Continuous butanol production from whey permeate with immobilized Clostridium beyerinckii LMD 27.6

Summary The aim of this study was to find the conditions necessary for the continuous butanol production from whey permeate with Clostridium beyerinckii LMD 27.6, immobilized in calcium alginate beads. The influence of three parameters on the butanol production was investigated: the fermentation temperature, the dilution rate (during start-up and at steady state) and the concentration of calcium ions in the fermentation broth. It was found that both a fermentation temperature of 30° C and a dilution rate of 0.1 h^-1 or less during the start-up phase are required to achieve continuous butanol production from whey permeate. Butanol can be produced continuously from whey permeate in reactor productivities sixteen times higher than those found in batch cultures with free C. beyerinckii cells on whey media.     

Butanol production of Clostridium acetobutylicum grown on sugars found in hemicellulose hydrolysates

Summary Clostridium acetobutylicum was grown on a variety of different media and assayed for enhanced butanol production. Butanol values of about 90% of theoretical were obtained when glucose was used as the substrate. Five other sugars were assayed and the order of butanol production using these substrates was cellobiose>mannose>arabinose>xylose >galactose. The addition of calcium carbonate to the media to enhance xylose utilized was beneficial although lower butanol values were obtained.     

Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum

Fermentation of whey by Clostridium acetobutylicum yielded butanol and acetone in a ratio of approximately 100:1. This ratio amounted to only 2:1 in synthetic media with glucose, lactose, or glucose plus galactose as substrates. Removal of citrate from whey and addition of minerals resulted in an increase in the amount of acetone produced. Experiments carried out in a chemostat with a low-phosphate synthetic medium revealed that the butanol/acetone ratio could be increased from 2:1 to 3.8:1 by cofermentation of l-lactate and from 2:1 to 8:1 by iron limitation. The performance of the fermentation in a low-iron glucose medium above pH 5.1 yielded l-lactate as the main product.     

Production of butyric acid by batch fermentation of cheese whey withClostridium beijerinckii

Summary The effect of pH on the fermentation of butyric acid byClostridium beijerinckii using cheese whey as a substrate was studied. Maximum concentrations of the acid were produced when the pH was controlled at 5.5. Raising or lowering of pH was found to reduce the total acid formation. This particular strain ofC. beijerinckii produced insignificant amounts of butanol in all the pure culture cases investigated. A comparative study of the fermentation in a synthetic glucose medium and in cheese whey showed the whey to produce more butyric acid.     

Alcohol production by selected yeast strains in lactase-hydrolyzed acid whey

Ethanol production by Kluyveromyces fragilis and Saccharomyces cerevisiae was studied using cottage cheese whey in which 80 to 90% of the lactose present had been prehydrolyzed to glucose and galactose. Complete fermentation of the sugar by K. fragilis required 120 hr at 30°C in lactase-hydrolyzed whey compared to 72 hr in nonhydrolyzed whey. This effect was due to a diauxic fermentation pattern in lactase-hydrolyzed whey with glucose being fermented before galactose. Ethanol yields of about 2% were obtained in both types of whey when K. fragilis was the organism used for fermentation. Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K. fragilis, but galactose was fermented only when S. cerevisiae was pregrown on galactose. Slightly lower alcohol yields were obtained with S. cerevisiae, owing to the presence of some lactose in the whey which was not fermented by this organism. Although prehydrolysis of lactose in whey and whey fractions is advantageous in that microbial species unable to ferment lactose may be utilized, diauxie and galactose utilization problems must be considered.     

Production of acetone - butanol from acid whey

Summary Clostridium acetobutylicum ATCC 824 was used to produce butanol and acetone by fermenting acid whey. Results showed that both autoclaving and agitation played roles in solvent production. Maximum production was obtained in 120 h using autoclaved, pH adjusted (6.0) acid whey at 37 °C in a fermentor that was not agitated.     

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0?C5?g/L) butyric acid and (0?C10?g/L) glucose enhances the butanol production. The combination of 5?g/L butyric acid with 5 and 10?g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10?g/L shifted the optimal butyric acid concentrations to 10 and 15?g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13?g/L of butanol in the presence of 20?g/L of glucose, and 0.7?g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.     

Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol

The study was focused on developing a continuous method to produce an alcohol mixture suitable to be used as a gasoline supplement. The immobilized column reactor with wood pulp fibers was successfully used for the continuous production of butanol and isopropanol using Clostridium beijerinckii DSM 6423. A sugar mixture (glucose, mannose, galactose, arabinose and xylose) representing lignocellulose hydrolysate was used as a substrate for the production of solvents. The effect of dilution rate on solvent production was studied during continuous operation. The maximum total solvent concentration of 11.99 g/l was obtained at a dilution rate of 0.16 h^?1. The maximum solvent productivity (5.58 g/l h) was obtained at a dilution rate of 1.5 h^?1. The maximum solvent yield of 0.45 g/g from sugar mixture was observed at 0.25 h^?1. The system will be further used for the solvent production using wood hydrolysate as a substrate.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

The effect of fermentable carbohydrate on sporulation and butanol production by Clostridium acetobutylicum P262

Summary This study was conducted to determine whether or not a variation in the type of carbohydrate fermented by Clostridium acetobutylicum could be exploited to inhibit sporulation during the butanol-producing phase of fermentation and thus enhance butanol production. C. acetobutylicum P262 was found to ferment a wide variety of carbohydrates, but butanol production was not necessarily enhanced when percentage sporulation was low. Butanol concentration was more related to the total amount of acidic end-products (acetic and butyric acid) reutilized by the microorganism for solvent production and to the type and amount of carbohydrate utilized. Fermentation of cellobiose led to conditions resulting in complete acid reutilization and the highest butanol concentration (10.4–10.6 g/l). In cultures containing a mixture of glucose and cellobiose, glucose repression of cellobiose utilization resulted in lower butanol concentrations (6.6–7.5 g/l). Sporulation was dependent on the type of carbohydrate utilized by the microorganism. Glucose had a greater enhancing effect on the sporulation process (22–42%) than starch (9–12%) or cellobiose (22–34%). It was concluded that whereas the type of carbohydrate fermented had a specific effect on the extent of sporulation of a culture, conditions of low sporulation did not enhance butanol concentration unless carbohydrate utilization and the reutilization of acidic products were high.Correspondence to: W. M. Ingledew     

Effects of different replicons in conjugative plasmids on transformation efficiency,plasmid stability,gene expression and n-butanol biosynthesis in <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis>

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum)

Factors that may initiate the metabolic transition for butanol production were investigated in batch cultures of Clostridium beijerinckii (synonym, Clostridium butylicum) VPI 13436. Cultures maintained at pH 6.8 produced nearly as much butanol as those incubated without pH control, indicating that neither a change in the culture pH nor acid conditions per se are always required to initiate solvent formation. Acetate and butyrate levels at the onset of butanol production were dependent on the pH at which the cultures were maintained. Cultures maintained at pH 6.8 could be accelerated into solvent production by artificially lowering the pH to 5.0 or by the addition of acetate plus butyrate without a pH change (but neither acid alone was effective). Solvent production was associated with slower rates of growth and general metabolism, and it did not show a requirement for mature spore formation. We speculate that a slowdown in metabolism, which may be brought about by several conditions, is mechanistically related to the onset of butanol production. Extracts of solvent-producing cells contained acetoacetate decarboxylase activity as well as higher NADP⁺-linked butanol dehydrogenase and lower hydrogenase activities than extracts of acid-producing cells. Solvent production did not appear to involve an enhanced ability to catalyze H₂ oxidation.     

Fermentation of cheese whey by a mixed culture ofClostridium beijerinckii andBacillus cereus

Summary Fermentation of cheese whey to produce butanol and butyric acid was carried out using a mixed culture ofClostridium beijerinkii andBacillus cereus. Fermentation selectivities were studied by controlling the pH of the system. Controlled pH values higher than 6.5 as well as those below 5.0 were not conducive to butanol production. Maximum product formation was obtained by controlling the pH at 5.5. When compared with the results obtained using the pure culture ofC. beijerinckii, a higher butanol concentration was obtained in the mixed culture without sacrificing the level of butyric acid formed.     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

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.     

Butanol production by Clostridium acetobutylicum in a continuous packed bed reactor

In this study, we report on a butanol production process by immobilized Clostridium acetobutylicum in a continuous packed bed reactor (PBR) using Tygon^® rings as a carrier. The medium was a solution of lactose (15–30 g/L) and yeast extract (3 g/L) to emulate the cheese whey, an abundant lactose-rich wastewater. The reactor was operated under controlled conditions with respect to the pH and to the dilution rate. The pH and the dilution rate ranged between 4 and 5, the dilution rate between 0.54 and 2.4 h^?1 (2.5 times the maximum specific growth rate assessed for suspended cells). The optimal performance of the reactor was recorded at a dilution rate of 0.97 h^?1: the butanol productivity was 4.4 g/Lh and the selectivity of solvent in butanol was 88%_w.