to shock + OLP increased TDNom to 74.9. When enough solubilized chicken feathers were added to match the protein content of corn grain, TDNom increases to 75.5, which is only 12.6 less than corn grain.

Development of highly digestible animal feed from lignocellulosic biomass Part 1: Oxidative lime pretreatment (OLP) and ball milling of forage sorghum.

To feed a growing population, alternative sources of animal feed (e.g., lignocellulose) are needed to replace grains (e.g., corn). Oxidative lime pretreatment (OLP) increases lignocellulose digestibility by removing lignin and hemicellulose acetyl content. Adding a mechanical pretreatment (e.g., ball milling) further improves digestibility. This study determines the effectiveness of OLP and ball milling to enhance the ruminant digestibility of lignocellulose. For forage sorghum, the 48-h in vitro TDN were 40, 64, and 84 g nutrients digested/100 g organic matter (OM) for raw, short-term OLP, and short-term OLP + ball milling, respectively. In terms of compositional changes, OLP increases NDF and decreases non-fiber carbohydrate (NFC) and crude protein (CP), all of which would normally be associated with a decrease in digestibility. However, because OLP and ball milling beneficially change composition (lignin removal) and structural features (reduced crystallinity), digestibility actually increases. Although ball milling increases digestibility according to standard laboratory assays, it reduces particle size possibly allowing fine particles to escape from the rumen before they are digested, thus limiting its practical application. Nonetheless, this study indicates that mechanical pretreatment greatly increases digestibility, and therefore it is desirable to identify an effective mechanical treatment that retains fiber integrity.

Fundamental factors affecting biomass enzymatic reactivity.

Poplar wood was treated with peracetic acid, KOH, and ball milling to produce 147 model lignocelluloses with a broad spectrum of lignin contents, acetyl contents, and crystallinity indices (CrIs), respectively. An empirical model was identified that describes the roles of these three properties in enzymatic hydrolysis. Lignin content and CrI have the greatest impact on biomass digestibility, whereas acetyl content has a minor impact. The digestibility of several lime-treated biomass samples agreed with the empirical model. Lime treatment removes all acetyl groups and a moderate amount of lignin and increases CrI slightly; lignin removal is the dominant benefit from lime treatment.

Laboratory method for high-solids countercurrent fermentations.

Equipment and procedures were developed to study the conversion of lignocellulosic biomass to carboxylic acids using high-solids countercurrent fermentations. Countercurrent fermentations of cattle manure yielded a rapid fermentation (maximum 2.98 g of total acid/[L x d]) with high acid concentrations (maximum of 32.5 g of total acid/L), but the acid yield tended to be low (maximum of 0.24 g of total acid/g of volatile solids). Countercurrent fermentations of a mixture of 80% municipal solid waste/20% sewage sludge fermented more slowly (maximum of 1.98 g of total acid/[L x d]) with a lower acid concentration (maximum of 26.5 g of total acids/L), but higher acid yields were achieved (maximum of 0.34 g of total acid/g of volatile solids).

Lime pretreatment of switchgrass.

Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of switchgrass. After studying many conditions, the recommended pretreatment conditions are: time = 2 h, temperature = 100 degrees C and 120 degrees C, lime loading = 0.1 g Ca(OH)(2)/g dry biomass, water loading = 9 mL/g dry biomass. Studies on the effect of particle size indicate that there was little benefit of grinding below 20 mesh; even coarse particles (4-10 mesh) digested well. Using the recommended pretreatment conditions, the 3-d reducing sugar yield was five times that of untreated switchgrass, the 3-d total sugar (glucose + xylose) yield was seven times, the 3-d glucose yield was five times, and the 3-d xylose yield was 21 times. A material balance study showed that little glucan (approx 10%) was solubilized as a result of the lime pretreatment, whereas about 26% of xylan and 29% of lignin became solubilized.

Oxidative lime pretreatment of high-lignin biomass: poplar wood and newspaper.

Lime (Ca[OH]2) and oxygen (O2) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150 degrees C, 6 h, 0.1 g of Ca(OH)2/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of -10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140 degrees C, 3 h, 0.3 g of Ca(OH)2/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO2 carbonation.

Biomass conversion to mixed alcohol fuels using the MixAlco process.

