Preliminary study on bioethanol from fresh water algae, <Emphasis Type="Italic">Cladophora glomerata</Emphasis> (Sarai Kai) by the fungus, <Emphasis Type="Italic">Monascus</Emphasis> sp. NP1

This work focused on the characteristics of ethanol regulation from Monascus sp. NP1. in glucose liquid medium, a saccharification method using algae and bioethanol production from Cladophora glomerata by the fungus. The results showed that when the fungus was grown in glucose (2, 20, 40 and 50%) medium under 110 rpm rotary culture at 30 °C, the ethanol concentration at 120 h increased from 2 to 20% glucose, where it peaked. It then decreased gradually to 40%, with production stopping at 50% glucose. This result indicated the glucose regulation of ethanol production by the fungus. Ethanol present in 20% glucose medium was identified by retention time and co-injection with a standard to demonstrate that the product was ethanol. Its yield was 285 mM [13 g L^?1 or 65 mg (g of glucose substrate)^?1] with a low interference of by-products. Three-millimetre-long pieces of dried algae were cut and exposed to concentrations of 1, 2, 3, 4, 5 and 6 g in 65 mL of 0.3 N hydrochloric acid or sulfuric acid before autoclaving (121 °C, 15 psi, 15 min). The amount of reducing sugar was greater than that of the control (without acid treatment) and varied with the increasing quantity of algae. The best condition was sulfuric acid and 6 g dried algae. The type of acid appeared to affect saccharification. During 12 days of fermentation in algal extraction (2 g reducing sugar per millilitre algal extraction), the mould could produce twofold more ethanol yield [34–55 mg (100 g dried weight algae)^?1] than the yeast, Saccharomyces cerevisiae TISTR 5049.     

Comparison of bioethanol production from cultivated versus wild <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> and <Emphasis Type="Italic">Gracilaria gigas</Emphasis>

The seaweed genus Gracilaria is a potential candidate for the production of bioethanol due to its high carbohydrate content. Gracilaria is abundant throughout the world and can be found in both wild and cultivated forms. Differences in the ecological factors such as temperature, salinity, and light intensity affecting wild and cultivated specimens may influence the biochemical content of seaweeds, including the carbohydrate content. This study aimed to investigate the proximate composition and potential bioethanol production of wild and cultivated G. gigas and G. verrucosa. Bioethanol was produced using separate hydrolysis fermentation (SHF), employing a combination of enzymatic and acid hydrolysis, followed by fermentation with Saccharomyces cerevisiae ATCC 200062. The highest carbohydrate content was found in wild G. gigas. The highest galactose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L^?1, respectively), as well as the highest production of bioethanol (3.56 ± 0.02 g L^?1), were also found in wild G. gigas. Thus, we conclude that wild G. gigas is the most promising candidate for bioethanol production. Further research is needed to optimize bioethanol production from wild G. gigas. Domestication of wild G. gigas is a promising challenge for aquaculture to avoid overexploitation of this wild seaweed resource.     

Mating of natural <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for improved glucose fermentation and lignocellulosic inhibitor tolerance

Natural Saccharomyces cerevisiae isolates from vineyards in the Western Cape, South Africa were evaluated for ethanol production in industrial conditions associated with the production of second-generation biofuels. The strains displayed high phenotypic diversity including the ability to grow at 45 °C and in the presence of 20% (v/v) ethanol, strain YI13. Strains HR4 and YI30 were inhibitor-tolerant under aerobic and oxygen-limited conditions, respectively. Spore-to-spore hybridization generated progeny that displayed heterosis, including increased ethanol productivity and improved growth in the presence of a synthetic inhibitor cocktail. Hybrid strains HR4/YI30#6 and V3/YI30#6 were able to grow at a high salt concentration (2 mol/L NaCl) with V3/YI30#6 also able to grow at a high temperature (45 °C). Strains HR4/YI30#1 and #3 were inhibitor-tolerant, with strain HR4/YI30#3 having similar productivity (0.36 ± 0.0036 g/L per h) as the superior parental strain, YI30 (0.35 ± 0.0058 g/L per h). This study indicates that natural S. cerevisiae strains display phenotypic variation and heterosis can be achieved through spore-to-spore hybridization. Several of the phenotypes (temperature-, osmo-, and inhibitor tolerance) displayed by both the natural strains and the generated progeny were at the maximum conditions reported for S. cerevisiae strains.     

