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High biomass yields have been documented for Tripidium spp. (Erianthus spp., Saccharum spp.), but targeted breeding for bioenergy applications has been limited. Advanced, interspecific hybrids between Tripidium ravennae and T. arundinaceum were planted in replicated field plots in 2016. Comparative feedstock evaluations examined biomass yields, cytogenetics, plant fertility, and compositional analyses relative to Miscanthus × giganteus. Dry biomass yields varied as a function of year and accession and increased each year ranging from 3.4 to 10.6, 8.6 to 37.3, and 23.7 to 60.6 Mg/ha for Tripidium hybrids compared to 2.3, 16.2 and 27.9 Mg/ha for M. × giganteus in 2016, 2017, and 2018, respectively. Cytology and cytometry confirmed that Tripidium hybrids were tetraploid with 2n = 4x = 40 (2C genome size = 5.06 pg) and intermediate between T. ravennae with 2n = 2x = 20 (2C genome size = 2.55 pg) and T. arundinaceum with 2n = 6x = 60 (2C genome size = 7.61 pg). Plant fertility characteristics varied considerably with some accessions producing no viable seeds or fewer than that observed for M. × giganteus. Accessions varied significantly for flowering culm number and height and dates of peak anthesis ranging from 14 September to 2 October. Variations in yield and compositional analyses contributed to variations in theoretical ethanol yields ranging from 10,181 to 27,546 L/ha for Tripidium accessions compared to 13,095 L/ha for M. × giganteus. Relative feed value (RFV) indices for winter‐harvested Tripidium accessions varied from 52.8 to 60.0 compared to M. × giganteus with 45.4. RFV for summer‐harvested Tripidium accessions varied from 71.6 to 80.5 compared to M. × giganteus with 61.0. These initial findings for Tripidium hybrids, including high biomass yields, cold hardiness, and desirable traits for multiple markets (e.g., forage, bioenergy, bioproducts), are promising and warrant further development of Tripidium as a temperate bioenergy feedstock.

Current research on bioenergy crops shows that perennial grasses can yield substantial amounts of dry biomass with relatively low inputs of water and fertilizer. In order to minimize competition with food production, it has been suggested that bioenergy crops could be directed to land areas less suitable for commodity crops, commonly referred to as “marginal” lands. These are land units with inherent limitations to vegetative growth and production, which may be due to several factors (soil physical and chemical properties, climatic conditions, etc.). However the term “marginal” is an adjective with imprecise meaning, and objective criteria for determining “marginal” lands for siting bioenergy crops are necessary. Here we propose that such criteria may be based on soil survey classifications and realistic yield estimates, and we show an example of its use to justify site selection for bioenergy crops in different regions of North Carolina.

Near infrared spectroscopy is widely used for compositional analysis of bulk materials because it is inexpensive, fast, and non-destructive. However, the chemometric techniques required to produce near infrared calibrations are varied and complex. While there are a number of commercial applications capable of implementing these techniques, there has also been a recent proliferation of R packages for chemometrics. The R programming language has greater capabilities for data processing, automation of multiple analyses, and user development of new techniques than many of the closed-source, graphical user interface-based commercial chemometrics applications do. The R project is thus a powerful, open-source option for generating and testing near infrared calibrations, albeit with a longer learning curve than many of the commercial chemometric applications. The calibration techniques available in R have been widely demonstrated in both the primary literature and introductory texts, but less so the steps between the acquisition of the data and the calibration. This tutorial seeks to bridge that gap by demonstrating a practical approach to data transfer and handling, using R and several packages available on the Comprehensive R Archive Network ( https://cran.r-project.org/ ), and then illustrates the use of the resulting data framework in the generation of near infrared calibrations.

Anthocyanins from purple-fleshed sweetpotatoes constitute highly valued natural colorants and functional ingredients. In the past, anthocyanin extraction conditions and efficiencies using a single acidified solvent have been assessed. However, the potential of solvent mixes that can be generated by fermentation of biomass-derived sugars have not been explored. In this study, the effects of single and mixed solvent, time, temperature, sweetpotato genotype and preparation, on anthocyanin and phenolic extraction were evaluated. Results indicated that unconventional diluted solvent mixes containing acetone, butanol, and ethanol were superior or equally efficient for extracting anthocyanins when compared to commonly used concentrated extractants. In addition, analysis of anthocyanidins concentrations including cyanidin (cy), peonidin (pe), and pelargonidin (pl), indicated that different ratios of pn/cy were obtained depending on the solvent used. These results could be useful when selecting processing conditions that better suit particular end-use applications and more environmentally friendly process development for purple sweetpotatoes.

The accurate characterization of biomass is critical for development of bioenergy feedstocks and their utilization. Most analytical approaches involve acid hydrolysis of the polysaccharides in biomass, leaving most of the lignin as insoluble residue. A limitation of this approach is that the same conditions used to hydrolyze polysaccharides also degrade the liberated monosaccharides. The NREL-compiled procedures account for this effect with “Sugar Recovery Standards”, in which a solution of the expected monosaccharides is prepared and subjected to the dilute-hydrolysis portion of the procedure; however, this tends to overestimate monosaccharide degradation and introduce bias between polysaccharides of different lability. The following recommended method modifications are intended to reduce these errors: (1) quantification of immediate degradation products of monosaccharides and their stoichiometric addition to the monosaccharide yield; (2) the adjustment of this combined yield with sugar recovery standard...