The MixAlco process is a patented technology that converts any biodegradable material (e.g., sorted municipal solid waste, sewage sludge, industrial biosludge, manure, agricultural residues, energy crops) into mixed alcohol fuels containing predominantly 2-propanol, but also higher alcohols up to 7-tridecanol. The feedstock is treated with lime to increase its digestibility. Then, it is fed to a fermentor in which a mixed culture of acid-forming microorganisms produces carboxylic acids. Calcium carbonate is added to the fermentor to neutralize the acids to their corresponding carboxylate salt. The dilute (approximately 3%) carboxylate salts are concentrated to 19% using an amine solvent that selectively extracts water. Drying is completed using multi-effect evaporators. Finally, the dry salts are thermally converted to ketones which subsequently are hydrogenated to alcohols. All the steps in the MixAlco process have been proven at the laboratory scale. A techno-economic model of the process indicates that with the tipping fees available in New York (126 dollars/dry tonne), mixed alcohol fuels may be sold for 0.04 dollars/L (0.16 dollars/gal) with a 60% return on investment (ROI). With the average tipping fee in the United States rates (63 dollars/dry tonne), mixed alcohol fuels may be sold for 0.18 dollars/L (0.69 dollars/gal) with a 15% ROI. In the case of sugarcane bagasse, which may be obtained for about 26 dollars/dry ton, mixed alcohol fuels may be sold for 0.29 dollars/L (1.09 dollars/gal) with a 15% ROI.

Analysis of an algae-based CELSS. Part 2: options and weight analysis.

Life support components are evaluated for application to an idealized closed life support system which includes an algal reactor for food production. Weight-based trade studies are reported as "break-even" time for replacing food stores with a regenerative bioreactor. It is concluded that closure of the life support gases (oxygen recovery) depends on the carbon dioxide reduction chemistry and that an algae-based food production can provide an attractive alternative to re-supply for longer duration missions.

Analysis of an algae-based CELSS. Part 1: model development.

A steady state chemical model and computer program have been developed for a life support system and applied to trade-off studies. The model is based on human demand for food and oxygen determined from crew metabolic needs. The model includes modules for water recycle, waste treatment, CO2 removal and treatment, and food production. The computer program calculates rates of use and material balance for food. O2, the recycle of human waste and trash, H2O, N2, and food production supply. A simple non-iterative solution for the model has been developed using the steady state rate equations for the chemical reactions. The model and program have been used in system sizing and subsystem trade-off studies of a partially closed life support system.

The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar.

The use of alcohol/water/catalyst mixtures to delignify wood allows the lignin to be recovered in a usable form while leaving the carbohydrate fraction relatively intact. The effects of temperature, reaction time, and the type of solvent and catalyst on the delignification of milled poplar wood were investigated. The lignin, cellulose, and hemicellulose composition of the pretreated material was measured for each treatment condition. In addition, the pretreated samples were subjected to enzymatic hydrolysis using the cellulases produced by the thermophilic bacterium Thermomonospora sp. YX. The extent of enzymatic hydrolysis was characterized using an empirical model, and the results were used to examine the effectiveness of the pretreatment.

Parametric analysis of the errors associated with the Michaelis-Menten equation.

Based on the well-known mechanism describing Michaelis-Menten kinetics, three rate expressions may be developed: the exact solution (Model 1), a rate equation resulting from the pseudo-steady-state assumption (Model 2), and Model 2 with the additional assumption that the amount of free substrate is approximately equal to the total amount of substrate (Model 3). Although Model 1 is the most precise, it must be integrated numerically and it requires three experimentally determined parameters. Models 2 and 3, however, are simpler and require only two parameters. Using dimensionless forms of the three models, we have evaluated the errors in the two simplified models relative to the exact solution using a wide range of parameter values. The choice of model for reactor design depends on the initial substrate to enzyme ratio (alpha(0)), and on the ratio of the Michaelis-Menten constant to the enzyme concentration (sigma). Based on a 2% model error criteria, when alpha(0) > 15 or sigma >or= 100, Model 3 is adequate; if 5 < alpha(0) < 15, or if sigma >or= 10, then Model 2 may be used; and if alpha(0) < 5 and sigma < 10, then the exact solution (Model 1) is required.

Oxygen transfer in a pulse bioreactor.

Oxygen transfer in a novel pulse bioreactor has been evaluated. The agitator consists of a series of alternately fixed and movable parallel plates mounted so that the movable plates vibrate at 30 Hz causing a pulsating fluid motion. Pure oxygen, at pressures up to 5 atm, diffuses through silicone rubber tubing that also vibrates at 30 or 60 Hz. The main feature of this bioreactor is high oxygen transfer with low shear to prevent damage to fragile animal cell membranes. We estimate that sufficient oxygen can be supplied to support over 10(8) cells/mL of human diploid foreskin cells growing on microcarriers.