Inactivation of the transcription factor <Emphasis Type="Italic">mig1</Emphasis> (<Emphasis Type="Italic">YGL035C</Emphasis>) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves tolerance towards monocarboxylic weak acids: acetic,formic and levulinic acid

Toxic concentrations of monocarboxylic weak acids present in lignocellulosic hydrolyzates affect cell integrity and fermentative performance of Saccharomyces cerevisiae. In this work, we report the deletion of the general catabolite repressor Mig1p as a strategy to improve the tolerance of S. cerevisiae towards inhibitory concentrations of acetic, formic or levulinic acid. In contrast with the wt yeast, where the growth and ethanol production were ceased in presence of acetic acid 5 g/L or formic acid 1.75 g/L (initial pH not adjusted), the m9 strain (Δmig1::kan) produced 4.06?±?0.14 and 3.87?±?0.06 g/L of ethanol, respectively. Also, m9 strain tolerated a higher concentration of 12.5 g/L acetic acid (initial pH adjusted to 4.5) without affecting its fermentative performance. Moreover, m9 strain produced 33% less acetic acid and 50–70% less glycerol in presence of weak acids, and consumed acetate and formate as carbon sources under aerobic conditions. Our results show that the deletion of Mig1p provides a single gene deletion target for improving the acid tolerance of yeast strains significantly.     

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.     

Biomass and terpenoids produced by mutant strains of <Emphasis Type="Italic">Arthrospira</Emphasis> under low temperature and light conditions

The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14?°C, 43.75 μmol photons m^?2 s^?1 and performed breed conservation at 2.5?°C, 8.75 μmol photons m^?2 s^?1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16?°C and 43.75 μmol photons m^?2 s^?1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057?±?80 mg l^?1, 68.7?±?2.5 mg l^?1 day^?1, 1839?±?44 mg l^?1, 60.6?±?1.8 mg l^?1 day^?1, and 2113?±?64 mg l^?1, 77.7?±?2.5 mg l^?1 day^?1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10?°C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28?°C, achieving 1140 mg l^?1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-d-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.     

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for second-generation bioethanol production

Background

Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results

Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.

Conclusions

This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

     

Endogenous arabitol and mannitol improve shelf life of encapsulated <Emphasis Type="Italic">Metarhizium brunneum</Emphasis>

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6?±?0.2 and 2.1?±?0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1?±?4.4 to 55.0?±?6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5?±?3.3% at 5 °C followed by 54.5?±?1.6% at 18 °C and 19.4?±?1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3?±?7.6 to 98.0?±?1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.     

Batch ethanol production from cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz.) flour using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells immobilized in calcium alginate

The objective of this research was to saccharify cassava flour by acid-acid and acid-enzyme hydrolysis and further conversion of the resulting sugar into ethanol by fermenting with the immobilized (in Ca-alginate) cells of Saccharomyces cerevisiae. The saccharification resulted in higher total sugar recovery by acid-enzyme hydrolysis (72.88 %) than by enzyme-enzyme hydrolysis (58.1 %). Further study on ethanol production was carried out using the hydrolysate obtained from acid-enzyme hydrolysis. The growth of the yeast started in the log phage and maximum ethanol (189?±?3.1 g ethanol/kg flour) production was achieved with 94.74?±?2.187 % sugar conversion during the stationary phase.     

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> IR-2 for high-temperature ethanol production

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 °C and over-expression of all PPP genes at 36 °C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 °C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.     

Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion

To increase thermotolerance and ethanol tolerance in Saccharomyces cerevisiae strain YZ1, the strategies of high-energy pulse electron beam (HEPE) and three rounds of protoplast fusion were explored. The YF31 strain had the characteristics of resistant to high-temperature, high-ethanol tolerance, rapid growth and high yield. The YF31 could grow on plate cultures up to 47?°C, containing 237.5?g?L^?1 of ethanol. In particular, the mutant strain YF31 generated 94.2?±?4.8?g?L^?1 ethanol from 200?g glucose L^?1 at 42?°C, which was 2.48 times the production of the wild strain YZ1. Results demonstrated that the variant phenotypes from the strains screening by HEPE irradiation could be used as parent stock for yeast regeneration and the protoplast fusion technology is sufficiently powerful in combining suitable characteristics in a single strain for ethanol fermentation.     

