Evaluating the performance of carboxylate platform fermentations across diverse inocula originating as sediments from extreme environments.

To test the hypothesis that microbial communities from saline and thermal sediment environments are pre-adapted to exhibit superior fermentation performances, 501 saline and thermal samples were collected from a wide geographic range. Each sediment sample was screened as inoculum in a 30-day batch fermentation. Using multivariate statistics, the capacity of each community was assessed to determine its ability to degrade a cellulosic substrate and produce carboxylic acids in the context of the inoculum sediment chemistry. Conductance of soils was positively associated with production of particular acids, but negatively associated with conversion efficiency. In situ sediment temperature and conversion efficiency were consistently positively related. Because inoculum characteristics influence carboxylate platform productivity, optimization of the inoculum is an important and realistic goal.

Comparison of three screening methods to select mixed-microbial inoculum for mixed-acid fermentations.

Using a mixed culture of microorganisms, the carboxylate platform converts biomass into hydrocarbons and chemicals. To develop a method that identifies the highest performing inoculum for carboxylate fermentations, five bacterial communities were screened and ranked by three fermentation performance tests: (1) 30-day batch screen, (2) 28-day continuum particle distribution model (CPDM), and (3) 5-month continuous countercurrent fermentation trains. To screen numerous inocula sources, these tests were used sequentially in an aseptic environment. For the batch-fermentation screen, Inoculum 1 achieved the highest conversion. For the CPDM evaluation, the operating map for Inoculum 1 had the highest performance. For the continuous countercurrent fermentation, the train resulting from Inoculum 1 was among the best performers. This study suggests that the three screens are a useful and predictive method for choosing optimal inocula sources. The bacterial community with optimal performance in these three screens could be considered for use in commercial-scale fermentations.

Mesophilic and thermophilic conditions select for unique but highly parallel microbial communities to perform carboxylate platform biomass conversion.

The carboxylate platform is a flexible, cost-effective means of converting lignocellulosic materials into chemicals and liquid fuels. Although the platform's chemistry and engineering are well studied, relatively little is known about the mixed microbial communities underlying its conversion processes. In this study, we examined the metagenomes of two actively fermenting platform communities incubated under contrasting temperature conditions (mesophilic 40°C; thermophilic 55 °C), but utilizing the same inoculum and lignocellulosic feedstock. Community composition segregated by temperature. The thermophilic community harbored genes affiliated with Clostridia, Bacilli, and a Thermoanaerobacterium sp, whereas the mesophilic community metagenome was composed of genes affiliated with other Clostridia and Bacilli, Bacteriodia, γ-Proteobacteria, and Actinobacteria. Although both communities were able to metabolize cellulosic materials and shared many core functions, significant differences were detected with respect to the abundances of multiple Pfams, COGs, and enzyme families. The mesophilic metagenome was enriched in genes related to the degradation of arabinose and other hemicellulose-derived oligosaccharides, and the production of valerate and caproate. In contrast, the thermophilic community was enriched in genes related to the uptake of cellobiose and the transfer of genetic material. Functions assigned to taxonomic bins indicated that multiple community members at either temperature had the potential to degrade cellulose, cellobiose, or xylose and produce acetate, ethanol, and propionate. The results of this study suggest that both metabolic flexibility and functional redundancy contribute to the platform's ability to process lignocellulosic substrates and are likely to provide a degree of stability to the platform's fermentation processes.

Comparison of mixed-acid fermentations inoculated with six different mixed cultures.

The MixAlco™ process biologically converts biomass to carboxylate salts that may be converted to a variety of chemicals and fuels. This study examines the fermentation performance of six different mixed cultures, and how the performance was affected by the bacterial composition of each community. All six countercurrent fermentations had very similar performance, but were dissimilar in microbial community composition. The acid concentrations varied by only 12% between fermentation trains and the conversions varied only by 6%. The microbial communities were profiled using 16S rRNA tag-pyrosequencing, which revealed the presence of dynamic communities that were dominated by bacteria resembling Clostridia, but they shared few taxa in common. Yue-Clayton similarity calculations of the communities revealed that they were extremely different. The presence of different but functionally similar microbial communities in this study suggests that it is the operating parameters that determine the fermentation end-products.

Propagated fixed-bed mixed-acid fermentation: Part I: Effect of volatile solid loading rate and agitation at high pH.

Countercurrent fermentation is a high performing process design for mixed-acid fermentation. However, there are high operating costs associated with moving solids, which is an integral component of this configuration. This study investigated the effect of volatile solid loading rate (VSLR) and agitation in propagated fixed-bed fermentation, a configuration which may be more commercially viable. To evaluate the role of agitation on fixed-bed configuration performance, continuous mixing was compared with periodic mixing. VSLR was also varied and not found to affect acid yields. However, increased VSLR and liquid retention time did result in higher conversions, productivity, acid concentrations, but lower selectivities. Agitation was demonstrated to be important for this fermentor configuration, the periodically-mixed fermentation had the lowest conversion and yields. Operating at a high pH (∼9) contributed to the high selectivity to acetic acid, which might be industrially desirable but at the cost of lower yield compared to a neutral pH.

