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.     

Production of ethanol and biomass from thin stillage by Neurospora intermedia: A pilot study for process diversification

Dry mill ethanol processes produce ethanol and animal feed from whole grains, where the wastewater after the distillation and separation of solid materials is called “thin stillage.” In this work, similar production of ethanol (3.5 g/L) and biomass (5 g/L) from thin stillage was obtained during batch cultivation of the edible fungus Neurospora intermedia in a 2‐m high airlift reactor and bubble column. The fungal biomass, containing 50% w/w protein and 12% w/w lipids, was rich in essential amino acids and omega‐3 and ‐6 fatty acids. In a continuous mode of fermentation, dilution rates of up to 0.2 h^?1 could be applied without cell washout in the bubble column at 0.5 vvm. At 0.1 h^?1, around 5 g/L of ethanol and 4 g/L of biomass containing ca. 50% w/w protein were produced. The fungus was able to assimilate saccharides in the liquid fraction as well as sugar backbones such as xylan and arabinan in the solid fraction. The inclusion of the current process could potentially lead to the production of 11 000 m³ of ethanol (5.5% improvement vs. normal industrial process) and around 6300 tons of high‐quality biomass for animal feed at a typical facility producing 200 000 m³ ethanol per year.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

Continuous ethanol production using induced yeast aggregates

Summary The induction of yeast cell aggregates in a column reactor was initiated by packing yeast cell paste of Saccharomyces uvarum into the column, and then YMP broth was fed into the column from the bottom at a linear flow rate of 2.5 cm/h. Thereafter, yeast cells aggregated in the column within 48 h without a supply of oxygen. When this yeast aggregate column reactor was used for continuous ethanol production, a final ethanol concentration of 10.8% (w/v) was obtained from 23% (w/v) of glucose in a YMP broth with a dilution rate of 0.05 h^-1, and 4.9% (w/v) was obtained from 10% (w/v) of glucose with a dilution rate of 0.6 h^-1. The theoretical yield was above 97% in both cases. The ethanol production rates were 13 g¹ h^-1 l^-1 and 90 g¹ h^-1 l^-1 for producing 10.8% (w/v) and 4.9% (w/v) of ethanol respectively. This column reactor was maintained at a steady state for more than one month.     

Effect of cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation

Summary An overflow filtration unit for cell recycle with Clostridium acetobutylicum was developed. A cellulose-triacetate ultrafiltration membrane with a cut-off volume of 20 000 MW was found to work best. C. acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH value of 4.4 with cell recycle, the cell dry weight in the culture vessel reached 13.1 g/l at a dilution rate of D=0.10 h^-1 and 37°C. 377 mM of glucose were fermented to 190 mM butanol, 116.2 mM acetone and 25.8 mM ethanol. Total acids were 47.6 mM. The butanol productivity was 1.41 g/l/h. At a dilution rate of 0.40 h^-1 the butanol productivity was increased to 4.1 g/l/h but glucose consumption was decreased to 285 mM and butanol, acetone and ethanol production to 138.2, 97.5, 16.5 mM, respectively.     

The development of the photosynthetic apparatus and energy transduction in malate-limited phototrophic cultures of Rhodobacter capsulatus

