Preservation of Microalgae, Lignocellulosic Biomass Blends by Ensiling to Enable Consistent Year-Round Feedstock Supply for Thermochemical Conversion to Biofuels.

Seasonal variation in microalgae productivity is a significant barrier to economical production of algae biofuels and chemicals. Summer production can be 3-5 times higher than in the winter resulting in uneven feedstock supplies at algae biorefineries. A portion of the summer production must be preserved for conversion in the winter in order to maintain a biorefinery running at capacity. Ensiling, a preservation process that utilizes lactic acid fermentation to limit microbial degradation, has been demonstrated to successfully stabilize algae biomass (20% solids) and algae-lignocellulosic blends (40% algae-60% lignocellulosic biomass, dry basis) for over 6 months, resulting in fuel production cost savings with fewer emissions. Preservation of algae as blends could be beneficial to biorefineries that utilize thermochemical approaches to fuel production as co-processing of algae and lignocellulosic biomass has been observed to enhance biocrude yield and improve oil quality. This study conducts a resource assessment of biomass residues in the southern United States to identify materials available during peak algae productivity and in sufficient quantity to meet the algae storage needs of an algae biofuel industry. Eight feedstocks met the quantity threshold but only three, distillers grains, haylage, and yard waste, were also available in season. Storage experiments utilizing both freshwater and marine strains of microalgae - Scenedesmus acutus, Chlorella vulgaris, Chlorella zofingiensis, Nannochloropsis gaditana, and Porphyridium purpureum - and yard waste were conducted for 30 days. Storage losses were less than 10% in all but one case, and the pH of all but one blend was reduced to less than 4.7, indicating that yard waste is a suitable feedstock for blending with algae prior to storage. To better understand whether the benefits to conversion realized by processing blends might be affected by storage, elemental analysis and bomb calorimetry of pre- and post-storage algae-yard waste blends were conducted to characterize changes occurring during storage. Storing algae biomass as blends with lignocellulosic biomass could be an effective method of mitigating seasonal variability in algae biomass production while retaining the synergistic effect of co-processing algae blends in thermochemical conversion.

Methyl Ketones from Municipal Solid Waste Blends by One-Pot Ionic-Liquid Pretreatment, Saccharification, and Fermentation.

The conversion of municipal solid waste (MSW) and lignocellulosic biomass blends to methyl ketones (MKs) was investigated by using bioderived ionic liquid (bionic liquid)-based hydrolysates followed by fermentation with an engineered Escherichia coli strain. The hydrolysates were produced by a one-pot process using six types of MSW-biomass blends, choline-based bionic liquids, and commercial enzymes. Based on the sugar yields, one blend (corn stover/MSW=95:5, w/w) and two bionic liquids {cholinium lysinate ([Ch][Lys]) and cholinium aspartate ([Ch]2 [Asp])} were selected for scale-up studies. Maximum yields of 82.3 % glucose and 54.4 % xylose were obtained from the selected blend in the scale-up studies (6 L), which was comparable with 83.6 % glucose and 52.8 % xylose obtained at a smaller scale (0.2 L). Comparable or higher yields of medium-chain (C11 -C17 ) MKs were achieved by using the MSW-biomass blend-derived hydrolysates, relative to the sugar controls (glucose and xylose) with similar sugar feeding concentrations. Up to 1145 mg L-1 of MKs was produced by using MSW-biomass-derived hydrolysates, and the MK titer decreased to 300 mg L-1 when the bionic-liquid concentration in the hydrolysate increased from 1 to 2 %, indicative of bionic-liquid inhibition. Technoeconomic analysis was conducted to investigate the economic potential of using the selected MSW-biomass blend as a feedstock to produce MKs.

Ternary ionic liquid-water pretreatment systems of an agave bagasse and municipal solid waste blend.

