Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Xylan-hydrolyzing thermotolerant <Emphasis Type="Italic">Candida tropicalis</Emphasis> HNMA-1 for bioethanol production from sugarcane bagasse hydrolysate

Sugarcane bagasse is one of the low-cost substrates used for bioethanol production. In order to solubilize sugars in hemicelluloses like xylan, a new thermotolerant isolate of Candida tropicalis HNMA-1 with xylan-hydrolyzing ability was identified and characterized. The strain showed relative tolerance to high temperature. Our results demonstrated 0.211 IU ml^?1 xylanase activity at 40 °C compared to 0.236 IU ml^?1 at 30 °C. The effect of high temperature on the growth and fermentation of xylose and sugarcane bagasse hydrolysate were also investigated. In both xylose or hydrolysate medium, increased growth was recorded at 40 °C. Meanwhile, the efficiency of ethanol fermentation was adversely affected by temperature since yields of 0.088 g g^?1 and 0.076 g g^?1 in the xylose medium, in addition to 0.090 g g^?1 and 0.078 g g^?1 in the hydrolysate medium were noticed at 30 °C and 40 °C, respectively. Inhibitory compounds in the hydrolysate medium demonstrated negative effects on fermentation and productivity, with maximum ethanol concentration attained after 48 h in the hydrolysate, as opposed to 24 h in the xylose medium. Our data show that the newly thermotolerant isolate, C. tropicalis HNMA-1, is able to efficiently ferment xylose and hydrolysate, and also has the capacity for application in ethanol production from hemicellulosic sources.     

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (g ethanol g^?1 biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82 g g^?1 h^?1). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80 g L^?1) and the RHO2-3RD strain was able to tolerate up to 1.5 g L^?1 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22 %, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.     

Screening and Mutation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature,Glucose and Ethanol

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33?±?1.34 to 122.22?±?2.80 g/L at 30–45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18?±?2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.     

Sea ice microbial dynamics over an annual ice cycle in Prydz Bay, Antarctica

Microbial community dynamics within the fast sea ice of Prydz Bay (68°S?78°E) were investigated over an annual cycle at two sites (1 and 3?km offshore) between April and November 2008. There are few long-term sea ice studies, and few that cover the phase of winter darkness when autotrophic processes are curtailed. Mean chlorophyll a concentrations in the ice column ranged between 0.76 and 44.8?μg?L^?1 at the 1-km site (Site 1) and 3.11–144.6?μg?L^?1 at the 3-km site (Site 2). Highest chlorophyll a usually occurred at the base of the ice. Bacterial concentrations ranged between 0.30 and 2.08?×?10⁸?cells?L^?1, heterotrophic nanoflagellates (HNAN) between 0.21?×?10⁵ and 2.98?×?10⁵?cells?L^?1 and phototrophic nanoflagellates (PNAN) 0–1.06?×?10⁵?cells?L^?1. While HNAN occurred throughout the year, PNAN were largely absent in winter. Dinoflagellates were a conspicuous and occasionally an abundant element of the community (maximum 17,460?cells?L^?1), while ciliates were sparse. The bacterial community showed considerable morphological diversity with a dominance of filamentous forms. Bacterial production continued throughout the year ranging between 0 and 22.92?μg?C?L^?1?day^?1 throughout the ice column. Lowest rates occurred between late June and early August. The sea ice sustained an active and diverse microbial community through its annual extent. The data suggest that during winter darkness the microbial community is dominated by heterotrophic processes, sustained by a pool of dissolved organic carbon.     

Comparison of mutant and parent strains of Clostridium acetobutylicum: butyrate uptake at different temperatures

A high butanol producing mutant strain of Clostridium acetobutylicum ATCC 4259 was obtained by chemical mutagenesis. Both mutant and parent strains were evaluated for butyrate uptake using the culture effluents of solventogenic fermentor-2 of the two-fermentor continuous system. Batch incubation of fermentor-2 culture effluents at 37?°C indicated lower butyrate uptake rates for mutant and parent strain, at 0.05 and 0.03?g?l^?1?h^?1, respectively. Increased butyrate uptake rates of 0.33 and 0.26 g l^?1 h^?1 for mutant and parent strain, respectively, were observed when effluents were batch incubated at lower temperature of 30?°C. Butyrate conversion efficiency, at 5?±?0.1 g l^?1 of externally added butyrate, were 98.8% and 96.9% for mutant and parent strain, respectively. Butyrate up to the externally added concentration of 11.4 g l^?1 did not inhibit butyrate uptake. The maximum butyrate consumption at a slightly reduced uptake rate was seen at 10.2 g l^?1 butyrate concentration at 27?°C. Based on the results under different temperatures, the electron flow pattern has been computed and the mechanism for butyrate uptake has been hypothesized.     

High yield production of extracellular recombinant levansucrase by Bacillus megaterium

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH?6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg?L^?1) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U?mL^?1 on fructose and 17.2 U?mL^?1 on glycerol). This was further increased in high cell density fed-batch processes up to 55 U?mL^?1, reflecting a levansucrase concentration of 0.52 g?L^?1. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.     

