Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

Effect of temperature on sucrose to ethanol conversion by Zymomonas mobilis strains

Summary Two strains of Zymomonas mobilis were tested for their ability to ferment sucrose to ethanol at elevated temperatures (30–42.5°C). The optimal temperature for efficient sucrose to ethanol conversion was 35°C with 22–27 h fermentation time and 75% conversion efficiency. Increases in magnesium concentration improved one of the strains at 40°C from 38 to 76% ethanol yield efficiency.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

A continuous thermophilic cellulose fermentation in an upflow reactor by aClostridium thermocellum-containing mixed culture

Summary A continuous thermophilic cellulose fermentation by aCl. thermocellum-containing mixed culture was carried out in an upflow reactor for a period of 100 days. The cellulose conversion rate was finally 0.35 g.1^–1.h^–1. Evidence that the fermentation process was influenced by both pH and dilution rate was given by the changes of concentration of the main fermentation products, acetic acid and ethanol. The role of cellodextrins and glucose as reactive intermediates in the process of cellulose breakdown was established.     

The effect of temperature on the ethanol tolerance of the yeast,Saccharomyces uvarum

The optimum temperature for fermentation by Saccharomyces uvarum was found to be higher than that for its growth. Fermentation continued at temperatures above the growth maximum (40°C). S.uvarum was most resistant to growth inhibition by ethanol at temperatures 5°C and 10°C below its growth optimum (35°C). Fermentation became more resistant to ethanol inhibition with increasing temperature.     

Solvent production byClostridium pasteurianum in media of high sugar content

Summary The fermentation end products ofClostridium pasteurianum ATCC 6013 are normally acetic and butyric acids. When grown in media of high sugar content however, significant quantities of solvents (acetone, butanol and ethanol) were produced. Solvent production was not stimulated by added acetic and butyric acids, nor was the effect due to a low water activity of the mediumper se.     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad     

The effect of temperature on the kinetics of ethanol production by a thermotolerant strain of Kluveromyces marxianus

Summary The batch fermentation kinetics of a novel thermotolerant strain of the yeast Kluveromyces marxianus were evaluated between 30°C and 48°C. The most significant effects of elevated temperature were reductions in overall biomass and ethanol yields. Decreases in the concentration of ethanol attained, and the presence of unutilized substrate suggested increased ethanol inhibition at the higher temperatures studied.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Selection of yeast able to produce ethanol from glucose at 40° C

Summary A total of 55 yeast strains selected from 7 genera known to ferment carbohydrates to ethanol were screened for their ability to ferment glucose to ethanol in shaken flask culture at 37°, 40° and 45°C. Yields of more than 50% of the theoretical maximum were obtained with 28 strains at 37°C, but only 12 at 40°C. Only 6 could grow at 45°C, but they produced poor yields. In general Kluyveromyces strains were more thermotolerant than Saccharomyces and Candida strains, but Saccharomyces strains produced higher ethanol yields. The 8 strains with the highest yields at 40°C were evaluated in batch fermentations. Three of these, two Saccharomyces and one Candida, were able to meet minimum commercial targets set at 8% (v/v) ethanol from 14% (w/v) glucose at 40°C.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

The effect of pH and temperature manipulation on metabolite composition during acidogenesis in a hybrid anaerobic digester

Summary A synthetic medium containing 9 g/l sucrose was hydrolyzed in a novel hybrid reactor. A minimum hydraulic retention time (HRT) of 9.9 h, with a gas production rate of 1.07 m³/m³·d, was obtained without continuous neutralization. A viable anaerobic cell count of 10⁹ organisms/ml was obtained in the reactor fluid. The results showed that both pH and temperature significantly influenced the type and concentration of the various metabolites formed. These include ethanol, formic, acetic, propionic and butyric acids as primary metabolites and caproic acid as secondary metabolite. From the results obtained, it is suggested that to obtain the energetically most favourable products, a substrate pH of 6.5 and a temperature of 35°C must be used in anaerobic acidogenic digesters.     

