Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation

Cocultivation of cellulolytic and saccharolytic microbial populations is a promising strategy to improve bioethanol production from the fermentation of recalcitrant cellulosic materials. Earlier studies have demonstrated the effectiveness of cocultivation in enhancing ethanolic fermentation of cellulose in batch fermentation. To further enhance process efficiency, a semicontinuous cyclic fed-batch fermentor configuration was evaluated for its potential in enhancing the efficiency of cellulose fermentation using cocultivation. Cocultures of cellulolytic Clostridium thermocellum LQRI and saccharolytic Thermoanaerobacter pseudethanolicus strain X514 were tested in the semicontinuous fermentor as a model system. Initial cellulose concentration and pH were identified as the key process parameters controlling cellulose fermentation performance in the fixed-volume cyclic fed-batch coculture system. At an initial cellulose concentration of 40 g liter⁻¹, the concentration of ethanol produced with pH control was 4.5-fold higher than that without pH control. It was also found that efficient cellulosic bioethanol production by cocultivation was sustained in the semicontinuous configuration, with bioethanol production reaching 474 mM in 96 h with an initial cellulose concentration of 80 g liter⁻¹ and pH controlled at 6.5 to 6.8. These results suggested the advantages of the cyclic fed-batch process for cellulosic bioethanol fermentation by the cocultures.     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.     

Application of a Doehlert experimental design to the optimization of microbial degradation of crude oil in sea water by continuous culture

Summary Crude oil was degraded by a mixed bacterial community grown in continuous culture on sea water. The fermentation process included an emulsification step prior to the introduction of the substrate in the reactor, with external cell recycling by a tangential-flow filtration system. Optimization of the fermentation technique was achieved by using the surface response methodology (Doehlert experimental design). Besides reducing the number of experiments, this approach allowed optimal experimental conditions to be chosen, for the particular goal: percent degradation of crude oil (80%), biomass (7.6 g·l^-1) and degradation rate (0.73 g·l^-1·h^-1). This biodegradation process could be used as a tool to fight against pollutions by petroleum products.     

Continuous enzymatic saccharification of cellulose with culture filtrates of trichoderma viride QM 6a.

Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr^?1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.     

Production of Extracellular Polysaccharide by Zoogloea ramigera

In batch cultures of Zoogloea ramigera the maximum rate of exopolysaccharide synthesis occurred in a partly growth-linked process. The exopolysaccharide was attached to the cells as a capsule. The capsules were released from the cell walls after 150 h of cultivation, which caused the fermentation broth to be highly viscous. Ultrasonication could be used to release capsular polysaccharide from the microbial cell walls. Treatment performed after 48 to 66 h of cultivation revealed exopolysaccharide concentration and apparent viscosity values in accordance with values of untreated samples withdrawn after 161 h of cultivation. The yield coefficient of exopolysaccharide on the basis of consumed glucose was in the range of 55 to 60% for batch cultivations with an initial glucose concentration of 25 g liter⁻¹. An exopolysaccharide concentration of up to 38 g liter⁻¹ could be attained if glucose, nitrogen, and growth factors were fed into the batch culture. The oxygen consumption rate in batch fermentations reached 25 mmol of O₂ liter⁻¹ h⁻¹ during the exopolysaccharide synthesis phase and then decreased to values below 5 mmol of O₂ liter⁻¹ h⁻¹ during the release phase. The fermentation broth showed pseudoplastic flow behavior, and the polysaccharide was not degraded when growth had ceased.     

Cellulose degradation and glucose accumulation byClostridium thermocellum ATCC 27405 under different cultural conditions

Effect of various cultural parameters on cellulose degradation, glucose accumulation and ethanol production byClostridium thermocellum ATCC 27405 were investigated. Optimum pH values for glucose accumulation and ethanol production were determined as 7 and 10, respectively. Highest amount of ethanol (0.92 g/l) was obtained from the culture which contains 10 g urea/l with 34.5% decrease in glucose accumulation. Addition of 100 mM phosphate to the medium increased ethanol production while cellulose degradation and sugar accumulation decreased by 34 and 99%, respectively. Among minerals tested, Mg⁺² was found to be the most important element which affects cellulose degradation. When the medium contained no Mg⁺², residual cellulose concentration was 4.3 g cellulose/l. When the cultural parameters were optimised, glucose accumulation started at early days of fermentation and glucose concentration was 60% higher than that of the control at the 10^th day of fermentation.     