Endocellulase Production by <Emphasis Type="Italic">Cotylidia pannosa</Emphasis> and its Application in Saccharification of Wheat Bran to Bioethanol

For efficient bioconversion of lignocellulosic materials to bioethanol, the study screened 19 white-rot fungal strains for their endocellulolytic activity and saccharification potential. Preliminary qualitative and quantitative screening revealed Cotylidia pannosa to be the most efficient endocellulase producing fungal strain when compared to the standard strain of Trichoderma reesei MTCC 164. Ensuing initial screening, the production of endocellulase was further optimized using submerged fermentation to recognize process parameters such as temperature, time, agitation pH, and supplementation of salts in media required for achieving maximum production of endocellulase. The strain C. pannosa produced the maximum amount of endocellulase (8.48 U/mL) under submerged fermentation with wheat bran (2%) supplemented yeast extract peptone dextrose (YEPD) medium after an incubation time of 56 h at 30 °C and pH 5.0 at an agitation rate of 120 rpm with a saccharification value of 50.5%. The fermentation of wheat bran hydrolysate with Saccharomyces cerevisiae MTCC 174 produced 4.12 g/L of bioethanol after 56 h of incubation at 30 °C. The results obtained from the present investigation establish the potential of white-rot fungus C. pannosa for hydrolysis and saccharification of wheat bran to yield fermentable sugars for their subsequent conversion to bioethanol, suggesting its application in efficient bioprocessing of lignocellulosic wastes.     

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch and fed-batch cultures

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45–50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90–95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47–0.50 g/g), and a final ethanol concentration of 100–110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.     

The Influences of Chromium Supplementation on Glycemic Control,Markers of Cardio-Metabolic Risk,and Oxidative Stress in Infertile Polycystic ovary Syndrome Women Candidate for In vitro Fertilization: a Randomized,Double-Blind,Placebo-Controlled Trial

This study was carried out to investigate the effects of chromium intake on glycemic control, markers of cardio-metabolic risk, and oxidative stress in infertile polycystic ovary syndrome (PCOS) women candidate for in vitro fertilization (IVF). This randomized double-blind, placebo-controlled trial was done among 40 subjects with infertile PCOS candidate for IVF, aged 18–40 years old. Individuals were randomly allocated into two groups to take either 200 μg/day of chromium (n?=?20) or placebo (n?=?20) for 8 weeks. Biochemical parameters were assessed at baseline and at end-of-trial. Compared with the placebo, taking chromium supplements led to significant reductions in fasting plasma glucose (??2.3?±?5.7 vs. +?0.9?±?3.1 mg/dL, P?=?0.03), insulin levels (??1.4?±?2.1 vs. +?0.4?±?1.7 μIU/mL, P?=?0.004), homeostatic model of assessment for insulin resistance (??0.3?±?0.5 vs. +?0.1?±?0.4, P?=?0.005), and a significant increase in quantitative insulin sensitivity check index (+?0.004?±?0.008 vs. ??0.001?±?0.008, P?=?0.03). In addition, chromium supplementation significantly decreased serum triglycerides (??19.2?±?33.8 vs. +?8.3?±?21.7 mg/dL, P?=?0.004), VLDL- (??3.8?±?6.8 vs. +?1.7?±?4.3 mg/dL, P?=?0.004) and total cholesterol concentrations (??15.3?±?26.2 vs. ??0.6?±?15.9 mg/dL, P?=?0.03) compared with the placebo. Additionally, taking chromium supplements was associated with a significant increase in plasma total antioxidant capacity (+?153.9?±?46.1 vs. ??7.8?±?43.9 mmol/L, P?<?0.001) and a significant reduction in malondialdehyde values (?0.3?±?0.3 vs. +?0.1?±?0.2 μmol/L, P?=?0.001) compared with the placebo. Overall, our study supported that chromium administration for 8 weeks to infertile PCOS women candidate for IVF had beneficial impacts on glycemic control, few variables of cardio-metabolic risk, and oxidative stress.     