Culture medium optimization of a new bacterial extracellular polysaccharide with excellent moisture retention activity

A new kind of extracellular polysaccharide (EPS) from Pseudomonas fluorescens PGM37 was obtained and culture media was optimized using the statistical methods single factor experiments and response surface methodology (RSM) design. As a result, the optimum cultivation conditions initial pH value, medium volume, inoculum size, temperature, and rotation speed were 7.5, 100 mL/250 mL, 5 %, 28 °C, and 180 rpm, respectively. The optimized media: sucrose 36.23 g?L^?1, yeast extract 3.32 g?L^?1, sodium chloride 1.13 g?L^?1, and calcium chloride 0.20 g?L^?1. The maximum predicted yield of EPS was 10.1163 g?L^?1 under these conditions. The validation data was 10.012 g?L^?1, which could strongly confirm the correlation between the experimental and theoretical values. Gas Chromatography analysis revealed that the polymer was made up of mannose and glucose in the ratio of 1:1. Infrared spectroscopy showed that the polysaccharide had β-D-pyranoid configuration and contained no other substituent. Graded by different multiples of alcohol after specific degradation by enzyme and then detected by LC-ESI-MS, the EPS structure was β-D-Glcp-(1, 4)-β-D-Manp-(1, 4)-β-D-Glcp-(1, 4)-β-D-Manp. The moisture retention ability of the EPS was found to be superior to glycerol and only a little inferior to hyaluronic acid (HA), which presented potential application value in cosmetics and clinical medicine fields.     

Chemical composition and ethanol production potential of Thai seaweed species

This study evaluated the potential use of several Thai seaweed species for ethanol production. The high biomass of the green algae Ulva intestinalis and Rhizoclonium riparium and the red algae Gracilaria salicornia and Gracilaria tenuistipitata in an earthen pond culture led us to select these species for our study. The seaweed species were analyzed for chemical composition, resulting in ash contents of 37.62?±?0.15 % and fiber of 11.93?±?0.16 %, with the highest values in R. riparium. Low lipid values were found in all species, with the highest value (p?<?0.05) in G. salicornia (1.69?±?0.07 %) and the lowest in R. riparium (0.28?±?0.01 %) and G. tenuistipitata (0.26?±?0.01 %). The highest carbohydrate contents were found in G. tenuistipitata (54.89 %), and the lowest were in R. riparium (29.53 %). G. tenuistipitata (8.58?±?0.36 %) and U. intestinalis (8.24?±?0.28 %) had higher sulfate contents compared with G. salicornia (4.69?±?0.04 %) and R. riparium (1.97?±?0.20 %). The monosugar algal tissue components were analyzed by HPLC; rhamnose, xylose, fucose, arabinose, mannose, glucose, and galactose were used as reference sugars. Total sugar was found to be highest in G. tenuistipitata (98.21 %). Arabinose, glucose, and galactose were the main sugar components in all species. Glucose obtained from G. tenuistipitata (6.55 %) and R. riparium (6.52 %) was higher than in G. salicornia (0.27 %) and U. intestinalis (2.78 %). G. tenuistipitata fermentation gave a higher yield of ethanol (4.17?×?10^?3 g ethanol g^?1 sugars; 139.12 μg ethanol g^?1 glucose) than R. riparium (0.086?×?10^?3 g ethanol g^?1 sugars; 33.84 μg ethanol g^?1 glucose), U. intestinalis (0.074?×?10^?3 g ethanol g^?1 sugars; 9.98 μg ethanol g^?1 glucose), and G. salicornia (0.031?×?10^?3 g ethanol g^?1 sugars; 1.43 μg ethanol g^?1 glucose).     

Effects of different nitrogen, phosphorus, and iron concentrations and salinity on lipid production in newly isolated strain of the tropical green microalga, Scenedesmus dimorphus KMITL

The fatty acid composition, the effect of different concentrations of nitrogen (16.5-344 mg ?L^?1), phosphorus (9–45 mg? L^?1), iron (9–45 mg? L^?1) and salinity levels (0–20 psu) on lipid production in the green microalga Scenedesmus dimorphus KMITL, a new strain isolated from a tropical country, Thailand, were studied. The alga was isolated from a freshwater fish pond, and cultured in Chlorella medium by varying one parameter at a time. The main fatty acid composition of this strain was C16–C18 (97.52 %) fatty acids. A high lipid content was observed in conditions of 16.5 mg? L^?1-N, or 22 mg ?L^?1-P, or 45 mg ?L^?1-Fe, or 5 psu salinity, which accumulated lipids to 20.3?±?0.4, 19.4?±?0.2, 24.7?±?0.5, and 14.3?±?0.2 % of algal biomass, respectively. Increasing lipid content and lipid productivity was noted when the alga was cultured under high iron concentration and high salinity, as well as under reduced phosphorus conditions, whereas nitrogen limitation only resulted in an increased lipid content.     

Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L^?1 yeast extract and 300 g L^?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h^?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L^?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.     

α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg?g cells^?1, pH?3.5, 30 °C, high dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g?l^?1), Y. lipolytica VKM Y-2412 produced up to 102.5 g?l^?1 of KGA with the mass yield coefficient of 0.95 g?g^?1 and the volumetric KGA productivity (Q _KGA) of 0.8 g?l^?1?h^?1.     

Influence of pH on butyrate uptake and solvent fermentation by a mutant strain of Clostridiumacetobutylicum

The effect of pH (between 4.4 and 6.6) on butyrate uptake by the mutant strain of Clostridium acetobutylicum was studied using the fermentation broth from fermentor-2 (solventogenic stage) of a two-fermentor continuous system. Low pH (?1 butyrate, under batch incubation at 30?°C, was not inhibited at pH?>?5.2, however, at pH??1. Batch incubation at relatively higher temperatures (35° and 37?°C) indicated a similar trend i.e., a pH of >5.5 was required for uptake of >8?g l^?1 butyrate. Optimization studies for butyrate uptake by C. acetobutylicum suggested a direct correlation between minimum pH and butyrate concentration or temperature. The role of undissociated butyric acid appears to be critical in regulation of butyrate uptake.     

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20–50?°C), gelatinization time (30–60 min) and broken rice concentration in culture medium (40–80 g L^?1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L^?1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31?°C temperature, 45 min gelatinization time and 79 g L^?1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.     

n-Butanol Production from Acid-Pretreated Jatropha Seed Cake by Clostridium acetobutylicum

n-Butanol fermentation using Clostridium strains suffers from low titers due to the inability of the strains to tolerate n-butanol. The current study demonstrates a process to get high titer of n-butanol in a single batch mode from the renewable feedstock jatropha seed cake by employing Clostridium acetobutylicum. Chemical mutagenesis was done for improvement of the strain for better n-butanol tolerance and production. Optimization of the parameters resulted in 13.2 g L^?1 of n-butanol in 120 h using acid-treated jatropha seed cake hydrolysate (7 %?w/v) in anaerobic sugar medium. The process was scaled up to 15 L level, yielding 18.6 g L^?1 of n-butanol in 72 h. The strain was found to be tolerant up to 30 g L^?1 n-butanol under optimized conditions. The n-butanol tolerance was accompanied by over-expression of the stress response protein, GroEL, change in fatty acid profile, and ability to accumulate rhodamine 6G in the strain. The study has a significant impact on economically producing n-butanol from biomass.     

Environmental controls on growth and lipid content for the freshwater diatom, Fragilaria capucina: A candidate for biofuel production

The use of microalgae for biofuel production has the potential to reduce fossil fuel consumption. Ideal candidate species of microalgae for bio-oil production need both relatively high growth rates and lipid content. Here, we report on the effects of temperature, nutrients (N, Si), and salinity on growth rates and lipid content of the common freshwater diatom, Fragilaria capucina (Desm), isolated from western Lake Erie. At low NaCl salinity, growth rate increased rapidly from 10 to 20°C, and then further increased slowly from 20 to 30°C, with a maximum specific growth rate of 0.61?day^?1. Growth rate declined with increasing salinity (e.g., reduced by ca. 50 and 100% at 137 and 274?mmol?L^?1 NaCl, respectively), and increased with increased N and Si concentration until ca. 100?μmol?L^?1 for each (with >85% of maximum growth rate at 10?μmol?L^?1). Lipid content (% total lipid per dry mass) in nutrient-replete cultures was 14% and (1) increased to >30% at low N and, especially, low Si; (2) was lower at 30°C vs. 20 or 10°C; and (3) decreased with salinity. Thus, F. capucina accumulates lipid to high levels even under N, Si, and temperature levels that permit a high growth rate for this species, and hence, this species is a candidate for use in biofuel production.     

Formation,regeneration, and transformation of protoplasts of Streptomyces diastatochromogenes 1628

Toyocamycin exhibits effective biological activities for use against plant pathogenic fungi thanks to its structural similarity to nucleoside. It has been recognized as a promising agricultural antibiotic utilized in controlling the occurrence of plant diseases. In our previous study, a strain that was isolated was identified and designated as Streptomyces diastatochromogenes whose major secondary metabolite was toyocamycin, but the production was largely limited. Protoplast transformation is a useful technique in the improvement of streptomycete. In this study, we optimized some key factors necessary for protoplast formation, regeneration, and transformation of S. diastatochromogenes. When mycelium was cultivated in CP medium with 1 % glycine, harvested at 48 h old, and then treated with 3 mg lysozyme/mL in P buffer for 1 h, the greatest regeneration frequency (42.5 %) of protoplasts was obtained. By using 1?×?10⁹/mL protoplasts with polyethylene glycol 1000 at a concentration of 30 % (w/v), the best performance of protoplast transformation efficiency was 4.8?×?10³/μg DNA transformants.