Thermodynamic prediction of hydrogen production from mixed-acid fermentations.

The MixAlco™ process biologically converts biomass to carboxylate salts that may be chemically converted to a wide variety of chemicals and fuels. The process utilizes lignocellulosic biomass as feedstock (e.g., municipal solid waste, sewage sludge, and agricultural residues), creating an economic basis for sustainable biofuels. This study provides a thermodynamic analysis of hydrogen yield from mixed-acid fermentations from two feedstocks: paper and bagasse. During batch fermentations, hydrogen production, acid production, and sugar digestion were analyzed to determine the energy selectivity of each system. To predict hydrogen production during continuous operation, this energy selectivity was then applied to countercurrent fermentations of the same systems. The analysis successfully predicted hydrogen production from the paper fermentation to within 11% and the bagasse fermentation to within 21% of the actual production. The analysis was able to faithfully represent hydrogen production and represents a step forward in understanding and predicting hydrogen production from mixed-acid fermentations.

Effect of biodiesel glycerol type and fermentor configuration on mixed-acid fermentations.

The MixAlco process biologically converts biomass to carboxylate salts that may be converted to a variety of chemicals and fuels. This study examines the viability of different types of glycerol as a potential feedstock. Batch fermentations of crude biodiesel glycerol were compared to distilled and refined glycerol. Continuous fermentations were performed in a CSTR and a packed-bed fermentor with refined glycerol. While crude and distilled glycerol are difficult to process industrially, all types of glycerol performed well during MixAlco fermentations, producing acid concentrations above 22 g/L and conversions of greater than 65%. The CSTR configuration produced excellent acid concentrations (16 g/L) while the packed-bed configuration produced high amounts of cell material for use in cell extract products or starter cultures.

Structure and dynamics of the microbial communities underlying the carboxylate platform for biofuel production.

The carboxylate platform utilizes a mixed microbial community to convert lignocellulosic biomass into chemicals and fuels. While much of the platform is well understood, little is known about its microbiology. Mesophilic (40 degrees C) and thermophilic (55 degrees C) fermentations employing a sorghum feedstock and marine sediment inoculum were profiled using 16S rRNA tag-pyrosequencing over the course of a 30-day incubation. The contrasting fermentation temperatures converted similar amounts of biomass, but the mesophilic community was significantly more productive, and the two temperatures differed significantly with respect to propionic and butyric acid production. Pyrotag sequencing revealed the presence of dynamic communities that responded rapidly to temperature and changed substantially over time. Both temperatures were dominated by bacteria resembling Clostridia, but they shared few taxa in common. The species-rich mesophilic community harbored a variety of Bacteroidetes, Actinobacteria, and gamma-Proteobacteria, whereas the thermophilic community was composed mainly of Clostridia and Bacilli. Despite differences in composition and productivity, similar patterns of functional class dynamics were observed. Over time, organisms resembling known cellulose degraders decreased in abundance, while organisms resembling known xylose degraders increased. Improved understanding of the carboxylate platform's microbiology will help refine platform performance and contribute to our growing knowledge regarding biomass conversion and biofuel production processes.

Effects of temperature and pretreatment conditions on mixed-acid fermentation of water hyacinths using a mixed culture of thermophilic microorganisms.

The MixAlco process biologically converts biomass to carboxylate salts that may be chemically converted to a wide variety of chemicals and fuels. This study investigated the use of water hyacinths as a feedstock, comparing digestibility after each of four different pretreatments at two fermentation temperatures (40 and 55 degrees C). Water hyacinths were treated with excess lime (0.3g Ca(OH)(2)/g dry biomass). Short-term treatment occurred for 1 and 2h at 100 degrees C. Long-term treatment occurred for 4 and 6 weeks at 50 degrees C. Treated water hyacinths were fermented with marine microorganisms for 28 days and acid concentration (g/L), conversion (g volatile solids (VS) digested/g VS fed), and selectivity (g acid/g VS digested) were measured. All pretreatments out performed fresh feedstock fermentations. The 40 degrees C fermentations exhibited greater acid yields and selectivity than the 55 degrees C. The 1-h hot-lime pretreatment exhibited the best overall outcomes at approximately 250%, 200%, and 125% increases relative to the fresh water hyacinths in total acid, conversion, and selectivity, respectively. The results show that with a gentle 1-h hot-lime pretreatment, water hyacinths can be fermented to produce liquid fuels, thus creating an economic value to water hyacinths that are cleared from choked waterways.