BACKGROUND: Pretreatment is necessary to reduce biomass recalcitrance and enhance the efficiency of enzymatic saccharification for biofuel production. Ionic liquid (IL) pretreatment has gained a significant interest as a pretreatment process that can reduce cellulose crystallinity and remove lignin, key factors that govern enzyme accessibility. There are several challenges that need to be addressed for IL pretreatment to become viable for commercialization, including IL cost and recyclability. In addition, it is unclear whether ILs can maintain process performance when utilizing low-cost, low-quality biomass feedstocks such as the paper fraction of municipal solid waste (MSW), which are readily available in high quantities. One approach to potentially reduce IL cost is to use a blend of ILs at different concentrations in aqueous mixtures. Herein, we describe 14 IL-water systems with mixtures of 1-ethyl-3-ethylimidazolium acetate ([C2C1Im][OAc]), 1-butyl-3-ethylimidazolium acetate ([C4C1Im][OAc]), and water that were used to pretreat MSW blended with agave bagasse (AGB). The detailed analysis of IL recycling in terms of sugar yields of pretreated biomass and IL stability was examined. RESULTS: Both biomass types (AGB and MSW) were efficiently disrupted by IL pretreatment. The pretreatment efficiency of [C2C1Im][OAc] and [C4C1Im][OAc] decreased when mixed with water above 40%. The AGB/MSW (1:1) blend demonstrated a glucan conversion of 94.1 and 83.0% using IL systems with ~10 and ~40% water content, respectively. Chemical structures of fresh ILs and recycle ILs presented strong similarities observed by FTIR and 1H-NMR spectroscopy. The glucan and xylan hydrolysis yields obtained from recycled IL exhibited a slight decrease in pretreatment efficiency (less than 10% in terms of hydrolysis yields compared to that of fresh IL), and a decrease in cellulose crystallinity was observed. CONCLUSIONS: Our results demonstrated that mixing ILs such as [C2C1Im][OAc] and [C4C1Im][OAc] and blending the paper fraction of MSW with agricultural residues, such as AGB, may contribute to lower the production costs while maintaining high sugar yields. Recycled IL-water mixtures provided comparable results to that of fresh ILs. Both of these results offer the potential of reducing the production costs of sugars and biofuels at biorefineries as compared to more conventional IL conversion technologies.Graphical abstractSchematic of ionic liquid (IL) pretreatment of agave bagasse (AB) and paper-rich fraction of municipal solid waste (MSW).

Scale-up and process integration of sugar production by acidolysis of municipal solid waste/corn stover blends in ionic liquids.

BACKGROUND: Lignocellulosic biorefineries have tonnage and throughput requirements that must be met year round and there is no single feedstock available in any given region that is capable of meeting the price and availability demands of the biorefineries scheduled for deployment. Significant attention has been historically given to agriculturally derived feedstocks; however, a diverse range of wastes, including municipal solid wastes (MSW), also have the potential to serve as feedstocks for the production of advanced biofuels and have not been extensively studied. In addition, ionic liquid (IL) pretreatment with certain ILs is receiving great interest as a potential process that enables fractionation of a wide range of feedstocks. Acid catalysts have been used previously to hydrolyze polysaccharides into fermentable sugars following IL pretreatment, which could potentially provide a means of liberating fermentable sugars from lignocellulose without the use of costly enzymes. However, successful optimization and scale-up of the one-pot acid-assisted IL deconstruction for further commercialization involve challenges such as reactor compatibility, mixing at high solid loading, sugar recovery, and IL recycling, which have not been effectively resolved during the development stages at bench scale. RESULTS: Here, we present the successful scale-up demonstration of the acid-assisted IL deconstruction on feedstock blends of municipal solid wastes and agricultural residues (corn stover) by 30-fold, relative to the bench scale (6 vs 0.2 L), at 10% solid loading. By integrating IL pretreatment and acid hydrolysis with subsequent centrifugation and extraction, the sugar and lignin products can be further recovered efficiently. This scale-up development at Advanced Biofuels/Bioproducts Process Demonstration Unit (ABPDU) will leverage the opportunity and synergistic efforts toward developing a cost-effective IL-based deconstruction technology by drastically eliminating enzyme, reducing water usage, and simplifying the downstream sugar/lignin recovery and IL recycling. CONCLUSION: Results indicate that MSW blends are viable and valuable resource to consider when assessing biomass availability and affordability for lignocellulosic biorefineries. This scale-up evaluation demonstrates that the acid-assisted IL deconstruction technology can be effectively scaled up to larger operations and the current study established the baseline of scaling parameters for this process.

Degradation of phenolic compounds by the lignocellulose deconstructing thermoacidophilic bacterium Alicyclobacillus Acidocaldarius.