Increased interest in biomass cultivation requires detailed analysis of spatial production potential of possible biorefinery locations, with emphasis on feedstock production cost minimization. Integrated assessment of publicly available spatial data on current crop production, soil type, and yield potential, coupled with techno-economic production cost estimates, can support a functional method for rapid analysis of potential biorefinery sites. A novel predictive model was developed to determine cropland conversion using a probabilistic profit based equation for multiple biomass crops: giant reed, miscanthus, switchgrass, and sorghum (with either canola or barley as a winter crop). The three primary regions of North Carolina (Mountains, Piedmont, and Coastal Plain) were used as a case study and with a single parameter uncertainty analysis was completed. According to the model, the county chosen to represent the Coastal Plain (Duplin County) had the largest potential acreage that would be converted (15,071 ha, 7.1% total land, 9.3% of cropland) primarily to sorghum with canola as a winter crop. Large portions were also predicted to convert to giant reed and switchgrass, depending on the price and yield parameters used. The Piedmont (Granville County, 7697 ha, 5.5% total land, 6.9% cropland) and Mountain (Henderson County, 2117 ha, 2.2% total land, 2.3% cropland) regions were predicted to convert primarily to switchgrass acreage for biomass production, with much less available biomass overall compared to the Coastal Plain. This model provided meaningful insight into regional cropping systems and feedstock availability, allowing for improved business planning in designated regions. Determination of cropland conversion is imperative to develop realistic biomass logistical operations, which in conjunction can assist with rapid determination of profitable biomass availability. After this rapid analysis method is conducted in-depth on-ground biorefinery feasibility analysis can occur, ensuring resource are used only in locations with a high potential for available low cost biomass feedstocks.

Clostridium autoethanogenum is an autotrophic acetogen also able to utilize xylose as a carbon source. Here, we evaluated this organism's industrially relevant capability since the transformation of sugars derived from lignocellulosic materials to biofuels is of great interest. Initially, the organism was challenged with different initial xylose levels to determine xylose utilization and maximum inhibitory concentrations for C. autoethanogenum when grown in batch cultures. A critical factor that could limit xylose utilization is culture pH, and this factor was separately examined as a means to provide C. autoethanogenum with more suitable environmental conditions. Finally, optimum pH conditions for growth were tested to assess how efficiently initial xylose concentrations could be fully utilized. C. autoethanogenum specific growth rate was not impacted when cells were exposed to initial xylose concentrations up to 30 g L−1, while cultures amended with concentrations as high as 45 g L−1 showed a delay in growth and in formation of final fermentation product concentrations. Intermittent adjustment of the culture pH to 5.8 allowed for complete substrate utilization when step-wise fed-batch xylose additions were performed. Under these conditions the cultures showed a unique biphasic growth which enhanced biomass and end product formation. In C. autoethanogenum cultures exposed to an initial xylose concentration as high as 30 g L−1, pH adjustment to 5.8 supported full utilization of all available xylose and generation of up to 2.4 g L−1 ethanol and 9.5 g L−1 acetate. This work demonstrated that C. autoethanogenum can effectively biotransform xylose at concentrations typically present in lignocellulosic hydrolysate streams.

Biomass logistics operations account for a major portion of the feedstock cost of running a biorefinery, and make up a significant portion of total system operational costs. Biomass is a bulky perishable commodity that is required in large quantities year round for optimal biorefinery operations. As a proof of concept for a decision making tool for biomass production and delivery, a heuristic was developed to determine biorefinery location, considering city size, agricultural density, and regional demographics. Switchgrass and sorghum (with winter canola) were selected to examine as viable biomass feedstocks based on positive economic results determined using a predictive model for cropland conversion potential. Biomass harvest systems were evaluated to examine interrelationships of biomass logistical networks and the least cost production system, with results demonstrating a need to shift to maximize supply-driven production harvest operations and limit storage requirements. For this supply-driven production harvest operations approach a harvest window from September until March was selected for producing big square bales of switchgrass for storage until use, forage chopped sorghum from September to December, and forage chopped switchgrass from December to March. A case study of the three major regions of North Carolina (Mountains, Piedmont, and Coastal Plain) was used to assess logistical optimization of the proposed supply-driven production harvest system. Potential biomass production fields were determined within a hundred mile radius of the proposed biorefinery location, with individual fields designated for crop and harvest system by lowest transportation cost. From these selected fields, crops and harvest system regional storage locations were determined using an alternate location-allocation heuristic with set storage capacity per site. Model results showed that the supply-driven production harvest system greatly reduced system complexity, maximized annual usage of high cost specialized equipment, and reduced logistical operations cost. The siting method and developed model shows promise and can be used for computational analysis of potential biorefinery site biomass production systems before costly on the ground logistical analysis.

Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO2 and H2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO2/H2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H2, CO2 and CO). This was further confirmed using 13C-Tracer experiments feeding 13CO2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.

