Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.     

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.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

<Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> mutant with high inhibitor tolerance obtained by low-energy ion implantation

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l⁻¹ of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g⁻¹. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l⁻¹ of ABE with a yield of 0.34 g g⁻¹, including 2.2 g l⁻¹ acetone, 6.8 g l⁻¹ butanol, and 0.5 g l⁻¹ ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.     

Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle

The performance of an innovative two-stage continuous bioreactor with cell recycle—potentially capable of giving very high ethanol productivity—was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l⁻¹ and ethanol concentrations of 31 and 65 g l⁻¹ were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l⁻¹ in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l⁻¹ h⁻¹) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.     

Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification

Saccharomyces cerevisiae was transformed for higher ethanol tolerance by error-prone whole genome amplification. The resulting PCR products were transformed back to the parental strain for homologous recombination to create a library of mutants with the perturbed genomic networks. A few rounds of transformation led to the isolation of mutants that grew in 9% (v/v) ethanol and 100 g glucose l⁻¹ compared to untransformed yeast which grew only at 6% (v/v) ethanol and 100 g glucose l⁻¹.     

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

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.     

Methanol utilizing <Emphasis Type="Italic">Desulfotomaculum</Emphasis> species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65°C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l⁻¹, while on H₂/CO₂, no apparent inhibition occurred up to a concentration of 500 mg l⁻¹. When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l⁻¹. These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H₂/CO₂ to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.     

Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina

One-hundred and five fungal strains, belonging to 46 different species, were screened for exopolysaccharide production. Phytopathogenicity and, in particular, inability to produce conidia, were physiological characteristics positively associated and correlated with the fungal ability to produce polysaccharides. Among the 29 positive strains, Botryosphaeria rhodina DABAC-P82 was the most interesting reaching, when grown on optimal nitrogen source and concentration (NaNO₃ and 2.0 g l⁻¹, respectively) and culture medium pH (3.7), 17.7 g l⁻¹ of exopolysaccharide production after only 24 h of fermentation; yield and productivity were 0.69 g g⁻¹ and 0.73 g l⁻¹ h⁻¹, respectively. The purified polysaccharide was characterised as a homopolysaccharide of glucose with a molecular weight of 4.875·10⁶ Da. Studies of structural analysis indicated the presence of β-1,3 and β-1,6 linkages; the EPS structure was very similar to that of scleroglucan. This revised version was published online in August 2006 with corrections to the Cover Date.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

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.     

Production,formulation and antagonistic activity of the biocontrol like-yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> against <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Aureobasidium pullulans (de Bary) Arnaud (Ach 1-1) was grown in a glucose fed-batch fermentor to 106 g dry wt l⁻¹ in 48 h. The cells were dried in a fluidized bed dryer with a final viability of 62%. After 7 months at 4°C, the viability was 28% of the initial value (= 2.3 × 10¹⁰ c.f.u. g⁻¹ dry matter). A protection level of 89% was achieved with the biomass preparation at 1 × 10⁸ c.f.u. ml⁻¹ after 28 and 7 days for apples stored respectively at 5 and 25°C against Penicillium expansum. Our process is suitable to produce large quantities of the strain Ach 1-1 as biological control agent for apple preservation.     

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.