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[Introduction] In this paper, the thermal performance of a parabolic trough direct steam generation system (DSGS) is investigated. With the considerations of the stratified flow phenomenon and real flow patterns in the complex two-phase flow, the heat transfer progress in the DSG system is numerically studied, and the thermodynamic performances are revealed. [Method] Based on the flow pattern inside the trough collector, the two-phase heat transfer model in the trough-type heat collecting region was established, and the DSG process in the trough collector was simulated by MatLab programming. [Result] The variation of heat transfer characteristics of DSG technology along the length of the tube is revealed. The stratification phenomenon occurs in the two-phase flow region where the dryness is between 0.7 and 0.8. The occurrence of delamination reduces the heat transfer between the working medium and metal tube, which weakens the heat transfer effect. [Conclusion] This study provides a reference for the design and operation of DSG power stations, and verifies that the optimal arrangement of DSG collectors is recirculating.

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Filamentous fungi are one of the platforms for producing fermented products. The specific characteristic of their submerged fermentation is the aggregation of mycelia that is affected by environmental conditions, leading to significantly different rheology for fermentation broth. Such a rheological change not only affects the transfer of mass, heat and momentum, but also the biosynthesis of target products and the efficiency of their production. In this article, strategies for morphological regulation of filamentous fungi are reviewed, and the impact of calcium signal transduction and chitin biosynthesis on apical growth of hyphae and branching of mycelia for their aggregation are further commented.

Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.

By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288C-HO, the recombinant strain S288C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses.

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

Production of bioenergy and bio-based chemicals by using fermentable sugars released from low-cost renewable lignocellulosic biomass has received great attention. Efficient cellulolytic enzymes are crucial for lignocellulose bioconversion, but high cellulase production cost is limiting the bioconversion efficiency of cellulosic biomass and industrial applications of lignocellulose biorefinery. Studies on induction and regulation of cellulase in filamentous fungi will help to further develop superior fungal strains for efficient cellulase production and reduce cellulase production cost. With the advances in high-throughput sequencing and gene manipulation technology using fungal strains, an in-depth understanding of cellulase induction and regulation mechanisms of enzyme expression has been achieved. We reviewed recent progresses in the induction and regulation of cellulase expression in several model filamentous fungi, emphasizing sugar transporters, transcription factors and chromatin remodeling. Future prospects in application of artificial zinc finger proteins for cellulase induction and regulation in filamentous fungi were discussed.

Microalgae have been identified as promising candidates for biorefinery of value-added molecules. The valuable products from microalgae include polyunsaturated fatty acids and pigments, clean and sustainable energy (e.g. biodiesel). Nevertheless, high cost for microalgae biomass harvesting has restricted the industrial application of microalgae. Flocculation, compared with other microalgae harvesting methods, has distinguished itself as a promising method with low cost and easy operation. Here, we reviewed the methods of microalgae harvesting using flocculation, including chemical flocculation, physical flocculation and biological flocculation, and the progress and prospect in bio-flocculation are especially focused. Harvesting microalgae via bio-flocculation, especially using bio-flocculant and microalgal strains that is self-flocculated, is one of the eco-friendly, cost-effective and efficient microalgae harvesting methods.

Chromosomal integration enables stable phenotype and therefore has become an important strategy for breeding of industrial Saccharomyces cerevisiae strains. pAUR135 is a plasmid that enables recycling use of antibiotic selection marker, and once attached with designated homologous sequences, integration vector for stable expression can be constructed. Development of S. cerevisiae strains by metabolic engineering normally demands overexpression of multiple genes, and employing pAUR135 plasmid, it is possible to construct S. cerevisiae strains by combinational integration of multiple genes in multiple sites, which results in different ratios of expressions of these genes. Xylose utilization pathway was taken as an example, with three pAUR135-based plasmids carrying three xylose assimilation genes constructed in this study. The three genes were sequentially integrated on the chromosome of S. cerevisiae by combinational integration. Xylose utilization rate was improved 24.4%-35.5% in the combinational integration strain comparing with that of the control strain with all the three genes integrated in one location. Strain improvement achieved by combinational integration is a novel method to manipulate multiple genes for genetic engineering of S. cerevisiae, and the recombinant strains are free of foreign sequences and selection markers. In addition, stable phenotype can be maintained, which is important for breeding of industrial strains. Therefore, combinational integration employing pAUR135 is a novel method for metabolic engineering of industrial S. cerevisiae strains.