The question was studied whether limited availability of the carbon source controls the development of the photosynthetic apparatus in Rhodobacter capsulatus. The organisms were grown phototrophically in a chemostat limited by malate as the sole source of reducing equivalents and carbon. The incident light-energy flux, representing the only energy source, was kept constant. Steady state levels of protein and dry weight of cells as well as molar growth yield coefficients (Y) decreased with increasing dilution rate (D, representing the growth rate, ) up to about D=0.14 h^-1. At higher D-values biomass levels as well as Y stayed largely constant. The specific rate of malate consumption leading to biomass production increased linearly while the rate representative of processes other than conversion of carbon into biomass increased almost exponentially with . Specific bacteriochlorophyll (Bchl) contents of cells as well as the specific rate of Bchl synthesis were rather low at low D-values. They increased as D was increased. Light energy fluxes required to half-maximally saturate proton extrusion by whole cells decreased when D was increased up to 0.1 h^-1; at higher D-values, however, they reached constancy. Maximal rates of proton extrusion as well as of photophosphorylation calculated on a Bchl basis decreased when D was increased up to 0.14 h^-1 and reached constancy at higher D-values. The results suggest that the availability of the growth limiting substrate controls the formation of the photosynthetic apparatus and, consequently, its functional properties including the efficiency of light-energy transduction. A relationship is assumed between malate conversion into biomass, i.e. Y-values, and the efficiency of light-energy transduction.Abbreviations ALA 5-aminoleyulinic acid - Bchl bacteriochlorophyll - D dilution rate [h^-1] - R Rhodobacter - Y molar growth yield coefficient - growth rate [h^-1]     

Production of acetic acid by Acetogenium kivui

Summary The formation of acetic acid by the thermophilic nonsporeforming homoacetogenic bacterium Acetogenium kivui was studied under various conditions. In pH-controlled batch fermentation at pH 6.4 this bacterium was able to produce up to 625 mM of acetic acid from glucose within 50–60 h. The value of _max obtained was about 0.17 h^-1, the yield was about 2.55 mol of acetic acid per mol of glucose utilized. In continuous fermentation both substrate concentration and dilution rate (D) influenced the yield of acetate and the stationary concentration: a glucose concentration of 67 mM at D=0.09 h^-1 resulted in 2.82 mol acetate/mol glucose and 190 mM acetate at a production rate of 17.1 mM/1 h. When the dilution rate was increased the production rate reached a maximal value of 43.2 mM/1 h at D=0.32 h^-1. At a glucose concentration of 195 mM the dependence of yield upon dilution rate followed a similar pattern and an acetate concentration of 420 mM could be obtained. Enzymatic studies indicate that in A. kivui pyruvate ferredoxin-oxidoreductase and acetate kinase are inhibited at acetate concentrations higher than 800 mM. Based on these results a fed-batch fermentation was developed, which allowed to produce more than 700 mM acetic acid within 40–50 h.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle

Summary To increase the solvent productivity of the acetone-butanol fermentation, a continuous culture of Clostridium acetobytylicum with cell recycling was used. At a dry cell mass concentration of 8 g l^-1 and a dilution rate of D=0.64 h^-1, a solvent productivity of 5.4 g l^-1 h^-1 was attained. To prevent degeneration of the culture, which occurs with high concentrations of solvents (acetone, butanol and ethanol), different reactor cascades were used. A two-stage cascade with cell recycling and turbidostatic cell concentration control turned out to be the best solution, the first stage of which was kept at relatively low cell and product concentrations. A solvent productivity of 3 and 2.3 g l^-1 h^-1, respectively, was achieved at solvent concentrations of 12 and 15 g l^-1.Symbols D Dilution rate (h^-1) - r _p solvent productivity (g l^-1 h^-1) - s residual glucose concentration (g l^-1) - V _R reactor volume (l) - V _O overall volume (l) - x (dry) cell mass concentration (g l^-1) - Y _P/S solvent yield (g g^-1)     

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

Summary The present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (_max) of 0.1 h^-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the _max was 0.13 h^-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (_max liquid=0.13 h^-1, _max vapour=0.12 h^-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h^-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO₂ and unidentified byproducts.     