A simultaneous saccharification fermentation (SSF) system was studied for ethanol production in flour industrial sweetpotato (ISP) feedstocks (lines: white DM02-180 and purple NC-413) as an integrated cost saving process, and to examine the feasibility of extracting anthocyanins from flour purple ISPs under a simultaneous extraction and fermentation (SEF) system. Furthermore, a separate hydrolysis fermentation (SHF) configuration was carried out to establish a baseline in sugar consumption and ethanol production from the ISP lines. The thermotolerant ethanol producing yeast strain Kluyveromyces marxianus NCYC 851 and the mesophilic Saccharomyces cerevisiae Ethanol Red were evaluated, using commercial alpha amylases for hydrolysis of available ISP starch to sugars. Fermentation by S. cerevisiae during SHF had an ethanol yield of 0.32 g/g dry ISP, a 1.1-fold increase above that produced by K. marxianus. Subsequent studies showed that ethanol yield could be increased in a SSF system with a maximum ethanol yield of 0.39 g/g dry ISP achieved, a 15% increase compared with using a SHF system when S. cerevisiae was used. Simultaneous extraction and fermentation of flour purple-fleshed NC-413 ISPs was studied to evaluate the effect of pH on extraction of total monomeric anthocyanins (TMA) and ethanol production. On average, maximum ethanol yield ranged from 0.31 to 0.34 g/g dry ISP and TMA concentration ranged from 45 to 64 mg cyanidin-3-glu/100 g dry powder (10–22 mg/100 g fresh weight) with the greatest ethanol production coming from non-adjusted pH fermentations. The highest anthocyanin recovery, 64 mg cyanidin-3-glu/100 g dry powder was obtained at 35 °C and pH 4.5 using S. cerevisiae Ethanol Red. This study showed the feasibility of extracting anthocyanins and producing ethanol simultaneously in one unit operation without the need of purified solvents.

This paper describes the economic, environmental, and energy issues of the farm to biorefinery continuum related to production of ethanol from soluble sugars recovered from sweet sorghum using the BE3 (bioenergy economics, energy, and environmental) model methodology. A comparative analysis of five process configurations was conducted to determine how process decentralization affects the total production system. An increased integration of on-farm processing resulted in a moderate increase in the breakeven sales price of ethanol ($0.08/L), however the substantial increase in value-added agricultural practices (approximately 180%) can offer greater returns to the farm operation. Benefits outside the scope of this analysis related to decentralized processing include: increased rural development, reductions in transportation requirements, additional income to farmers, and dissipation of some environmental impacts. Using a single parameter sensitivity analysis for those process configurations the greatest economic impacts were found to be related to conversion efficiency, crop yield, and press efficiency. Conservative values were used throughout the process modeling procedure (e.g. crop yield, Brix level of juice, conversion efficiency, and by-product usage), yet with system optimization, breakeven sales price could be significantly decreased.

Sweet and forage varieties of sorghum (Sorghum bicolor (L.) Moench) can produce large quantities of soluble sugars, starch, and fiber in a single crop and so are highly suited for bioenergy production. To develop this potential, it is critical to reliably quantify the components of the biomass: primarily carbohydrates (monosaccharides, sucrose, starch, and holocellulose) as well as lignin and extractives. Techniques compiled by the National Renewable Energy Laboratory (NREL) are commonly used for these analyses; however, some characteristics of sorghum require some adaptation of those methods. Here, we present an analysis of some of these characteristics and a modified analytical procedure to account for them. In particular, greater accuracy can be obtained by separating the pith and rind portions of the stalk prior to analysis. Several important properties of the stalk differ between the pith and rind, and in many cases, the magnitude of that difference varies among cultivars. As we demonstrate, analyzing the stalk without separating the pith and rind can lead to systematic bias in the measurement of these properties and lignocellulosic composition; if separation is not possible, the bias can be mitigated by exercising care in ensuring a representative subsampling of ground material. Some modifications to initial Soxhlet extraction procedures also improve the accuracy and practicality of the biomass analysis.

Liquefaction, saccharification, and fermentation of FTA-94 industrial sweetpotatoes (ISPs) were examined using α-amylase and glucoamylase for the production of ethanol. Starch degradation and sugars produced over time were examined for (1) α-amylase (Liquozyme SC) at different loading rates (0.045, 0.45, and 4.5% KNU-S/g dry ISP) during liquefaction; and (2) three glucoamylases (Spirizyme Fuel, Spirizyme Plus Tech, and Spirizyme Ultra) at different loading rates (0.5, 1.0, and 5.0 AGU/g dry ISP) during saccharification. The majority of starch, 47.7 and 65.4% of dry matter, was converted during liquefaction of flour and fresh sweetpotato preparations, respectively, with the addition of 0.45 KNU-S/g dry ISP of Liquozyme SC after 2 h (66.4 and 80.1% initial starch in dry matter, respectively). The enzymes used during saccharification increased starch breakdown, but was more effective in conversion of short chain carbohydrates to fermentable sugars. The addition of 5.0 AGU/g of Spirizyme Ultra after 48 h produced 795.4 and 685.3 mg glucose/g starch with flour and fresh preparations, respectively. Yeast fermentation on hydrolyzed starch was examined over time with and without the addition of salt nutrients. Yeast converted all fermentable sugar (e.g. glucose, fructose, maltose) and produced 62.6 and 33.6 g ethanol/L of hydrolysate for flour (25% w/v, substrate loading) and fresh (12.5% w/v, substrate loading) ISP, respectively, after 48 h without salt addition.

A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in a mixotrophic medium containing fructose and syngas. Whole-genome sequencing identified four unique single nucleotide polymorphisms (SNPs) in the C. ljungdahlii OTA1 genome. Among these, two SNPs were found in the gene coding for AcsA and HemL, enzymes involved in acetyl-CoA formation from CO/CO2. Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2, demonstrating that its activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes coding for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-D-ribose catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-D-ribose were likely to have minor effects on biomass and fermentation product yields. In addition, in silico flux balance analysis simulating mixotrophic and heterotrophic conditions showed no change in flux to ethanol when flux through HemL was changed whereas limited flux through AcsA increased the ethanol flux for both simulations. In characterizing the effects of the SNPs identified in the C. ljungdahlii OTA1 genome, a non-autotrophic hyper ethanol-producing strain of C. ljungdahlii was identified that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.