Zinc-finger proteins have been widely studied due to their highly conserved structures and DNA-binding specificity of zinc-finger domains. However, researches on the zinc-finger proteins from microorganisms, especially those from prokaryotes, are still very limited. This review focuses on the latest progress on microbial zinc-finger proteins, especially those from prokaryotes and the application of artificial zinc-finger proteins in the breeding of robust strains. Artificial zinc-finger proteins with transcriptional activation or repression domain can regulate the global gene transcription of microbial cells to acquire improved phenotypes, such as stress tolerance to heat, ethanol, butanol, and osmotic pressure. Using the zinc-finger domain as DNA scaffold in the construction of enzymatic system can enhance the catalytic efficiency and subsequently the production of specific metabolites. Currently, zinc-finger domains used in the construction of artificial transcription factor are usually isolated from mammalian cells. In the near future, novel transcription factors can be designed for strain development based on the natural zinc-finger domains from different microbes, which may be used to regulate the global gene expression of microbial cells more efficiently.

Industrial microorganisms are subject to various stress conditions, including products and substrates inhibitions. Therefore, improvement of stress tolerance is of great importance for industrial microbial production. Acetic acid is one of the major inhibitors in the cellulosic hydrolysates, which affects seriously on cell growth and metabolism of Saccharomyces cerevisiae. Studies on the molecular mechanisms underlying adaptive response and tolerance of acetic acid of S. cerevisiae benefit breeding of robust strains of industrial yeast for more efficient production. In recent years, more insights into the molecular mechanisms underlying acetic acid tolerance have been revealed through analysis of global gene expression and metabolomics analysis, as well as phenomics analysis by single gene deletion libraries. Novel genes related to response to acetic acid and improvement of acetic acid tolerance have been identified, and novel strains with improved acetic acid tolerance were constructed by modifying key genes. Metal ions including potassium and zinc play important roles in acetic acid tolerance in S. cerevisiae, and the effect of zinc was first discovered in our previous studies on flocculating yeast. Genes involved in cell wall remodeling, membrane transport, energy metabolism, amino acid biosynthesis and transport, as well as global transcription regulation were discussed. Exploration and modification of the molecular mechanisms of yeast acetic acid tolerance will be done further on levels such as post-translational modifications and synthetic biology and engineering; and the knowledge obtained will pave the way for breeding robust strains for more efficient bioconversion of cellulosic materials to produce biofuels and bio-based chemicals.

Propionic acid, a major inhibitor to yeast cells, was accumulated during continuous ethanol fermentation from corn meal hydrolysate by the flocculating yeast under stillage backset conditions. Based on its inhibition mechanism in yeast cells, strategies were developed for alleviating this effect. Firstly, high temperature processes such as medium sterilization generated more propionic acid, which should be avoided. Propionic acid was reduced significantly during ethanol fermentation without medium sterilization, and concentrations of biomass and ethanol increased by 59.3% and 7.4%, respectively. Secondly, the running time of stillage backset should be controlled so that propionic acid accumulated would be lower than its half inhibition concentration IC50 (40 mmol/L). Finally, because low pH augmented propionic acid inhibition in yeast cells, a higher pH of 5.5 was validated to be suitable for ethanol fermentation under the stillage backset condition.

Ethanol tolerance is related to the expression of multiple genes, and genome-based engineering approaches are much more efficient than manipulation of single genes. In this study, ultraviolet (UV) mutagenesis, dielectric barrier discharge (DBD) air plasma mutagenesis, and artificial transcription factor (ATF) technology were adopted to treat an industrial yeast strain S. cerevisiae Sc4126 to obtain mutants with improved ethanol tolerance. Mutants with high ethanol tolerance were obtained, and the ratio of positive mutants was compared. Among the three approaches, the rate of positive mutation obtained by ATF technology was 10- to 100-folds of that of the two other methods, with highest genetic stability, suggesting the ATF technology promising for rapid alteration of phenotypes of industry yeast strains for efficient ethanol fermentation.