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

Summary Cryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h^-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h^-1 in nitrogen-limited (Y _L/S=0.16 g/g) as in carbon-limited (Y _L/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h^-1) and a slight decrease was observed at D=0.08 h^-1, while the specific lipid productivity, q _L, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near _max, whereas q _L was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (/mole) was also observed with increasing lipid content in the cells.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776

Summary Enzymatic hydrolysates of hemicellulose from steam-pretreated aspenwood were more fermentable than the acid hydrolysate after rotoevaporation or ethyl acetate extraction treatments to remove acetic acid and sugar- and lignin-degradation products prior to fermentation by Pichia stipitis CBS 5776. Total xylose and xylobiose utilization from 5.0% (w/v) ethyl acetate extracted enzymatic hydrolysate was observed with an ethanol yield of 0.47 g ethanol/g total available substrate and an ethanol production rate of 0.20 g·l^-1 per hour in 72 h batch fermentation.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Acetaldehyde mediates growth stimulation of ethanol-stressed <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: evidence of a redox-driven mechanism

The ability of acetaldehyde (90 mg l⁻¹) to stimulate ethanol-stressed S. cerevisiae fermentations is examined and reasons for the effect explored. Alternative metabolic electron acceptors generated similar stimulatory effects to acetaldehyde, decreasing the ethanol-induced growth lag phase from 9 h to 3 h, suggesting a redox-driven effect. The exposure to ethanol caused an instant 60% decline in intracellular NAD⁺ which was largely prevented by the addition of acetaldehyde. Furthermore, the exposure to ethanol affected glycolysis by decreasing the rate of glucose utilisation from 0.33 g glucose g⁻¹ biomass h⁻¹ to 0.11 g glucose g⁻¹ biomass h⁻¹, while the addition of acetaldehyde to an ethanol stressed culture increased this rate to 0.14 g glucose g⁻¹ biomass h⁻¹.     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Phenol inhibition kinetics for growth of Acetobacter aceti on ethanol

Acetobacter aceti have been grown on ethanol under inhibitory conditions created by high concentrations of phenol. A defined medium with no vitamin or amino acid supplements has been used such that ethanol was the sole carbon substrate. The culture temperature was maintained at 30 °C while the pH was manually controlled to fall within the range 4.5–6.0 during ethanol consumption. Growth on ethanol at a few thousand milligrams per litre (below the known inhibitory level) resulted in a maximum specific growth rate of 0.16 h⁻¹ with a 95% yield of acetic acid, followed immediately by acetic acid consumption at a growth rate of 0.037 h⁻¹. Phenol was found to inhibit growth by decreasing both the specific growth rate and the biomass yield during ethanol consumption. On the other hand, the yield of acetic acid during ethanol consumption and the yield of biomass during acetic acid consumption remained constant, independent of phenol inhibition. A model is presented and is shown to represent the phenol-inhibited growth behaviour of A. aceti during both ethanol and acetic acid consumption. Received: 6 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Fine-tuning ethanol oxidation pathway enzymes and cofactor PQQ coordinates the conflict between fitness and acetic acid production by Acetobacter pasteurianus

The very high concentrations required for industrial production of free acetic acid create toxicity and low pH values, which usually conflict with the host cell growth, leading to a poor productivity. Achieving a balance between cell fitness and product synthesis is the key challenge to improving acetic acid production efficiency in metabolic engineering. Here, we show that the synergistic regulation of alcohol/aldehyde dehydrogenase expression and cofactor PQQ level could not only efficiently relieve conflict between increased acetic acid production and compromised cell fitness, but also greatly enhance acetic acid tolerance of Acetobacter pasteurianus to a high initial concentration (3% v/v) of acetic acid. Combinatorial expression of adhA and pqqABCDE greatly shortens the duration of starting-up process from 116 to 99 h, leading to a yield of 69 g l^-1 acetic acid in semi-continuous fermentation. As a final result, average acetic acid productivity has been raised to 0.99 g l^-1 h^-1, which was 32% higher than the parental A. pasteurianus. This study is of great significance for decreasing cost of semi-continuous fermentation for producing high-strength acetic acid industrially. We envisioned that this strategy will be useful for production of many other desired organic acids, especially those involving cofactor reactions.     

Improvement of acetic acid tolerance and fermentation performance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by disruption of the <Emphasis Type="Italic">FPS1</Emphasis> aquaglyceroporin gene

The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.