Sweet sorghum (Sorghum bicolor (L.) Moench) is particularly suitable as a feedstock for a variety of bioprocesses, largely because of its high yields of both lignocellulosic biomass and fermentable saccharides. Sweet sorghum is less economically important for refined sugar production than other sugar crops, e.g., sugar beet and sugarcane, but can produce more raw fermentable sugar under marginal conditions than those crops. In this review, the agronomic requirements of sorghum (viz., water, soil, and nutrient requirements), cultural practices, and plant morphology are discussed from a bioprocessing perspective. Historically, sugar extraction from the plant in the form of juice has been of primary interest; these methods, along with modern developments are presented. Recently, the direct yeast fermentation of sorghum juice for ethanol production has been studied. Additionally, the bagasse resulting from the juice extraction has been used for a variety of potential products: forage, silage, combustion energy, synthesis gas, and paper. The bagasse contains high levels of relatively low crystallinity cellulose, along with relatively labile lignin, and so is itself of interest as a fermentation feedstock. Whole sorghum stalk, and its bagasse, have been subjected to studies of a wide array of pretreatment, enzymatic hydrolysis, and fermentation processes. The potential fermentation products of sweet sorghum are wide ranging; those demonstrated include ethanol, acetone, butanol, various lipids, lactic acid, hydrogen, and methane. Several potential native products of the plant, in addition to cellulose for paper production, are also identified: waxes, proteins, and allelopathic compounds, such as sorgoleone.

This research focused on advanced oxidation of toluene by evaluating Ni-olivine catalysts in combination with ozone. Specifically, our objectives were to: (1) synthesize catalysts via electroless plating (ELP) and thermal impregnation (TI) techniques to impregnate nickel onto the olivine surface; (2) characterize Ni-olivine catalysts by Brunauer-Emmett-Teller specific surface area, electron microscopy, energy dispersive spectroscopy, and x-ray photoelectron spectroscopy to understanding the mechanisms of toluene oxidation; (3) determine the catalytic activity of the newly synthesized Ni-olivine catalysts in the oxidation of toluene; and (4) evaluate the influence of varying toluene and ozone concentrations on Ni-olivine oxidation efficiencies. Testing was performed in a continuous packed bed reactor (200°C). Toluene (200 to 600 ppmv) and ozone (70 to 800 ppmv) were injected into the heated reactor, while inlet and outlet concentrations were measured using gas chromatography. Results indicated that 90% of toluene was oxidized within a 1 s residence time using ELP and TI catalyst synthesis techniques and 70 ppmv ozone. Microscopic and spectroscopic analyses revealed porous structures and a nickel film uniformly coating the electroless plated olivine surface, mostly comprised of Ni-P-O, while the thermally impregnated Ni-olivine possessed sparingly deposited Ni2O3 compounds on the surface. An increase in ozone concentrations increased toluene oxidation efficiencies, whereas an increase in toluene concentrations temporarily decreased toluene oxidation efficiencies. Knowledge obtained from this research can be used for synthesizing advanced catalysts for toluene oxidation at significantly lower temperature.

The production of synthesis gas (syngas) involves the gasification of biomass under oxygen-limited conditions, which also produces tars. Tars pose significant problems for mechanical devices by depositing on piping, resulting in clogging and engine fouling. While recent research has shown that thermally impregnated Ni-olivine has been effective in reforming tars into H2 and CO, this technique possessed limited economic feasibility due to high input energy requirements. Thus, stable, active, and inexpensive catalysts are required for effective and efficient conditioning of syngas. This research compared the activity of Ni-olivine catalysts synthesized via electroless plating (ELP) (35°C) and thermal impregnation (TI) (1400°C) for oxiding toluene in a flow-through reactor. The objectives were to (1) determine the kinetics of toluene oxidation, (2) propose a reaction mechanism for toluene oxidation, and (3) investigate the effect of syngas on toluene oxidation. Conversion of toluene using Ni-olivine catalysts increased with increasing ozone concentration and temperature, as well as decreasing toluene molar flow rate, and facilitated the complete oxidation of toluene. The information obtained from this research is expected to provide opportunities for efficient cleanup of tars from biomass gasification facilities at lower temperatures.

Capsaicinoid extraction from peppers is typically performed using organic solvents, however, the extraction efficiencies can vary with peppers, their parts and pre-extraction processing. In the absence of in depth information on capsaicinoid extraction from habanero peppers, this work was undertaken to examine the processing parameters for solvent extraction of capsaicinoids from whole habanero peppers (Capsicum chinense) and their various parts. The effects of solvent type (ethanol, acetone and acetonitrile), pepper part(s) (seeds, shells), tissue preparation (freeze and oven drying), and time on capsaicinoid recovery (capsaicin and dihydrocapsaicin) were evaluated. Across all solvents, capsaicin yields were on average 16, 5 and 8 mg/g dry pepper part for seeds, shells and whole peppers, respectively. Dihydrocapsaicin yield ranged from 0.65 to 9.17 mg/g dry pepper depending on interaction between parts and preparation. Overall, higher yields of capsacinoids were obtained from oven-dried peppers using acetone as the solvent.

Lignocellulosic biomass has been identified among the renewable energy sources to have the highest potential to minimize dependency on dwindling supplies of fossil fuels. Conversion of this biomass to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas (a mixture of CO, CO2, N2 and H2) through gasification and transformed to fuels using microbial catalysts that can convert the CO, H2 and CO2 to fuels such as ethanol, butanol and hydrogen. Biomass gasification–fermentation processing systems have shown promise and companies are now entering the marketplace for commercial-scale ethanol production from synthesis gas. Isolation of new organisms capable of higher product yield, as well as functional implementation of bioreactors that enhance gas solubility for microbial fermentation, make this technology an attractive option for reducing our dependency on fossil fuels.

