Production of ethanol from mannitol by Zymobacter palmae

Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic mannitol medium with a yield of 0.38 g ethanol (g mannitol)⁻¹. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57. Received 27 June 1999/ Accepted in revised form 23 September 1999     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Kinetic study of mannitol production using cashew apple juice as substrate

The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, a kinetic study of mannitol production using cashew apple juice as substrate was studied. The carbohydrates of cashew apple juice are glucose and fructose. Sucrose addition favored the yield of mannitol (85%) at the expense of lower productivity. The best results were obtained applying only cashew apple juice as substrate, containing 50 g L⁻¹ of total reducing sugar (28 g L⁻¹ of fructose), yielding 18 g L⁻¹ of mannitol with 67% of fructose conversion into mannitol and productivity of 1.8 g L⁻¹ h⁻¹.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

Ethanol fermentation of mixed-sugars using a two-phase, fed-batch process: method to minimize D-glucose repression of Candida shehatae D-xylose fermentations

Candida shehatae cells pre-grown on D-xylose simultaneously consumed mixtures of D-xylose and D-glucose, under both non-growing (anoxic) and actively growing conditions (aerobic), to produce ethanol. The rate of D-glucose consumption was independent of the D-xylose concentration for cells induced on D-xylose. However, the D-xylose consumption rate was approximately three times lower than the D-glucose consumption rate at a 50% D-glucose: 50% D-xylose mixture. Repression was not observed (substrate utilization rates were approximately equal) when the percentage of D-glucose and D-xylose was changed to 22% and 78%, respectively. In fermentations with actively growing cells (50% glucose and D-xylose), ethanol yields from D-xylose increased, the % D-xylose utilized increased, and the xylitol yield was significantly reduced in the presence of D-glucose, compared to anoxic fermentations (Y_ETOH,xylose = 0.2–0.40 g g⁻¹, 75–100%, and Y_xylitol = 0–0.2 g g⁻¹ compared to Y_ETOH,xylose = 0.15 g g⁻¹, 56%, Y_xylitol = 0.51 g g⁻¹, respectively). To increase ethanol levels and reduce process time, fed-batch fermentations were performed in a single stage reactor employing two phases: (1) rapid aerobic growth on D-xylose (μ = 0.32 h⁻¹) to high cell densities; (2) D-glucose addition and anaerobic conditions to produce ethanol (Y_ETOH,xylose = 0.23 g g⁻¹). The process generated high cell densities, 2 × 10⁹ cells ml⁻¹, and produced 45–50 g L⁻¹ ethanol within 50 h from a mixture of D-glucose and D-xylose (compared to 30 g L⁻¹ in 80 h in the best batch process). The two-phase process minimized loss of cell viability, increased D-xylose utilization, reduced process time, and increased final ethanol levels compared to the batch process. Received 23 February 1998/ Accepted in revised form 15 July 1998     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Fermentation of pretreated sugarcane bagasse hemicellulose hydrolysate to ethanol by <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis>

Sugarcane bagasse hemicellulose hydrolysates, pretreated by either over-liming or electrodialysis and, supplemented with nutrient materials, were fermented to ethanol using Pachysolen tannophilus DW06. Compared with detoxification by over-liming, detoxification by electrodialysis decreased the loss of sugar and increased the acetic acid removal, leading to better fermentability. A batch culture with electrodialytically pretreated hydrolysate as substrate was developed giving 21 g ethanol l⁻¹ with a yield of 0.35 g g⁻¹ sugar and productivity of 0.59 g l⁻¹ h⁻¹.     

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol. Received: 2 July 1999 / Accepted in revised form: 12 October 1999     

Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast

The GPD2 gene, encoding NAD⁺-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P _PGK -gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P _PGK -GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (μ _max) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.     

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L⁻¹) was obtained in 9 h with 20 g L⁻¹ glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L⁻¹. Maximum bacteriocin activity (2.9–3.2 MBU L⁻¹) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h⁻¹ and specific bacteriocin production rate increased linearly with dilution rate. Received 31 July 1996/ Accepted in revised form 01 November 1996     

Glycolytic pathway and hydrogen yield studies of the extreme thermophile <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

NMR analysis of ¹³C-labelling patterns showed that the Embden–Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H₂ per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g⁻¹ h⁻¹ and 20 mmol l⁻¹ h⁻¹. An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.     

