The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

Summary Z.mobilis is strain ZM4 was grown on 250 g/l fructose and sucrose media in batch culture and on 100 and 150 g/l sucrose media in continuous culture. With fructose, a significant reduction in the growth rate and the cell yield was apparent although the other kinetic parameters were similar to those previously reported for fermentation of glucose. With sucrose the major differences were a reduction in ethanol yield, (due to levan formation) and a lower final ethanol concentration. Ethanol inhibition of sucrose metabolism occurred at relatively low ethanol concentrations compared to those inhibiting glucose metabolism.     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Alcoholic fermentation of cellulose hydrolysate by Zymomonas mobilis

Summary Growth and ethanol production by three strains of the bacterium Zymomonas mobilis were tested, at 40°C, in a medium containing cellulose hydrolysate (hexose fraction) as carbon source. The thermotolerant mutant C107 exhibited the best growth compared to wild type ZM4 and to the osmotolerant mutant SBE15. When cultivated in media supplemented with various nutrients, growth was only observed in presence of yeast extract (10 g/l) which acted both as a vitamin supplier and pH stabilizer. Using calcium pantothenate instead of yeast extract and sodium acetate to control pH resulted in growth inhibition by the high medium osmolality. Batch fermentation with pH control (KOH addition) showed good growth and ethanol production with the mineral medium.     

Butane 2,3-diol production by Aeromonas hydrophila grown on starch

Summary The conversion of soluble starch to butane 2,3-diol by Aeromonas hydrophila was investigated. The diol was produced optimally at pH 6.2 to 5.0. A significantly higher yield of the diol occurred in media buffered with potassium rather than sodium phosphates. The addition of relatively low concentrations of sodium acetate ( 1g/l) to the starch-based growth medium caused substantial increases in the yield of the diol, although no obvious proportional relationship between the amount of acetate added and the enhanced yield of the diol was recorded. The addition of 5g/l of sodium acetate caused severe growth inhibition and decreased the amount of butane 2,3-diol produced.     

Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

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 poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant strains of Escherichia coli

A recombinant strain of Escherichia coli was used to produce poly(4-hydroxybutyric acid), P(4HB), homopolyester by fed-batch culture in M9 mineral salts medium containing glucose and 4-hydroxybutyric acid as carbon sources. The final cell dry weight, P(4HB) concentration and P(4HB) content were 12.6 g/l, 4.4 g/l, and 36% of cell dry weight, respectively, in a 27-l stirred and aerated fermenter after 60 h of fed-batch fermentation at constant pH.     

Rapid detection of heterotrophic growth of Haematococcus pluvialis using indirect conductimetry

An indirect conductimetric method was developed for the rapid detection of heterotrophic growth of Haematococcus pluvialis. Selective HKU medium was superior to Whitley Impedance Broth. Acetate was better than glucose not only for the biomass production (30,200 cells ml on 1 g acetate l vs. 14,800 cells ml on 1 g glucose l ) but also for the time needed to detect significant growth (10.9 h on 1 g acetate l vs. 36.9 h on 1 g glucose l ). The largest maximum conductance change of -1455 S and the shortest detection time of 10.9 h were found in HKU medium containing 1 g acetate l , and the cell concentration increased by 5.5 times after 4 days of cultivation.     

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.     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

A one-step process for the production of single-cell protein and amyloglucosidase

Summary A new Rhizopus species was isolated from traditional Indonesian food, tempeh. The newly isolated species was similar in its morphological characteristics to Rhizopus oligosporus UQM 145F, but grew faster on potato-dextrose agar as well as in submerged culture. The new isolate was found to convert ground cassava tuber directly into single cell protein without pretreatment due to its high amyloglucosidase formation.From 100 g ground tuber, a dry biomass of 33.75 g containing 26.48% true protein together with 60 ml of highly active amyloglucosidase (282 units) was obtained in 12 h. The amyloglucosidase was recovered by ultrafiltration, releasing 26.226 millimol glucose/l/min from soluble starch. The crude enzyme exhibited a pH optimum between 4.6 and 5.0, a temperature optimum between 55 and 60° C and an apparent Km of 3.125 g/l. High substrate concentrations and ammonium sulphate are inhibitory to the enzyme.     

Increased production of bacteriocin ST4SA byEnterococcus mundtii ST4SA in modified corn steep liquor

Enterococcus mundtii ST4SA produces a broad-spectrum bacteriocin (bacST4SA), active against Gram-positive and Gramnegative bacteria. Growth in corn steep liquor (CSL) with a sugar content of 5.0 and 10.0 g/l yielded bacST4SA levels of 12800 AU/ml. A four-fold increase in bacST4SA production (51200 AU/ml) was recorded in CSL with a sugar content of 7.5 g/l supplemented with 6.5 g/l yeast extract (CSL-YE). Poor growth and low levels of bacST4SA production were observed when cells were grown in CSL-YE controlled at pH 5.5. Fermentation at pH 7.5 yielded 25600 AU/ml after 6 h, but the activity levels decreased to approximately 1000 AU/ml during the next 6 h. Adjustment of the culture pH from 6.5 to 5.5 after 6 h of fermentation extended bacST4SA activity (51200 AU/ml) over 8 h. Activity then decreased to 25600 AU/ml and was maintained this level for 10 h. Optimal levels of bacST4SA production (102400 AU/ml) were obtained after 6 h of fermentation in CSL-YE supplemented with 7.5 g/l glucose at the start of the fermentation. This level of production was maintained by changing the culture pH from 6.5 after 6 h of fermentation to pH 5.5. This study proved that bacST4SA could be produced at high levels in an inexpensive industrial medium byE. mundtii ST4SA.     

Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6.8, but not at pH 4.0, when incubated at 37 degrees C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30 degrees C at pH 4.0 was 0.03 mol/l and for Escherichia coli it was 0.09 mol/l. Fermented pig wastes in a digester, maintained at pH 5.9, were inoculated with Salm. typhimurium and then incubated at 37 degrees C for 24 h. The pH was adjusted to either 4.0 or 5.0 and after a further 48 h at 30 degrees C, Salm. typhimurium survived at pH 5.0 but not at pH 4.0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

喇叭石蕊共生菌藻液体培养的研究(英文)

对喇叭石蕊共生菌、藻液体培养条件进行了研究。结果表明:共生菌生长在以40g/L肌醇为碳源、2g/LL-谷氨酰胺为氮源、起始pH值为7.0的LB液体培养基中,培养温度为20℃时表现最佳。其共生藻的生长在以160g/L葡萄糖为碳源、1.75g/LNaNO3为氮源、起始pH值为5.0的BBM液体培养基中,培养温度为20℃时表现最佳。     

Optimization of Bifidobacterium longum growth by use of calcium carbonate-alginate beads

High concentrations of ammonium and sodium ions inhibited Bifidobacterium longum growth more than a high calcium ion concentration. The optimal pH for B. longum growth was determined to be 5.0 due to the lower accumulation of ammonium ion. To reduce the accumulation of ammonium ion and obtain an enhanced growth of B. longum, the pH of the culture containing immobilized calcium carbonate beads was controlled to 5.0 with ammonia water. The concentrations of ammonium, sodium, and calcium ions in the culture were maintained at the desired level. The maximum cell mass increased to 16.8 g/l, 1.23 times higher than cultures without calcium carbonate beads. The number of viable cells in the culture increased to 5.0 × 10¹⁰, 1.67 times more than cultures without calcium carbonate beads.