Agricultural biomass has shown potential to aid in the solution to the rising need for biofuels and other biobased products. This study focused on sweet sorghum, a crop that produces relatively high quantities of easy to process, directly fermentable aqueous sugar. Sweet sorghum is desirable because of the reduced steps and inputs required for complete conversion to ethanol compared to starch and lignocellulosic biomass, as well as its adaptability to temperate regions, drought tolerance, and photosynthetic efficiency. Laboratory fermentation studies were conducted to evaluate the processing parameters for ethanol production on fresh and frozen sorghum juice. The effects of pH level, yeast type, incubation time, and sterilization were investigated. Ethanol and individual sugar concentrations (sucrose, fructose, and glucose) were measured over time. Fresh sorghum juice stocks on average contained 50% w/w sucrose, 24% w/w glucose, and 26% w/w fructose of total initial sugars, while frozen sorghum juice stocks contained 27% w/w sucrose, 37% w/w glucose, and 36% w/w fructose of total initial sugars. Ethanol yields were higher for sterile treatments, with 7.86% v/v ethanol (62 g L-1) and 6.24% v/v ethanol (49.3 g L-1) within 3 days on average for fresh sterile and non-sterile treatments, respectively. Frozen juice samples resulted in 7.81% v/v ethanol (61.6 g L-1) and 7.10% v/v ethanol (56.1 g L-1) for sterile and non-sterile treatments, respectively, after 3 days of incubation. Sterile treatments typically resulted in >84% conversion of sugars to ethanol, while non-sterile treatments ranged between 60% and 75% conversion. Overall, sweet sorghum juice is capable of supporting yeast fermentation with minimal input, making it a suitable substrate for development of on-farm and large-scale industrial ethanol conversion.

Recent trends in health and wellness as well as fossil fuel dependent markets provide opportunities for agricultural crops as renewable resources in partial replacement of synthetic components in food, clothing and fuels. This investigation focused on purple-fleshed industrial sweetpotatoes (ISPs), a crop which is used for industrial purposes because it produces relatively high quantities of antioxidants in the form of anthocyanins as well as high starch content for potential hydrolysis into fermentable sugars. Laboratory extraction and enzymatic hydrolysis studies were conducted on purple-fleshed ISPs in order to evaluate the effects of solvent, extraction temperature and solid loading on recovery of anthocyanins and fermentable sugars. Total monomeric anthocyanin and phenolic concentrations of the extracts were measured. Residual solids from anthocyanin extraction were subsequently hydrolyzed for sugar production (maltotriose, maltose, glucose and fructose). Extraction temperature of 80 °C using acidified methanol at 3.3% (w/v) solid loading showed the highest anthocyanin recovery at 186.1 mg cyanidin-3-glucoside/100 g fw. Acidified solvents resulted in 10–45% and 16–46% more anthocyanins than non-acidified solvents of ethanol and methanol, respectively. On average, glucose production ranged from 268 to 395 mg/g dry ISP. Solid residues that went through extraction with acidified ethanol at 50 °C at 17% (w/v) solid loading had the highest average production of glucose at 395 mg/g dry ISP. Residues from methanol solvents had lower glucose production after hydrolysis compared to those of ethanol based extraction. Fermentation of produced sugars from ISP residues was limited, where 38% less ethanol was produced from extraction residues compared to treatments that did not undergo initial extraction. Overall, purple-fleshed ISPs are amenable to anthocyanin and phenolic extraction, making it a suitable substrate for development of industrial colorants and dyes. However, more research is needed to obtain a suitable extraction point when trying to achieve a high recovery of anthocyanins and effective starch conversion to fermentable glucose.

Effects of initial medium pH and gas flow rate on Clostridium ljungdahlii and Clostridium autoethanogenum in liquid batch, continuous gas fermentations were investigated. Synthesis gas components were supplied at varying flow rates (5, 7.5 and 10 mL/min) for C. ljungdahlii (pH 6.8 and 5.5) and C. autoethanogenum (pH 6.0). Growth on synthesis gas was slower than growth on sugars. For C. ljungdahlii, higher cell densities were achieved at pH 6.8 (579 mg/L) compared to pH 5.5 (378 mg/L). The ethanol concentration at pH 6.8 was also 110% greater than that at pH 5.5. The interaction of flow rate and pH was statistically significant with the greatest acetate production in the 10 mL/min, pH 6.8 treatment. The ethanol to acetate ratios were smaller at lower pH levels and higher flow rates. In C. autoethanogenum fermentations, higher flow rates resulted in greater end product formation with no significant effect on product ratios.

The potential of microbial pretreatment of cotton stalks by Phanerochaete chrysosporium to degrade lignin and facilitate fuel ethanol production was investigated under two culture conditions: submerged cultivation (SmC) and solid state (SSC) cultivation. Although microbial pretreatments showed significant lignin degradation (LD) (19.38% and 35.53% for SmC and SSC, respectively), a study on hydrolysis and fermentation of the microbial-pretreated cotton stalks showed no increase in cellulose conversion (10.98% and 3.04% for SmC and SSC pretreated samples, respectively) compared to untreated cotton stalks (17.93%). Solid state cultivation demonstrated better selectivity of 0.82 than 0.70 with submerged pretreatment. Washing of pretreated cotton stalks did not significantly increase cellulose conversion. However, heating and washing remarkably improved (P