Combined effect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast

Summary The internal pH of Saccharomyces cerevisiae IGC 3507 III (a respiratory-deficient mutant) was measured by the distribution of [¹⁴C]propionic acid, when the yeast was fermenting glucose at pH 3.5, 4.5 and 5.5 in the presence of several concentrations of acetic acid and ethanol. Good correlation was obtained between fermentation rates and internal pH. For all external pH values tested, the internal pH was 7.0–7.2 in the absence of inhibitors. The addition of acetic acid and/or ethanol resulted in a decrease of fermentation rate together with a drop in internal pH. Internal pH did not depend on the concentration of total external acetic acid but only on the concentration of the undissociated form of the acid. Ethanol potentiated the effect of acetic acid both with respect to inhibition of fermentation and internal acidification.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Catabolism of 3-indole acetic acid in protoplasts from etiolated seedlings of scots pine (Pinus sylvestris L.)

Protoplasts preparated from dark grown seedlings of Pinus sylvestris L. were incubated with 3-indole (1-¹⁴C) acetic acid and 3-indole (2-¹⁴C) acetic acid (IAA). Three catabolites were consistently produced in the (2-¹⁴C) IAA feeds, one of which co-chromatographed with 3-indole methanol on reversed phase high performance liquid chromatography (HPLC). Protoplasts feed with (1-¹⁴C) IAA produced only one labelled catabolite. The non-decarboxylated compound formed was highly polar on reversed phase HPLC, both in the ion suppression and the ion pair mode. The substance was not hydrolysable at pH 11 and 100° indicating that it is not a conjugated form. Effects of time of incubation, pH and the cofactors hydrogen peroxide and 2,4-dichlorphenol on the catabolic rate of IAA are discussed.Abbreviations BSA bovine serum albumin - DCP 2,4-dichlorophenol - HPLC high performance liquid chromatography - IAA 3-indole acetic acid - IAld 3-indole carboxaldehyde - ICA 3-indole carboxylic acid - IM 3-indole methanol - MES 4-morpholineethane sulfonic acid - MO 3-methyl-oxindol - MnO 3-methylene-oxidol - OxIAA 3-oxindole acetic acid - PEG polyethylene glycol     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Production of ethanol,organic acids and hydrogen: an opportunity for mixed culture biotechnology?

Anaerobic fermentation of biodegradable organic materials is usually carried out to obtain the final product, methane, a valuable energy source. However, it is also well known that various intermediates are produced in this process, e.g. ethanol, volatile organic acids and hydrogen. All these species have applications and value as fuels or chemicals. This paper shows a critical analysis of the potential of using anaerobic fermentation by mixed cultures to produce intermediates, e.g. ethanol, acetic, lactic and butyric acid and hydrogen, rather than methane. This paper discusses the current processes to produce these chemicals and compares them with the alternative approach of using open mixed cultures to produce them simultaneously via fermentation from renewable resources. None of these chemicals is currently produced via mixed culture fermentation: ethanol and lactic acid are usually produced in pure culture fermentation using food crops, e.g. corn or sugar cane, as starting materials; hydrogen, acetic and butyric acids are mainly produced via chemical synthesis from fossil fuel derived starting materials. A possible flow-sheet for the production of these chemicals from organic waste using mixed culture fermentation is proposed and the advantages and disadvantages of this process compared to current processes are critically discussed. The paper also discusses the research challenges which need to be addressed to make this process feasible.     

Temperature effects in ethanol fermentation high corn media

Summary A relationship between temperature and high ethanol yields has been found using whole corn mashes saccharified with Aspergillus oryzae wheat bran koji. Decreased ethanol yields were obtained at 34.5°C with high concentration corn mashes in contrast to high ethanol yields with the same medium at lower temperatures. The decreased yields appear to be related to mass and/or heat transfer problems rather than primary ethanol toxicity. Scale-up of the high corn medium will require a re-evaluation of alcohol fermentation technology.