Production of laccase and manganese peroxidase by<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> grown on wheat bran

The aim of this work was to study the growth and production of ligninolytic enzymes by Fomes sclerodermeus using a natural medium based on wheat bran as the principal substrate in a solid-state fermentation. Growth was monitored by measuring the chitin content in the substrate. The maximum rate of growth was observed between days 7 and 18. A 38% total dry-weight loss of the substrate was measured after 28 days of cultivation. Differential hydrolysis of the substrate revealed that cellulose was more extensively degraded than lignin. In the 28-day incubation period, the losses of cellulose and lignin were 38 and 15%, respectively. No lignin peroxidase activity was found in any of the media tested. The maximum manganese-dependent peroxidase activity recorded was 6.3 U g⁻¹ at 14 days, while the maximum laccase activity was 270 U g⁻¹ at 28 days post-inoculation. Addition of commonly used inducers such as copper or manganese did not produce a further increase in the enzyme activities, nor did addition of glucose, asparagine, or malt extract. Electronic Publication     

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.     

Production of cellulose from glucose by Acetobacter xylinum

Acetobacter xylinum 1FO 13693 was selected as the best cellulose-producing bacterium among 41 strains belonging to the genus Acetobacter and Agrobacterium. Cellulose was found to be produced at the liquid surface in static liquid cultivation. The rate of cellulose production depended proportionally on the surface-area of the culture medium and was unaffected by the depth and volume of the medium. The optimum pH for cellulose production was 4.0 to 6.0. Glucose, fructose and glycerol were preferred carbon sources for cellulose production. The yield of cellulose, relative to the glucose consumed, decreased with an increase in initial glucose concentration, and gluconic acid accumulated at a high initial glucose concentration. The decrease in cellulose yield could be due to some glucose being metabolized to gluconic acid. However, the accumulated gluconic acid did not affect cellulose production. The culture conditions of the bacterium for cellulose production were optimized. The maximum production rate of cellulose was 36 g/d·m², with a yield of 100% for added glucose under the optimal conditions.     

Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Estimating the maintenance energy and biomass concentration of Saccharomyces cerevisiae by continuous calorimetry

Continuous calorimetry has been applied to monitoring the heat evolution of Saccharomyces cerevisiae grown on d-glucose. The heat evolution, together with the energy and carbon balances, was used to evaluate the energetic efficiency of biomass, by-product biosynthesis, fermentative heat evolution as well as the maintenance energy of S. cerevisiae in ‘aerobic fermentation’ and ‘aerobic respiration’. In aerobic fermentation, under catabolite repression, the fraction of substrate energy converted to heat evolution, maintenance requirement, and biomass decreased with the increase of d-glucose concentration. The fraction of substrate energy converted to ethanol is the highest value and it could contribute up to 70% of the total substrate energy. In aerobic respiration, 43% of the total substrate energy was evolved as heat. While 50% of the total substrate energy was converted into biomass, only 7% of the total substrate energy was used for maintenance functions. The maintenance energy coefficient of S. cerevisiae was determined to be 0.427 MJ kg^?1 cell h^?1 (0.102 kcal g^?1 cell h^?1). For the first time, heat evolution together with yield-maintenance energy was used to predict biomass concentration during the fed-batch cultivation of S. cerevisiae.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Effect of caffeine concentration on biomass production, caffeine degradation, and morphology of Aspergillus tamarii

The aim of the present study was to evaluate the effect of the initial caffeine concentration (1–8 g/L) on growth and caffeine consumption by Aspergillus tamarii as well as pellet morphology, in submerged fermentation. Caffeine was used as sole nitrogen source. At 1 g/L of initial caffeine concentration, caffeine degradation was not affected, resulting in a production of 8.7 g/L of biomass. The highest biomass production (12.4–14.8 g/L) was observed within a range of 2 to 4 g/L of initial caffeine concentration. At these initial caffeine concentrations, after 96 h of fermentation, 41–51 % of the initial caffeine was degraded. Using an initial caffeine concentration of 2–3 g/L, the highest specific growth rate was observed (μ?=?0.069 1/h). Biomass production decreased at 8 g/L of initial caffeine concentration. A. tamarii formed mainly pellets at all concentrations tested. The size of the pellet decreased at a caffeine concentration of 8 g/L.     

Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase

Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated P_ADB3.8) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB). In the systems, cellulase was immobilized on pH-response copolymer P_MDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH₄)₂SO₄. Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.     

Cellulose Fermentation by an Asporogenous Mutant and an Ethanol-Tolerant Mutant of Clostridium thermocellum

Two mutants of Clostridium thermocellum were isolated after UV light mutagenesis. Mutant A1, selected as asporogenous, exhibited a fermentation pattern similar to that of the wild type. However, at pH 6.5, the mutant degraded 12% more cellulose than did the wild type, leading to enhanced ethanol production. Mutant 647, selected as ethanol tolerant, was able to grow in medium containing 4% ethanol. During the early stage of the exponential growth phase, ethanol was produced as the main product, up to a concentration of about 9 g/liter. After 3 days of culture, 48.3 g (89% of the initial amount) of degraded cellulose per liter was fermented into 12.7 g of ethanol per liter.