Determining the inhibition constants in the HCH-1 model of cellulose hydrolysis.

The products of cellulose hydrolysis, glucose and cellobiose, caused noncompetitive inhibition patterns to be exhibited when Thermomonospora sp. YX cellulase hydrolyzed dyed cellulose. The glucose binding constant, beta(1), was 0.00683 +/- 0.00031 L/g and the cellobiose binding constant, beta(2), was 0.095 +/- 0.0058 L/g. Thus, cellobiose was about 14 times more inhibitory than glucose.

The HCH-1 model of enzymatic cellulose hydrolysis.

Solka Floc BW200 was enzymatically hydrolyzed in a batch reactor using a commercial cellulase preparation. A total of 50 different hydrolysis conditions were run within a 10-fold range in enzyme concentration and a 30-fold range in cellulose concentration. The data were evaluated in three ways using five different models. Previous literature models were not as successful in correlating the data as the HCH-1 Model derived in this work.

A comparison of two empirical models for the enzymatic hydrolysis of pretreated poplar wood.

Two empirical models for the enzymatic hydrolysis of cellulose are used to analyze the same set of experimental data to determine if one model is superior to the other. Both models adequately describe the data. The parameters in both models may be correlated to the hydrolysis conditions of enzyme, substrate, and product inhibitor concentrations. Both empirical models have features which are consistent with theoretical models of cellulose hydrolysis.

ATP regeneration by thermostable ATP synthase.

We investigated the possibility of using thermostable ATP synthase (TF(0)F(1)) for a new ATP regeneration method. TF(0)F(1) was purified from a thermophilic bacterium, PS3, and reconstituted into liposomes. ATP synthesis experiments showed that TF(0)F(1) liposomes could synthesize ATP in micromole concentrations by acid-base change. The acid-base change was repeated six times over an 11-day period with no detectable loss of activity at the reaction temperature (45 degrees C). Given these encouraging results, we conceptualized and modeled a system to synthesize ATP using ATP synthase with energy supplied by acid-base change. In this system, liposomes containing ATP synthase are immobilized on small glass spheres that facilitate separation of buffers from the liposomes after the acid-base change. Compared to an alternate system that uses membranes to separate the buffers from the liposomes, the glass spheres reduce inefficient mixing of acidic and basic buffers during the acid-base change. To increase the ATP synthesis yield, this system uses electrodialysis to regenerate a potassium gradient after the acid-base change. It also employs water-splitting electrodialysis to regenerate KOH and HCl required to adjust the pH of acidic and basic buffers. All reagents are recycled, so electrical energy is the only required input.

Saccharification, fermentation, and protein recovery from low-temperature AFEX-treated coastal bermudagrass.

Coastal bermudagrass was pretreated by a low-temperature ammonia fiber explosion (AFEX) process, which soaked the grass in liquid ammonia and then explosively released the pressure. Saccharifying enzymes were systematically applied to the AFEX-treated grass corresponding to low, medium, and high loadings of cellulase/hemicellulase (from Trichoderma reesei), cellobiase, glucoamylase, and pectinase. Three-day sugar yields linearly correlated with the logarithm of the cellulase loading. Supplemental enzymes (cellobiase, pectinase) caused upward shifts in the lines. The linearity and upward shifts are consistent with the HCH-1 model of cellulose hydrolysis. The hydrolysis sugars were converted to ethanol using yeast (Saccharomyces cerevisiae). The solid residues were treated with proteases to attempt recovery of valuable proteins. The low-temperature AFEX pretreatment was able o nearly double sugar yields. At the highest cellulase loadings (30 IU/g), the best reducing sugar and ethanol yields were 53% and 44% of the maximum potential, respectively. Protein recovery was, at most, 59%.

Energy requirements for the size reduction of poplar and aspen wood.

The energy requirements associated with conventional mechanical size reduction of poplar and aspen wood are compared to a new method of size reduction employing a wood planer. Although the planer requires about 2.3 times less energy to achieve the same size reduction as conventional methods, large-scale equipment to implement this approach does not currently exist. Explosive depressurization was also compared to conventional mechanical size reduction. The conventional mechanical methods require roughly 70% more energy to achieve the same size reduction as explosive depressurization. Thus, explosive depressurization appears to be the preferred method and has the added benefit of altering the chemical structure of the wood to enhance the enzymatic hydrolysis of the cellulose fraction.