<Emphasis Type="Italic">Scopulibacillus cellulosilyticus</Emphasis> sp. nov., a cellulose-degrading bacterium isolated from tea

A Gram-stain positive, aerobic, non-motile, endospore-forming and rod-shaped strain (THG-NT9^T) was isolated from a green tea sample. Growth occurred at 20–45 °C (optimum 28–35 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–2.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-NT9^T were identified as Scopulibacillus daqui DSM 28236^T (98.6%), Scopulibacillus darangshiensis DSM 19377^T (97.4%), Pullulanibacillus pueri CGMCC 1.12777^T (96.7%) and Pullulanibacillus camelliae CGMCC 1.15371^T (96.3%). The DNA G?+?C content of strain THG-NT9^T was determined to be 47.5 mol %. DNA–DNA hybridization values between strain THG-NT9^T and S. daqui DSM 28236^T, S. darangshiensis DSM 19377^T, P. pueri CGMCC 1.12777^T, P. camelliae CGMCC 1.15371^T and Pullulanibacillus naganoensis DSM 10191^T were 41.3?±?0.1 (39.4?±?0.4% reciprocal analysis), 39.1?±?0.1 (37.3?±?0.1%), 21.4?±?0.7 (20.1?±?0.3%), 20.7?±?0.1 (20.1?±?0.4%) and 12.1?±?0.2% (8.3?±?0.2%). The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. The quinone was identified as MK-7. The major fatty acids were C_18:3 ω7c, iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The cell wall type was determined to be A1γ peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid plus alanine and glutamic acid and glucose as the cell wall sugar. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-NT9^T represents a novel species of the genus Scopulibacillus, for which the name Scopulibacillus cellulosilyticus sp. nov. is proposed. The type strain is THG-NT9^T (=?KCTC 33918^T?=?CGMCC 1.16305^T).     

Cloning,deletion, and overexpression of a glucose oxidase gene in <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. P6 for Ca<Superscript>2+</Superscript>-gluconic acid overproduction

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca²⁺-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca²⁺-GA (160.5?±?5.6 g/L) and higher GOD1 activity (1438.6?±?73.2 U/mg of protein) than its parent P6 strain (118.7?±?4.3 g/L of Ca²⁺-GA and 1100.0?±?23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8?±?6.0 g/L of Ca²⁺-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca²⁺-GA (186.8?±?6.0 g/L) than its wild-type strain P6.     

Effect of inorganic salt stress on the thermotolerance and ethanol production at high temperature of <Emphasis Type="Italic">Pichia kudriavzevii</Emphasis>

Application of cross-protection is expected to improve the thermotolerance of yeasts to enhance their ethanol production at high temperature. In this study, the effects of eight kinds of inorganic salts on the thermotolerance and ethanol production at high temperature in Pichia kudriavzevii were investigated. P. kudriavzevii showed strong thermotolerance and the ability to produce ethanol at high temperature, and higher ethanol production of P. kudriavzevii was observed at high temperature (37–42 °C) compared with that at 30 °C. Inorganic salt stresses induced obvious cross-protection of thermotolerance in P. kudriavzevii. The presence of 0.1 mol/L KNO₃ or Na₂SO₄ or 0.2 mol/L NaCl, KCl, NaNO₃, K₂SO₄ or MgCl₂ increased the yeast biomass in YEPD medium at 44 °C to 2.72–3.46 g/L, obviously higher than that in the absence of salt stress (2.17 g/L). The addition of NaCl, KCl, NaNO₃, KNO₃, Na₂SO₄, K₂SO₄, CaCl₂ and MgCl₂ significantly increased the ethanol production of P. kudriavzevii in YEPD fermentation medium at 44 °C by 37–58%. KCl and MgCl₂ exhibited the best performance on improving the thermotolerance and ethanol production, respectively, of P. kudriavzevii. A highly significant correlation (P?<?0.01) was obtained among ethanol production, biomass and glucose consumption, suggesting the important role of thermotolerance and glucose consumption in enhanced ethanol production. The combination of NaCl, KCl and MgCl₂ had a synergistic effect on the improvement of thermotolerance and ethanol production at high temperature in P. kudriavzevii. This study provides some important clues for improving ethanol production of thermotolerant yeasts at high temperature.     

