Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Kinetic evaluation of products inhibition to succinic acid producers <Emphasis Type="Italic">Escherichia coli</Emphasis> NZN111, AFP111, BL21, and <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> 130Z<Superscript>T</Superscript>

Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130Z^T and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8–17.6 g/L, 10–40 g/L, 9–18 g/L, and 10–80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.     

Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613

Conditions for the laboratory-scale production of acetoin plus diacetyl by Enterobacter Cloacae ATCC 27613 were studied. Thirty-five g acetoin plus diacetyl/50 g sucrose were obtained when fermentation was carried out in 2. 5 liter medium containing 12.5 g peptone and 12. 5 g yeast extract, at pH 7.0, in a 5 liter conical flask on a shaker (240rpm) at 28–30°C for 48 hr. Recovery of pure diacetyl was 85% of the total plus diacetyl.     

Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber

Fermentation of sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and butanol production (1.7 ± 0.2 g/L acetone butanol ethanol or ABE) by Clostridium beijerinckii BA101. Treatment of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 ± 0.5 g/L ABE and a yield of 0.39 ± 0.015. Fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell inhibition and resulted in the production of 8.6 ± 1.0 g/L ABE and used 24.6 g/L total sugars. ABE production from fermentation of 25 g/L glucose and 25 g/L xylose was 9.9 ± 0.4 and 9.6 ± 0.4 g/L, respectively, suggesting that the culture was able to utilize xylose as efficiently as glucose. Production of only 9.3 ± 0.5 g/L ABE (compared with 17.7 g/L ABE from fermentation of 55 g/L glucose-control) from the XAD-4 treated SACFH suggested that some fermentation inhibitors may still be present following treatment. It is suggested that inhibitory components be completely removed from the SACFH prior to fermentation with C. beijerinckii BA101. In our fermentations, an ABE yield ranging from 0.35 to 0.39 was obtained, which is higher than reported by the other investigators.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t-amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH₄NO₃ and 7.6 g of KNO₃ per liter as well as suitable levels of growth hormones. Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.     

高抗逆高丁比拜氏梭菌的选育及其性能考察简

以抗逆突变株Clostridium beijerinckii IB4为出发菌株,通过常压室温等离子体诱变（ ARTP ）,刃天青平板初筛,摇瓶发酵复筛,筛选出1株高抗逆高丁比的突变菌株C.beijerinckii IT111。发酵结果表明：该突变菌株利用多种C源时均展现其高丁醇比的特性,以玉米芯酸解糖液为C源时,溶剂产量达到10.5 g/L,丁醇8.0 g/L,丁醇比高达76％。抑制物抗逆性测试结果显示：糠醛和酸类对C.beijerinckii发酵影响较小,酚类物质对C.beijerinckii抑制作用较强,其中以香草醛为最。综上所述,C.beijerinckii IT111是1株极具潜力的利用木质纤维原料制备丁醇的菌株。     

In vitro inhibitory effects of rosemary and sage extracts on mycelial growth and sclerotial formation and germination of Sclerotinia sclerotiorum

The anti-fungal efficacy for two Labiate plants, rosemary (Rosmarinus officinalis L.) and Greek sage (Salvia fructicosa Mill.), against Sclerotinia sclerotiorum fungus (Lib.) de Bary has been investigated. The inhibitory effect of these plants as crude leaf ethanolic extract on the radial mycelial growth as well as on sclerotial production and germination was measured in vitro at various concentrations (stock?=?0.5?g dry leaf powder/ml ddH₂O) in the growth medium. In general, rosemary extract revealed a remarkable anti-fungal effect against the fungus, being more inhibitory than Greek sage in this respect. This was evident as total inhibition of radial mycelial growth by rosemary occurred at 10% extract concentration, while sage was half as potent producing such an effect at double the concentration (20%). Both rosemary and sage extracts were more inhibitory to sclerotial formation than to mycelial growth as the fungus ceased to produce any sclerotia at the lower concentrations of 5 and 5–10%, respectively. In addition, rosemary was highly effective in inhibiting sclerotia germination as total inhibition of germination occurred at 20% extract concentration at three?days and onward after incubation. Moreover, at this level, the survival of sclerotia was totally lost when examined after 12?days of incubation. For sage, inhibition of sclerotial germination/death was only 20% at 12th day of incubation. The results of this study indicate that the extracts of rosemary and Greek sage leaves could become natural alternatives to synthetic fungicides to manage diseases of S. sclerotiorum.     

Effects of Pesticides on Nitrite Oxidation by Nitrobacter agilis

The influence of pesticides on the growth of Nitrobacter agilis in aerated cultures and on the respiration of N. agilis cell suspensions and cell-free extracts was studied. Two pesticides, aldrin and simazine, were not inhibitory to growth of Nitrobacter, but five compounds [isopropyl N-(3-chlorophenyl) carbamate (CIPC), chlordane, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (DDD), heptachlor, and lindane] prevented growth when added to the medium at a concentration of 10 mug/ml. Whereas CIPC and eptam prevented nitrite oxidation by cell suspensions, the addition of DDD and lindane resulted in only partial inhibition of the oxidation. Heptachlor and chlordane also caused only partial inhibition of oxidation, but were more toxic with cell-free extract nitrite oxidase. None of the pesticides inhibited the nitrate reductase activity of cell-free extracts, but most caused some repression of cytochrome c oxidase activity. Heptachlor was the most deleterious compound.     

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.     

Microbiological studies on the formation of gramicidin S synthetases

Rapid and extensive growth of Bacillus brevis ATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol-ammonium sulfate-Tris-salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappearance.     

Cover Image

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.     

Utilization of cellulose from waste paper by Myrothecium verrucaria

Extensive screening studies on cellulolytic bacteria and fungi led to the selection of Myrothecium verrucaria as the organism producing the maximum rate of protein biosynthesis from ball-milled newspaper. Studies in aerated stirred-jar fermentors were carried out to determine the conditions for maximum protein synthesis rate and maximum final protein concentration. The optimum aeration rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate at 0.1% stimulated growth rate and protein production. The maximum rate of protein biosynthesis and the maximum protein yield were 0.3 g/liter/day and 1.42 g/liter, respectively, from medium G3 with 4% ball-milled newspaper. The final product, obtained by evaporation of the total culture, was 33.7 g from one liter of medium which originally contained 40 g of ball-milled newspaper and 11.3 g of other dissolved materials. The protein content of this final product was 3.3 g, calculated from total organic N × 6.25 or 1.42 g calculated from the biuret method. Both the synthesis rate and the final cell yield are below those obtainable by growing Fungi Imperfecti, yeasts or bacteria on soluble materials such as glucose.     

Formation of Short-Chain Fatty Acids from H2 and CO2 by a Mixed Culture of Bacteria

The biological utilization of CO₂ and H₂ for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37°C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.