The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization

Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l⁻¹ h⁻¹ at a dilution rate of 0.4 h⁻¹. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant K _S of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, μ_max, of 1.0 h⁻¹. Oxygen uptake rates of up to 2.9 g l⁻¹h⁻¹ were calculated and the yield coefficient, Y _X/O, was determined to be 0.29 g dry cell weight/g O₂. From an overall material balance the yield coefficient, Y _X/S, was estimated to be 0.60 g dry cell weight/g olive oil. Received: 8 January 1997 / Received revision: 30 April 1997 / Accepted: 4 May 1997     

Growth parameters of Acinetobacter calcoaceticus on acetate and ethanol

Summary The growth parameters of Acinetobacter calcoaceticus, relevant to its mass cultivation on acetate and ethanol, were determined in batch and continuous culture experiments. Acetic acid exhibited a more powerful inhibitory effect on the growth rate than ethanol. In batch culture, the acetate component of an acetate-ethanol substrate pair was preferentially utilized, but diauxic growth as such was not evident. The temperature optimum for growth was in the region of 29°–36°C, and the cell yield did not change appreciably over this temperature range. In carbon-limited chemostat cultures, the maximum specific growth rates on acetate and ethanol were 1.22 h⁻¹ and 0.96 h⁻¹ respectively, and the respective yield coefficients were 0.4 and 0.75. A high maintenance energy requirement was exhibited, especially during acetate-limited growth. The respiratory quotient was dependent on the growth rate, the significance of which is discussed. Part of the material included in this paper was presented at the VIth International Fermentation Symposium, London, Ontario, July 1980     

Production of acetone butanol (AB) from liquefied corn starch,a commercial substrate,using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> coupled with product recovery by gas stripping

A potential industrial substrate (liquefied corn starch; LCS) has been employed for successful acetone butanol ethanol (ABE) production. Fermentation of LCS (60 g l⁻¹) in a batch process resulted in the production of 18.4 g l⁻¹ ABE, comparable to glucose: yeast extract based medium (control experiment, 18.6 g l⁻¹ ABE). A batch fermentation of LCS integrated with product recovery resulted in 92% utilization of sugars present in the feed. When ABE was recovered by gas stripping (to relieve inhibition) from the fed-batch reactor fed with saccharified liquefied cornstarch (SLCS), 81.3 g l⁻¹ ABE was produced compared to 18.6 g l⁻¹ (control). In this integrated system, 225.8 g l⁻¹ SLCS sugar (487 % of control) was consumed. In the absence of product removal, it is not possible for C. beijerinckii BA101 to utilize more than 46 g l⁻¹ glucose. A combination of fermentation of this novel substrate (LCS) to butanol together with product recovery by gas stripping may economically benefit this fermentation. Mention of trade names of commercial products in this article/publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of ≥20 ml air l⁻¹ mash min⁻¹ showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.     

Acetaldehyde stimulation of the growth of Saccharomyces cerevisiae in the presence of inhibitors found inlignocellulose-to-ethanol fermentations

The addition of small quantities of acetaldehyde to fermentations containing inhibitory concentrations of furfural, acetate and other compounds typically present in lignocellulosic hydrolyzates significantly reduced the lag phase of yeast growth and stimulated ethanol production. Similar effects were observed when acetaldehyde (0.06 g l⁻¹) was added to fermentations of a birch wood hydrolyzate produced by steam/acid pretreatment. Acetaldehyde addition appears to have potential as a low-cost alternative (or adjunct) to current procedures for medium detoxification in lignocellulose-to-ethanol fermentations, particularly those in which high inhibitor concentrations are generated through recycling of the culture broth. Journal of Industrial Microbiology & Biotechnology (2000) 25, 104–108. Received 18 March 2000/ Accepted in revised form 02 June 2000     

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR_max) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR_max decreased. The molecular mass obtained at 3.0 mmol l⁻¹ h⁻¹ was 7.0 times higher (1,560 kDa) than at 9.0 mmol l⁻¹ h⁻¹ (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.