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.

Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.

Consolidated bioprocessing technology can be used for Kluyveromyces marxianus YX01 to produce ethanol from Jerusalem artichoke, which is one of the potential processes to produce biofuel from non-cereal crops. In this study, we combined the aeration rate with the substrate concentration to conduct cross-over experiments for K. marxinaus YX01, and studied ethanol fermentation and the influence of inulin enzyme activity. The substrate concentration had a little repressive effect on ethanol productivity. When substrate concentration reached 250 g/L under anaerobic conditions, ethanol concentration was 84.8 g/L, and ethanol yield was reduced from 86.4% (50 g/L substrate concentration) to 84.7% of the theoretical value. Aeration rate could accelerate K. marxinaus YX01 ethanol fermentation, but reduced ethanol yield. When substrate concentration reached 250 g/L under aeration at 1.0 vvm, ethanol yield was reduced from 84.7% under anaerobic conditions to 73.3% of the theoretical value. With increased concentration of the carbon source and reduced aeration rate, the inulinase of K. marxinaus YX01 reduced and the concentration of glycerol increased, however, the acetic acid increased with the increased concentration of the carbon source and aeration rate. When substrate concentration reached 250 g/L under anaerobic conditions, inulinase activity was only 6.59 U/mL; when substrate concentration reached 50 g/L under aeration at 1.0 vvm, inulinase activity was 21.54 U/mL.

We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(Lh). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.

The objective of this work is to investigate how dilution rate and carbon-to-nitrogen (C/N) ratio affects lipid accumulation by Rhodosporidium toruloides AS 2.138 9 in continuous culture. Under steady-state conditions, the increase in dilution rate led to the decrease in lipid content and lipid yield. The highest lipid yield and lipid content at D = 0.02 h(-1) were 0.18 g lipid/g sugar and 57.1%, respectively, while the highest lipid productivity and biomass productivity were obtained at D = 0.14 h(-1). The increase in C/N ratio led to the increase in lipid content. The highest lipid content of 38% was obtained at C/N = 237. The highest lipid yield of 0.12 g lipid/g sugar was obtained at C/N = 92. However, the highest lipid productivity of 0.12 g/(L x h) was obtained at C/N = 32. No significant changes were observed in terms of fatty acid composition of the lipid produced under different C/N ratios, and these three fatty acids, palmitic acid, stearic acid and oleic acid, took over 85% in all samples.

A mixture of fructose and glucose was developed to simulate the hydrolysate of Jerusalem artichoke tubers, the fructose-based feedstock suitable for butanol production. With the initial pH of 5.5 without regulation during mixed-sugar fermentation, as high as 23.26 g/L sugars were remained unconverted, and butanol production of 5.51 g/L were obtained. Compared with either glucose or fructose fermentation, the early termination of mixed-sugar fermentation might be caused by toxic organic acids and the low pH. When the pH of the fermentation system was controlled at higher levels, it was found that sugars utilization was facilitated, but less butanol was produced due to the over-accumulation of organic acids. On the other hand, when the pH was controlled at lower levels, more sugars were remained unconverted, although butanol production was improved. Based on these experimental results, a stage-wise pH regulation strategy, e.g., controlling the pH of the fermentation system at 5.5 untill the OD620 reached 1.0, and then the pH control was removed, was developed, which significantly improved the fermentation performance of the system, with only 2.05 g/L sugars unconverted and 10.48 g/L butanol produced.

A system coupling ethanol fermentation with microalgae culture was developed, in which CO2 produced during ethanol fermentation was used as carbon source for the growth of Tetraselmis subcordiformis, a microalgae accumulating starch intracellularly. The biomass concentration about 2.0 g DCW/L was achieved within the photobioreactor for the batch culture of 7 days, and intracellular starch accumulation was about 45%. Furthermore, ultrasonic pretreatment and enzymatic hydrolysis were applied to the microalgae biomass, and 71.1% of the intracellular starch was converted into glucose that was fermented sequentially to ethanol by Saccharomyces cerevisiae with an ethanol yield of 87.6% of the theoretical value, indicating that the microalgae biomass could be an alternative feedstock for ethanol production to save grain consumption, and in the meantime mitigate the CO2 emission.