White rot fungi degrade lignin and have biotechnological applications in conversion of lignocellulose to valuable products. Pretreatment is an important processing step to increase the accessibility of cellulosic material in plant biomass, impacting efficiency of subsequent hydrolysis and fermentation. This study investigated microbial pretreatment of cotton stalks by solid state cultivation (SSC) using Phanerochaete chrysosporium to facilitate the conversion into ethanol. The effects of substrate moisture content (M.C.; 65%, 75% and 80% wet-basis), inorganic salt concentration (no salts, modified salts without Mn(2+), modified salts with Mn(2+)) and culture time (0-14 days) on lignin degradation (LD), solids recovery (SR) and availability of carbohydrates (AOC) were examined. Moisture content significantly affected lignin degradation, with 75% and 80% M.C. degrading approximately 6% more lignin than 65% M.C. after 14 days. Within the same moisture content, treatments supplemented with salts were not statistically different than those without salts for LD and AOC. Within the 14day pretreatment, additional time resulted in greater lignin degradation, but indicated a decrease in SR and AOC. Considering cost, solid state cultivation at 75% M.C. without salts was the most preferable pretreatment resulting in 27.6% lignin degradation, 71.1% solids recovery and 41.6% availability of carbohydrates over a period of 14 days. Microbial pretreatment by solid state cultivation has the potential to be a low cost, environmentally friendly alternative to chemical approaches. Moisture relationships will be significant to the design of an effective microbial pretreatment process using SSC technology.

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO(2), H(2), N(2), with smaller amounts of CH(4), NO(x), O(2), C(2) compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.

Enzymes play an important role in the breakdown of lignin during microbial pretreatment of lignocellulosic feedstocks. However, quantification of the various enzyme activities with assays developed for enzyme extracts from pure cultures can be challenging. In this study, spectrophotometric assays used for the quantification of peroxidases in enzyme extracts from submerged (SmC) and solid-state (SSC) cultivation of P. chrysosporium on cotton stalks during 14 days pretreatment failed to detect lignin peroxidase (LiP) and manganese peroxidase (MnP) activities. However, results from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested presence of protein bands with molecular weights corresponding to MnP and LiP in the enzyme extracts from fungal pretreatment cultures. Addition of crude enzyme extracts from SmC and SSC treated samples to fresh cotton stalks showed 3.42% and 7.45% increase in lignin content, respectively. This slight increase may be attributed to components within crude extracts that polymerize the phenolic compounds instead of resulting in delignification. It can be inferred from this study that although qualitative methods for ligninolytic enzyme estimation provide useful information, it is essential to investigate alternative approaches to quantify ligninolytic enzymes during cultivation on natural lignocellulosic materials to overcome the limitations of existing assays.

Clostridium ljungdahlii is an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H 2 , CO, and fructose after 8% O 2 exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and that C. ljungdahlii 's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism of C. ljungdahlii and provides new genetic targets to generate C. ljungdahlii strains that produce more ethanol and are more tolerant to syngas contaminants.

During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20 kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequence datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.

The solid substrate cultivation (SSC) process is often limited by rapid increases in temperature as a result of metabolic activity and poor heat transfer properties of the solid substrate. High temperatures can adversely affect microbial growth and product formation. Reduction of substrate temperatures is important for improved productivity, yet effective and efficient control of heat transfer in a deep-bed SSC reactor has proven difficult. An internal cooling water coil was incorporated into a deep-bed reactor system and evaluated for its ability to reduce substrate temperature. Three operating treatments were investigated: cooling water circulation whenever the center bed temperature was above 30°C, timed cooling water operation on 10 min on/off intervals from the start of cultivation, and timed operation on 10 min on/off intervals after the expected start of microbial exponential growth. Enzyme yield, peak temperature, and net rate of metabolic heat accumulation within the substrate bed were measured during the cultivation of Trichoderma longibrachiatum on wheat bran for xylanase production. The cooling water reduced the temperatures observed in the substrate bed by 5°C on average and was effective in decreasing the rate of heat accumulation in the bed. Within a 10 min time interval, the internal heat exchanger demonstrated a net decrease in heat accumulation at an average rate of approximately 0.5°C/min. Despite the decrease in temperatures observed with the control strategies, the xylanase yields were not statistically different from the cultivations with no temperature control.

Fungal pretreatment, using lignin-degrading microorganisms to improve lignocellulosic feedstocks with minimal energy input, is a potential alternative to physiochemical pretreatment methods. Identifying the kinetics for fungal pretreatment during solid substrate cultivation is needed to help establish the processing conditions for effective scale up of this technology. In this study, a set of mathematical models were proposed for describing the interactions between holocellulose consumption, lignin degradation, cellulase, ligninolytic enzyme, and the growth of Phanerochaete chrysosporium during a 14 day fungal pretreatment process. Model parameters were estimated and validated by the System Biology Toolbox in MatLab. Developed models provided sufficiently accurate predictions for fungal growth (R 2 = 0.97), holocellulose consumption (R 2 = 0.97), lignin degradation (R 2 = 0.93) and ligninolytic enzyme production (R 2 = 0.92), and fair prediction for cellulase production (R 2 = 0.61). The models provide valuable information for understanding the interactive mechanisms in biological systems as well as for fungal pretreatment process scale up and improvement.

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(−1) of butanol and 26.3 g l(−1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(−1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.

Clostridium autoethanogenum is an anaerobic, autotrophic acetogen that is capable of converting CO and CO 2 into ethanol and acetate. Here we report the draft genome sequence of C. autoethanogenum JA1-1 strain DSM 10061 (4.5 Mbp; G+C content, 37.5%) and the findings obtained from annotation of the genome sequence.