The heterologous expression potential of an acid-tolerant <Emphasis Type="Italic">Talaromyces pinophilus</Emphasis> β-glucosidase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A filamentous fungus displaying high cellulase activity was isolated from a compost heap with triticale (a wheat-rye hybrid) as the main constituent. It was preliminarily identified as a Talaromyces pinophilus species. A 2577 base pair β-glucosidase gene was cloned from complementary DNA and heterologously expressed in Saccharomyces cerevisiae. The recombinant β-glucosidase production profile was assessed and compared to that of the Saccharomycopsis fibuligera β-glucosidase which served as a benchmark. The enzyme was also characterised in terms of pH and temperature tolerance as well as response to inhibitors. Maximal extracellular β-glucosidase activity of 0.56 nkat/mg total protein was measured using p-nitrophenyl-β-D-glucopyranoside as substrate. The recombinant protein displayed a pH optimum of 4.0, and good thermostability as 70% of maximal enzyme activity was retained after 1 h at 60 °C. Activity of the recombinant β-glucosidase was adversely affected by the presence of glucose and ethanol at higher concentrations while xylose had no effect. The expression of the T. pinophilus β-glucosidase did not reach the same titres as for the benchmark; however, in the context of constructing a yeast strain for bioethanol production in a consolidated bioprocess, the enzyme may still show good potential.     

Bioprospecting of thermo- and osmo-tolerant fungi from mango pulp–peel compost for bioethanol production

The persistent edaphic stress on microbial succession due to dynamic changes during composting was explored for selection of multi-stress tolerant microbe(s) desirable for ethanol production. A total of 23 strains were isolated from mango compost using four successive enrichments in YP broth (g l^?1): glucose, 100; 150; 250 with ethanol (40) and cycloheximide (0.4) at 40 °C, pH 6.0. Based on multi-gene ribotyping, 14 yeasts (61 %) of Saccharomycetaceae, 2 filamentous fungi (8.6 %) and 7 bacteria (30.4 %) were obtained. Phenetic and phylogenetic analysis of the 14 yeasts revealed 64.3 % tolerant to 500 g l^?1 glucose, growth at 45 °C and resemblance to Candida sp. (14.3 %), Kluyveromyces marxianus (35.7 %), Pichia kudriavzevii (21.4 %) and Saccharomyces cerevisiae (28.6 %). Assessment of the 14 yeasts in glucose fermentation medium (pH 4.5 at 40 °C) showed ethanol productivity of ≥92 % by 12 yeasts with theoretical yields of 90–97 %. Fermentation of molasses (150 g l^?1 glucose equivalent) by P. kudriavzevii D1C at 40 °C resulted in 73.70 ± 0.02 g l^?1 ethanol and productivity of 4.91 ± 0.01 g l^?1 h^?1. Assessment of P. kudriavzevii D1C revealed multi-stress tolerance towards 5-hydroxymethyl furfural, ethanol (20 %, v/v), high gravity and H₂O₂ (0.3 M) indicating suitability for ethanol production using high gravity molasses and pre-treated lignocellulosic biomass fermentation.     

Inheritance pattern and genetic correlations among growth and wood quality traits in Para rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis>) and implications for breeding

Inheritance pattern of wood traits viz. specific gravity, fibre dimensions and fibre-derived biometrical indices and their interactions among themselves and with that of growth are reported in Hevea brasiliensis. Girth (h² =???0.02?±?0.44 to h² =?0.35?±?0.24) showed moderate genetic control. Among wood traits, specific gravity (h²?=?0.15?±?0.31 to h² =?0.33?±?0.28) was found to be under moderate genetic control. Fibre traits viz., fibre length (h² =???0.26?±?0.30 to h² =?0.50?±?0.34), fibre diameter (h² =?0.19?±?0.49 to h² =?0.70?±?0.11), fibre lumen diameter (h² =???0.18?±?0.35 to h² =?0.56?±?0.47) and fibre wall thickness (h² =???5.17?±?5.26 to h² =?0.50?±?0.50) were under moderate to strong genetic control. Among fibre-derived indices, flexibility coefficient (h² =?0.48?±?0.21 to h² =?0.89?±?0.29) showed moderate to very strong genetic control. The Runkel ratio (h² =???0.40?±?0.27 to h² =?0.42?±?0.29) and slenderness ratio (h² =???0.36?±?0.29 to h² =?0.43?±?0.28) showed moderate genetic control. Girth showed very strong positive genetic correlation with fibre wall thickness and strong positive correlation with fibre width indicating scope of indirect selection potential for these traits. Wood specific gravity was not correlated with either girth or fibre traits. Hence, it would be possible to concomitantly improve growth and fibre traits without adversely affecting wood specific gravity. Moderate to very high estimates of heritability for fibre traits, girth and specific gravity indicated that considerable genetic gain can be realised for these traits. Implications of the above findings in genetic improvement of wood in Hevea are discussed.