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.

The flocculating yeast strain SPSC01 is a fusant strain of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The use of SPSC01 to absorb Cr(VI) from Cr(VI) containing aqueous solution would greatly reduce the cost of post-adsorption separation, since the superior flocculating property of SPSC01 would allow easy separation of the Cr(VI)-biomass from the solution. In order to investigate the effects of flocculating proteins on Cr(VI) reduction and absorption by SPSC01, the absorption behaviors of SPSC01 and its parental strains were compared. The results showed that Cr(VI) removal rate of SPSC01 was almost the same as that of S. pombe, which also has flocculating ability, but was faster than that of S. cerevisiae, which has no flocculating ability. When the system reached equilibrium, the amount of total Cr adsorbed by S. pombe, SPSC01 and S. cerevisiae were 68.8%, 48.6% and 37.5%, respectively. This showed that flocculation was beneficial to Cr(VI) reduction and adsorption, and suggested that focculating proteins may play a role in enhancing the Cr(VI) adsorption capacity of SPSC01 and S. pombe. We investigated the mechanism of Cr(VI) adsorption by SPSC01 using chemical modification and FTIR. The results indicated that the major functional groups (amino, carboxyl and amide) of surface proteins may contribute to the absorption of Cr(VI).

Improving stress tolerance of the microbial producers is of great importance for the process economy and efficiency of bioenergy production. Key genes influencing ethanol tolerance of brewing yeast can be revealed by studies on the molecular mechanisms which can lead to the further metabolic engineering manipulations for the improvement of ethanol tolerance and ethanol productivity. Trahalose shows protective effect on the cell viability of yeast against multiple environmental stress factors, however, further research is needed for the exploration of the underlying molecular mechanisms. In this study, the promoter region of the trehalose-6-phosphate synthase gene TPS1 was cloned from the self-flocculating yeast Saccharomyces cerevisiae flo, and a reporter plasmid based on the expression vector pYES2.0 on which the green fluorescence protein EGFP was directed by the TPS1 promoter was constructed and transformed to industrial yeast strain Saccharomyces cerevisiae ATCC4126. Analysis of the EGFP expression of the yeast transformants in presence of 7% and 10% ethanol revealed that the P(TPS1) activity was strongly induced by 7% ethanol, showing specific response to ethanol stress. The results of this study indicate that trehalose biosynthesis in self-flocculating yeast is a protective response against ethanol stress.

Butanol production from acid hydrolysate of Jerusalem artichoke juice by Clostridium acetobutylicum L7 was investigated, and it was found that natural components of the hydrolysate were suitable for solvent production with the species. With batch fermentation using the medium containing 48.36 g/L total sugars, 8.67 g/L butanol was produced at 60 h, and the ratio of butanol to acetone to ethanol was 0.58:0.36:0.06, which were similar to the fermentation with fructose as carbon source, but both of these two fermentations were slower than that with glucose as carbon source, indicating the fructose transport of the species might not be effective as that for glucose. When the total sugars of the medium were increased to 62.87 g/L, the residual sugars increased slightly from 3.09 g/L to 3.26 g/L, but butanol production of the fermentation system was improved significantly, with 11.21 g/L butanol produced and the ratio of butanol to acetone to ethanol at 0.64:0.29:0.05, which illustrated that an excess in sugars enhanced the butanol biosynthesis of the species by compromising its acetone production. When the sugar concentration of the medium was further increased, much more sugars were remained unconsumed, making the process economically unfavourable.

Biofuels are renewable and environmentally friendly, but high production cost makes them economically not competitive, and the development of robust strains is thus one of the prerequisites. In this article, strain improvement studies based on the information from systems biology studies are reviewed, with a focus on their applications on stress tolerance improvement. Furthermore, the contribution of systems biology, synthetic biology and metabolic engineering in strain development for biofuel production is discussed, with an expectation for developing more robust strains for biofuel production.