North Carolina has grown to the third largest pork exporter in the United States mainly through the use of concentrated animal feed operations. Hog farming produces a large quantity of nutrient rich waste material that is generally disposed through land application. Currently the fields are composed of primarily bermudagrass which is removed at regular intervals to reduce nutrient loads. This material has a limited market and due to the high density of hog farms the market is over saturated in many areas of North Carolina. Using high yielding bioenergy crops may increase nutrient removal rates, supplement farmer income, reduce bioenergy feedstock production costs, and provide beneficial bioenergy production characteristics. Sweet sorghum was chosen as the feedstock of choice due to its accelerated growth rate, high bioethanol production potential, and ease of crop management. Five scenarios have been used for comparison: sweet sorghum grown on effluent spray fields, traditional farm grown sweet sorghum, and bermudagrass grown on effluent spray fields (at replanting schedules of 20 year, 10 year, and 5 year). Economic, environmental, and energy assessments are calculated to the field edge due to the multiple processing possibilities related to sweet sorghum.

We describe a latex wet coalescence method for gas-phase immobilization of microorganisms on paper which does not require drying for adhesion. This method reduces drying stresses to the microbes. It is applicable for microorganisms that do not tolerate desiccation stress during latex drying even in the presence of carbohydrates. Small surface area, 10–65 μm thick coatings were generated on chromatography paper strips and placed in the head-space of vertical sealed tubes containing liquid to hydrate the paper. These gas-phase microbial coatings hydrated by liquid in the paper pore space demonstrated absorption or evolution of H2, CO, CO2 or O2. The microbial products produced, ethanol and acetate, diffuse into the hydrated paper pores and accumulate in the liquid at the bottom of the tube. The paper provides hydration to the back side of the coating and also separates the biocatalyst from the products. Coating reactivity was demonstrated for Chlamydomonas reinhardtii CC124, which consumed CO2 and produced 10.2 ± 0.2 mmol O2 m−2 h−1, Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47 ± 0.04 mmol H2 m−2 h−1, Clostridium ljungdahlii OTA1, which consumed 6 mmol CO m−2 h−1, and Synechococcus sp. PCC7002, which consumed CO2 and produced 5.00 ± 0.25 mmol O2 m−2 h−1. Coating thickness and microstructure were related to microbe size as determined by digital micrometry, profilometry, and confocal microscopy. The immobilization of different microorganisms in thin adhesive films in the gas phase demonstrates the utility of this method for evaluating genetically optimized microorganisms for gas absorption and gas evolution.

Microbial pretreatment of lignocellulosic feedstocks is an environment friendly alternative to physio-chemical pretreatment methods. A better understanding of the interactive fungal mechanisms in biological systems is essential for enhancing performance and facilitating scale-up and commercialization of this pretreatment technique. In this study, mathematical models were developed for describing cellulose and hemicellulose consumption, lignin degradation, cellulase and ligninolytic enzyme production and oxygen uptake associated with the growth of Phanerochaete chrysosporium during a 14-day shallow stationary submerged fungal pretreatment process on cotton stalks. Model parameters were estimated and validated by Statistics Toolbox in MatLab 7.1. Models yielded sufficiently accurate predictions for cellulose and hemicellulose consumption ( R 2 = 0.9772 and 0.9837), lignin degradation ( R 2 = 0.9879 and 0.8682) and ligninolytic enzyme production ( R 2 = 0. 8135 and 0.9693) under both 1-day and 3-day oxygen flushing conditions, respectively. The predictabilities for fungal growth ( R 2 = 0.6397 and 0.5750) and cellulase production ( R 2 = 0.0307 and 0.3046) for 1-day and 3-day oxygen flushing, respectively, and oxygen uptake ( R 2 = 0.5435) for 3-day oxygen flushing were limited.

Syngas fermentation is considered an alternate processing method for biofuel and biochemical production as part of thermochemical biomass conversion. Exposure of syngas fermenting microorganisms to sugars, either in the primary syngas fermentation or through pre-adaptation in the seed culture, has the potential to enhance overall fermentation performance and stress tolerance. In this rapid communication, Clostridium ljungdahlii was grown on different carbon sources including syngas only, syngas-fructose and fructose only to identify ideal pre-adaptation conditions for ethanol and acetate production from subsequent cultures grown in reactors containing syngas only or fructose-syngas substrates. In syngas only reactors, cultures pre-adapted to fructose had faster cell production rates (2X) and at least 83% higher ethanol and 16% higher acetate formation than cells pre-adapted on syngas or syngas-fructose. In syngas- fructose reactors, cultures did not show significant growth or acetate production differences under pre-adaptation treatments. Nevertheless, in these syngas-fructose reactors, cultures pre-adapted on syngas and syngas-fructose had nearly 20% higher ethanol production than those pre-adapted on fructose. Among pre-adaptation treatments, fructose had better results in syngas only reactors than syngas-fructose reactors. However, the presence of syngas in pre- adaptation cultures was better overall for ethanol production.

World energy consumption is expected to increase 44% in the next 20 years. Today, the main sources of energy are oil, coal, and natural gas, all fossil fuels. These fuels are unsustainable and contribute to environmental pollution. Biofuels are a promising source of sustainable energy. Feedstocks for biofuels used today such as grain starch are expensive and compete with food markets. Lignocellulosic biomass is abundant and readily available from a variety of sources, for example, energy crops and agricultural/industrial waste. Conversion of these materials to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas through gasification and transformed to fuels using chemical catalysts. Chemical conversion of synthesis gas components can be expensive and highly susceptible to catalyst poisoning, limiting biofuel yields. However, there are microorganisms that can convert the CO, H(2), and CO(2) in synthesis gas to fuels such as ethanol, butanol, and hydrogen. Biomass gasification-biosynthesis processing systems have shown promise as some companies have already been exploiting capable organisms for commercial purposes. The discovery of novel organisms capable of higher product yield, as well as metabolic engineering of existing microbial catalysts, makes this technology a viable option for reducing our dependency on fossil fuels.

