Effect of inoculum source on the enrichment of microbial communities on two lignocellulosic bioenergy crops under thermophilic and high-solids conditions.

AIMS: Culturing compost-derived microbial communities on biofuel feedstocks under industrial conditions is a technique to enrich for organisms and lignocellulolytic enzymes for bioenergy feedstock deconstruction. In this study, microbial communities from green waste compost (GWC) and grape pomace compost (GPC) were cultured on switchgrass and eucalyptus to observe the impact of inoculation on feedstock decomposition and microbial community structure. METHODS AND RESULTS: Respiration was monitored as a measure of microbial activity, and 16S ribosomal RNA gene sequencing was used to characterize microbial community structure. The enriched community structure and respiration were influenced by the choice of feedstock, compost type, and application of thermophilic, high-solids conditions. However, the effect of compost source was significantly less than the effects of the other culture variables. CONCLUSIONS: Although there are subtle differences in potentially lignocellulolytic taxa between GPC- and GWC-derived communities, these differences do not affect the decomposition rates for these communities on switchgrass or eucalyptus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are useful for designing future experiments to discover lignocellulolytic micro-organisms from compost. They suggest that such work may be better served by deemphasizing screening of compost sources and instead focusing on how compost-derived communities adapt to the feedstocks and process conditions relevant to biofuel production.

Identification of Desulfobacterales as primary hydrogenotrophs in a complex microbial mat community.

Hypersaline microbial mats have been shown to produce significant quantities of H2 under dark, anoxic conditions via cyanobacterial fermentation. This flux of a widely accessible microbial substrate has potential to significantly influence the ecology of the mat, and any consumption will affect the net efflux of H2 that might otherwise be captured as a resource. Here, we focus on H2 consumption in a microbial mat from Elkhorn Slough, California, USA, for which H2 production has been previously characterized. Active biologic H2 consumption in this mat is indicated by a significant time-dependent decrease in added H2 compared with a killed control. Inhibition of sulfate reduction, as indicated by a decrease in hydrogen sulfide production relative to controls, resulted in a significant increase in H2 efflux, suggesting that sulfate-reducing bacteria (SRB) are important hydrogenotrophs. Low methane efflux under these same conditions indicated that methanogens are likely not important hydrogenotrophs. Analyses of genes and transcripts that encode for rRNA or dissimilatory sulfite reductase, using both PCR-dependent and PCR-independent metatranscriptomic sequencing methods, demonstrated that Desulfobacterales are the dominant, active SRB in the upper, H2-producing layer of the mat (0-2 mm). This hypothesis was further supported by the identification of transcripts encoding hydrogenases derived from Desulfobacterales capable of H2 oxidation. Analysis of molecular data provided no evidence for the activity of hydrogenotrophic methanogens. The combined biogeochemical and molecular data strongly indicate that SRB belonging to the Desulfobacterales are the quantitatively important hydrogenotrophs in the Elkhorn Slough mat.

Thermophilic enrichment of microbial communities in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate.

AIMS: The aim of the study was to develop an approach to enrich ionic liquid tolerant micro-organisms that efficiently decompose lignocellulose in a thermophilic and high-solids environment. METHODS AND RESULTS: High-solids incubations were conducted, using compost as an inoculum source, to enrich for thermophilic communities that decompose switchgrass in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]). Ionic liquid levels were increased from 0 to 6% on a total weight basis incrementally. Successful enrichment of a community that decomposed lignocellulose at 55°C in the presence of 6% [C2mim][OAc] was achieved, when the [C2mim][OAc] level was increased stepwise from 2% to 4% to 5% to 6%. Pyrosequencing results revealed a shift in the community and a sharp decrease in richness, when thermophilic conditions were applied. CONCLUSIONS: A community tolerant to a thermophilic, high-solids environment containing 6% [C2mim][OAc] was enriched from compost. Gradually increasing [C2mim][OAc] concentrations allowed the community to adapt to [C2mim][OAc]. SIGNIFICANCE AND IMPACT OF THE STUDY: A successful approach to enrich communities that decompose lignocellulose under thermophilic high-solids conditions in the presence of elevated levels of [C2mim][OAc] has been developed. Communities yielded from this approach will provide resources for the discovery of enzymes and metabolic pathways relevant to biomass pretreatment and fuel production.

Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate.

AIMS: This work aimed to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. METHODS AND RESULTS: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. CONCLUSIONS: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.

Hydrogen transfer to carbonyls and imines from a hydroxycyclopentadienyl ruthenium hydride: evidence for concerted hydride and proton transfer.

Reaction of ([2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)CO)](2)H)Ru(2)(CO)(4)(mu-H) (6) with H(2) formed [2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)COH)Ru(CO)(2)H] (8), the active species in catalytic carbonyl reductions developed by Shvo. Kinetic studies of the reduction of PhCHO by 8 in THF at -10 degrees C showed second-order kinetics with Delta H(double dagger) = 12.0 kcal mol(-1) and Delta S(double dagger) = -28 eu. The rate of reduction was not accelerated by CF(3)CO(2)H, and was not inhibited by CO. Selective deuteration of the RuH and OH positions in 8 gave individual kinetic isotope effects k(RuH)/k(RuD) = 1.5 +/- 0.2 and k(OH)/k(OD) = 2.2 +/- 0.1 for PhCHO reduction at 0 degrees C. Simultaneous deuteration of both positions in 8 gave a combined kinetic isotope effect of k(OHRuH)/k(ODRuD) = 3.6 +/- 0.3. [2,5-Ph(2)-3,4-Tol(2)(eta(5)-C(4)COSiEt(3))Ru(CO)(2)H] (12) and NEt(4)(+)[2,5-Ph(2)-3,4-Tol(2)(eta(4)-C(4)CO)Ru(CO)(2)H](-) (13) were unreactive toward PhCHO under conditions where facile PhCHO reduction by 8 occurred. PhCOMe was reduced by 8 30 times slower than PhCHO; MeN=CHPh was reduced by 8 26 times faster than PhCHO. Cyclohexene was reduced to cyclohexane by 8 at 80 degrees C only in the presence of H(2.) Concerted transfer of a proton from OH and hydride from Ru of 8 to carbonyls and imines is proposed.

Format-independent data collection forms.

Good form design is generally acknowledged to be a key element of clinical trials and follow-up studies. Although some data are collected directly from automated equipment, most measurements, examination findings, and interview responses require at least one manual transcription, either onto paper forms or directly into a computer. Regardless of the transcription method adopted, there is broad agreement that the quality of study data depends strongly on the format and layout of the data collection instruments. The task of form design and production is complicated by two facts: first, a series of different forms is needed even when the period of follow-up is short; and second, form design is an ongoing process that starts before the first person is enrolled and continues over the course of the trial. This paper (1) presents a concept (format independence) aimed at simplifying the design and revision of data collection forms; (2) explains how a feature (styles) present in most word processing software can be used to implement the concept; (3) discusses the advantages and disadvantages of a format-independent approach as compared to more traditional form development tools; and (4) describes the environment that prompted this approach to form development.

Design, development, and implementation of a data processing system for multiple controlled trials and epidemiologic studies.

We were given the opportunity to design and implement a general data processing system to accommodate several different epidemiologic studies to be conducted by a new research group. A survey of 15 operating data centers was conducted in preparation for undertaking the design and development of our system. The results of the survey indicated that data processing activities can be classified, both conceptually and operationally, into three modules: data recording and data entry, data management, and data analysis, and that the data management functions were those amenable to generalization. Based on our survey and the varying needs of our studies, we selected a "mixed" hardware environment, using both a computer center mainframe and microcomputers. We created the systems using commercially available software, including a mainframe database manager and mainframe statistics packages, microcomputer data entry software, and a communications package to link the two environments. Our strategy was to buy software, when possible, rather than to build custom programs, and to let software tools govern hardware needs. Hardware independence, price, and functional capability directed our software choices, while hardware selection was constrained most importantly by available software, then by budget, by available computing resources, and finally by the marketplace. The system has been used successfully in three studies differing in design, size, data collection locale, and rate of data accrual.