Winter Rye as a Bioenergy Feedstock: Impact of Crop Maturity on Composition, Biological Solubilization and Potential Revenue

Background: Winter annual crops such as winter rye (Secale cereale L) can produce biomass feedstock on seasonally fallow land that continues to provide high-value food and feed from summer annuals such as corn and soybeans. As energy double crops, winter grasses are likely to be harvested while still immature and thus structurally different from the fully senesced plant material typically used for biofuels. This study investigates the dynamic trends in biomass yield, composition, and biological solubilization over the course of a spring harvest season. Results: The water soluble fraction decreased with increasing maturity while total carbohydrate content stayed roughly constant at about 65%. The protein mass fraction decreased with increasing maturity, but was counterbalanced by increasing harvest yield resulting in similar total protein across harvest dates. Winter rye was ground and autoclaved then fermented at 15 g/L total solids by either (1) Clostridium thermocellum or (2) simultaneous saccharification and cofermentation (SSCF) using commercial cellulases (CTec2 and HTec2) and a xylose-fermenting Saccharomyces cerevisiae strain. Solubilization of total carbohydrate dropped significantly as winter rye matured for both C. thermocellum (from approximately 80% to approximately 50%) and SSCF (from approximately 60% to approximately 30%). C. thermocellum achieved total solubilization 33% higher than that of SSCF for the earliest harvest date and 50% higher for the latest harvest date. Potential revenue from protein and bioethanol was stable over a range of different harvest dates, with most of the revenue due to ethanol. In a crop rotation with soybean, recovery of the soluble protein from winter rye could increase per hectare protein production by 20 to 35%. Conclusions: Double-cropping winter rye can produce significant biomass for biofuel production and feed protein as coproduct without competing with the main summer crop. During a 24-day harvest window, the total carbohydrate content remained relatively constant while the early-harvest yielded much higher carbohydrate solubilization for both C. thermocellum fermentation and SSCF. C. thermocellum fermentation achieved higher carbohydrate solubilization than SSCF across all growth stages tested. Although winter rye’s yield, composition, and biological reactivity change rapidly in the spring, it offers a substantial and stable income across the harvest season and thus flexibility for the farmer

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA

Achieving provider engagement: providers' perceptions of implementing and delivering integrated care

The literature on integrated care is limited with respect to practical learning and experience. Although some attention has been paid to organizational processes and structures, not enough is paid to people, relationships, and the importance of these in bringing about integration. Little is known, for example, about provider engagement in the organizational change process, how to obtain and maintain it, and how it is demonstrated in the delivery of integrated care. Based on qualitative data from the evaluation of a large-scale integrated care initiative in London, United Kingdom, we explored the role of provider engagement in effective integration of services. Using thematic analysis, we identified an evolving engagement narrative with three distinct phases: enthusiasm, antipathy, and ambivalence, and argue that health care managers need to be aware of the impact of professional engagement to succeed in advancing the integrated care agenda

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium Saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type.This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Background: The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber \u3e 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results: The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that \u3e 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions: This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading

Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in ∼50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO- and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors

Technoeconomic and life-cycle analysis of single-step catalytic conversion of wet ethanol into fungible fuel blendstocks

Technoeconomic and life-cycle analyses are presented for catalytic conversion of ethanol to fungible hydrocarbon fuel blendstocks, informed by advances in catalyst and process development. Whereas prior work toward this end focused on 3-step processes featuring dehydration, oligomerization, and hydrogenation, the consolidated alcohol dehydration and oligomerization (CADO) approach described here results in 1-step conversion of wet ethanol vapor (40 wt% in water) to hydrocarbons and water over a metal-modified zeolite catalyst. A development project increased liquid hydrocarbon yields from 36% of theoretical to >80%, reduced catalyst cost by an order of magnitude, scaled up the process by 300-fold, and reduced projected costs of ethanol conversion 12-fold. Current CADO products conform most closely to gasoline blendstocks, but can be blended with jet fuel at low levels today, and could potentially be blended at higher levels in the future. Operating plus annualized capital costs for conversion of wet ethanol to fungible blendstocks are estimated at $2.00/GJ for CADO today and$1.44/GJ in the future, similar to the unit energy cost of producing anhydrous ethanol from wet ethanol ($1.46/GJ). Including the cost of ethanol from either corn or future cellulosic biomass but not production incentives, projected minimum selling prices for fungible blendstocks produced via CADO are competitive with conventional jet fuel when oil is$100 per barrel but not at $60 per barrel. However, with existing production incentives, the projected minimum blendstock selling price is competitive with oil at$60 per barrel. Life-cycle greenhouse gas emission reductions for CADO-derived hydrocarbon blendstocks closely follow those for the ethanol feedstock