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 µM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Blending municipal solid waste with corn stover for sugar production using ionic liquid process.

Municipal solid waste (MSW) represents an attractive cellulosic resource for sustainable fuel production. However, its heterogeneity is the major barrier to efficient conversion to biofuels. MSW paper mix was generated and blended with corn stover (CS). It has been shown that both of them can be efficiently pretreated in certain ionic liquids (ILs) with high yields of fermentable sugars. After pretreatment in 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), over 80% glucose has been released with enzymatic saccharification. We have also applied an enzyme-free process by adding mineral acid and water directly into the IL/biomass slurry to induce hydrolysis. With the acidolysis process in 1-ethyl-3-methylimidazolium chloride ([C2C1Im]Cl), up to 80% glucose and 90% xylose are released. There is a correlation between the viscosity profile and hydrolysis efficiency; low viscosity of the hydrolysate generally corresponds to high sugar yields. Overall, the results indicate the feasibility of incorporating MSW as a robust blending agent for biorefineries.

Assessment of xylanase activity in dry storage as a potential method of reducing feedstock cost.

Enzymatic preprocessing of lignocellulosic biomass in dry storage systems has the potential to improve feedstock characteristics and lower ethanol production costs. To assess the potential for endoxylanase activity at low water contents, endoxylanase activity was tested using a refined wheat arabinoxylan substrate and three commercial endoxylanases over the water activity range 0.21-1.0, corresponding to water contents of 5% to >60% (dry basis). Homogeneously mixed dry samples were prepared at a fixed enzyme to substrate ratio and incubated in chambers at a variety of fixed water activities. Replicates were sacrificed periodically, and endoxylanase activity was quantified as an increase in reducing sugar relative to desiccant-stored controls. Endoxylanase activity was observed at water activities over 0.91 in all enzyme preparations in less than 4 days and at a water activity of 0.59 in less than 1 week in two preparations. Endoxylanase activity after storage was confirmed for selected desiccant-stored controls by incubation at 100% relative humidity. Water content to water activity relationships were determined for three lignocellulosic substrates, and results indicate that two endoxylanase preparations retained limited activity as low as 7% to 13% water content (dry basis), which is well within the range of water contents representative of dry biomass storage. Future work will examine the effects of endoxylanase activity toward substrates such as corn stover, wheat straw, and switchgrass in low water content environments.

Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens.

BACKGROUND: Chromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles). RESULTS: Core samples obtained from a Cr(VI) contaminated aquifer at the Hanford facility in Washington were enriched in Vogel Bonner medium at 10 Centigrade with 0, 25, 50, 100, 200, 400 and 1000 mg/l Cr(VI). The extent of Cr(VI) reduction was evaluated using the diphenyl carbazide assay. Resistance to Cr(VI) up to and including 1000 mg/l Cr(VI) was observed in the consortium experiments. Reduction was slow or not observed at and above 100 mg/l Cr(VI) using the enrichment consortium. Average time to complete reduction of Cr(VI) in the 30 and 60 mg/l Cr(VI) cultures of the consortium was 8 and 17 days, respectively at 10 Centigrade. Lyophilized consortium cells did not demonstrate adsorption of Cr(VI) over a 24 hour period. Successful isolation of a Cr(VI) reducing organism (designated P4) from the consortium was confirmed by 16S rDNA amplification and sequencing. Average time to complete reduction of Cr(VI) at 10 Centigrade in the 25 and 50 mg/l Cr(VI) cultures of the isolate P4 was 3 and 5 days, respectively. The 16S rDNA sequence from isolate P4 identified this organism as a strain of Arthrobacter aurescens, a species that has not previously been shown to be capable of low temperature Cr(VI) reduction. CONCLUSION: A. aurescens, indigenous to the subsurface, has the potential to be a predominant metal reducer in enhanced, in situ subsurface bioremediation efforts involving Cr(VI) and possibly other heavy metals and radionuclides.

Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus.

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 degrees C and a pH range from 6 to 10, with optimum activity occurring at 90 degrees C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 degrees C and 3 h at 90 degrees C. The enzyme also demonstrated excellent stability at 70 degrees C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A K(m) of 35.5 mM and a V(max) of 20.3 mM/min.mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.