World energy consumption is expected to increase 44% in the next 20 years. Today, the main sources of energy are oil, coal, and natural gas, all fossil fuels. These fuels are unsustainable and contribute to environmental pollution. Biofuels are a promising source of sustainable energy. Feedstocks for biofuels used today such as grain starch are expensive and compete with food markets. Lignocellulosic biomass is abundant and readily available from a variety of sources, for example, energy crops and agricultural/industrial waste. Conversion of these materials to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas through gasification and transformed to fuels using chemical catalysts. Chemical conversion of synthesis gas components can be expensive and highly susceptible to catalyst poisoning, limiting biofuel yields. However, there are microorganisms that can convert the CO, H(2), and CO(2) in synthesis gas to fuels such as ethanol, butanol, and hydrogen. Biomass gasification-biosynthesis processing systems have shown promise as some companies have already been exploiting capable organisms for commercial purposes. The discovery of novel organisms capable of higher product yield, as well as metabolic engineering of existing microbial catalysts, makes this technology a viable option for reducing our dependency on fossil fuels.

This study used the fungus, Phanerochaete chrysosporium, to pretreat cotton stalks with two methods, shallow stationary and agitated cultivation, at three supplemental salt concentrations. Pretreatment efficiencies were compared by evaluating lignin degradation, solid recovery and carbohydrate availability over a 14-day period. Shallow stationary cultivation with no salts gave 20.7% lignin degradation along with 76.3% solid recovery and 29.0% carbohydrate availability. The highest lignin degradation of 33.9% at a corresponding solid recovery and carbohydrate availability of 67.8% and 18.4%, respectively, was obtained through agitated cultivation with Modified NREL salts. Cultivation beyond 10 days did not significantly increase lignin degradation during 14 days of pretreatment. Manganese addition during shallow stationary and agitated cultivation resulted in higher solid recoveries of over 80% but lower lignin degradation. Although agitated cultivation resulted in better delignification, results indicate that pretreatment under submerged shallow stationary conditions provides a better balance between lignin degradation and carbohydrate availability.

Spore-forming solventogenic Clostridium spp. are receiving renewed attention due to their butanol production abilities. However, there is an absence of literature describing the preparation of dense, vigorous and homogeneous seed cultures of Clostridium spp., guaranteeing reproducibility during fermentation. Therefore, we performed a series of growth experiments of Clostridium beijerinckii NCIMB 8052 and its offspring SA-1 to evaluate the influence of inoculum age (harvest time) on the subsequent population's maximum specific growth rate, as a signal of population homogeneity. The organisms were cultivated in Reinforced Clostridial Medium and supplemented sweet sorghum juice. The best inoculum ages coincided with the late-exponential growth phase: between 9 and 11 h in the conditions tested. Additionally, the harvest time was delayed up to 4 h by pre-adapting the seed culture with 0.75 g L(-1) butyric acid. These findings were validated by performing a series of bench-top batch fermentations showcasing reproducibility in growth kinetics with 95% confidence limits. Overall, these experiments allowed us to understand the transient nature of seed cultures of C. beijerinckii NCIMB 8052 and SA-1, while enabling reproducibility and consistent culture performance.

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100 mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.

Solid substrate cultivation of thermophilic, anaerobic bacteria offers an alternative production method for many bio-based chemicals; however the process must be optimized for each substrate-organism fermentation. The effects of initial substrate moisture content (SMC, 30%, 50% and 70% wet-basis), supplemental nutrient concentration (SNC, 12%, 50% and 100%) and duration of cultivation time (6, 10 and 14 days), on product formation (lactate, ethanol and acetate) by Clostridium thermocellum 27405 were examined during growth on paper pulp sludge. Water activities at moisture contents above 30% wet-basis were essentially identical ( approximately 0.99), yet the water contents differed significantly, and affected the metabolic activity of C. thermocellum. Increases in initial substrate moisture content from 50% to 70% for cultures supplemented with 50% or 100% nutrients resulted in a 75-145 mM increase in total end products. At 70% SMC, the addition of 100% SNC generated a 56% increase in product formation above the addition of 50% nutrient supplementation. Increases in the quantity of free water present in the solid substrate cultivation system up to the water holding capacity of the paper pulp sludge led to improved performance of this anaerobic bacterium. While nutrient supplementation is common in the form of salts for many aerobic microorganisms, efficient metabolism for anaerobic C. thermocellum grown in SSC was highly dependent on added salts, vitamins and reducing agents. Further studies are needed to determine if this is a general effect for other anaerobes grown in solid substrate cultures.

This work describes an instrumentation and data acquisition system designed for a deep bed reactor used to cultivate Trichoderma longibrachiatum on wheat bran. The system allowed on–line measurements of substrate temperature, oxygen concentration within the reactor headspace, relative humidity and temperature of the inlet air, and inlet airflow rates while maintaining aseptic conditions and without disturbing the cultivation process. An error analysis for the instrumentation and data acquisition equipment was completed and provided insight into the reliability of the sensor readings. The collected data provided quantitative information about the reactor system dynamics which can be used to evaluate and apply environmental control schemes, gain knowledge on microbial growth characteristics, and develop and validate mathematical models describing heat and mass transfer interactions.