Biofuels and bioenergy not only benefit independence of energy supply, but also mitigate CO2 emissions. This special issue includes review reports and research articles involving various biofuels and bioenergy products and systems such as fuel ethanol, biodiesel, biogas, biohydrogen, microbial fuel cells and microbial electrolysis cells. Both fundamental research and technology development are highlighted. And in the meantime, challenges for large scale production and application of biofuels and bioenergy are discussed. Taking advantages of modern biotechnology advances, solutions to address these challenges are envisioned.

Prior research reported the oscillatory behavior characterized by long period and high amplitude during high gravity continuous ethanol fermentations at the dilution rate of 0.027 h(-1). In this paper, high gravity continuous ethanol fermentations using Saccharomyces cerevisia at different dilution rates were carried out. Similar oscillations were observed when the dilution rate was switched to 0.04 h(-1). Both oscillatory and steady processes can be achieved at dilution rates of 0.027 or 0.04 h(-1), which depends on the initial status of the fermentation system. However, compared to steady process at the same dilution rate of 0.04 h(-1), the average residual sugar concentration was lowered by 14.8% for the oscillatory process, while the average ethanol concentration and productivity were increased by 12.6% and 12.3%, respectively. Further investigation revealed that besides the lag time, oscillatory processes were different from steady ones in kinetics because a higher specific growth rate can be achieved at the same residual sugar and ethanol concentrations (increased by 53.8% in average).

Directed evolution of transcription factors can be employed to effectively improve the phenotypes which are controlled by multiple genetic loci. In this study, we used error-prone PCR for the directed evolution of SPT3, which is the component of yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex responsible for the transcription of stress-related genes, and studied its effect on the improvement of ethanol tolerance. Mutant library was constructed by ligating the error-prone PCR products with a modified pYES2.0 plasmid, and the expression plasmids were subsequently transformed to yeast industrial strain Saccharomyces cerevisiae 4126. One mutant strain M25 showing superior growth in presence of 10% ethanol was selected. M25 produced 11.7% more ethanol than the control strain harboring the empty vector when 125 g/L glucose was used as substrate. This study revealed that SPT3 is an important transcription factor for the metabolic engineering of yeast ethanol tolerance.

Directed evolution, which is also called molecular evolution, or artificial evolution, combines random mutagenesis and directed selection. In previous studies, it has been extensively applied for the improvement of enzyme catalytic properties and stability, as well as the expanding of substrate specificity. In recent years, directed evolution was also employed in metabolic engineering of promoters for improving their strength and function, and the engineering of global transcription machinery. These techniques contribute to breeding more tolerant strains against environmental stress, as well as strains with improved fermentation efficiency. In this article, we reviewed the applications of directed evolution in the metabolic engineering of promoters and global transcription machinery. These techniques enabled fine-tuning of gene expression and simultaneous alternation of multiple gene transcription inside the cells, and thus are powerful new tools for metabolic engineering.

In order to save energy consumption for the downstream processes, consecutive very-high-gravity batch fermentation was developed for ethanol production with the self-flocculating yeast Saccharomyces cerevisiae flo. The fermentation system exhibited a high ethanol productivity of 8.2 g/(L x h) with average ethanol concentration around 120 g/L. However, deterioration of the sedimentation performance of yeast flocs was observed as the consecutive fermentation process was prolonged, which significantly extended the time required for yeast flocs to separate from fermentation broth, and exaggerated the inhibition of high ethanol concentration on the yeast flocs, making them quickly lost viability and the fermentation system interrupted after 11 consecutive batches. Experimental results illustrated that decrease of the size of yeast flocs was the main reason, which could be prevented by stimulating the propagation of the yeast flocs. Thus, yeast was purged from the fermentation system at the end of each batch, and the concentration of yeast flocs within the fermentor was maintained at a relatively low level to stimulate their propagation. Although the ethanol productivity was decreased to 4.0 g/(L x h), the size of yeast flocs was stabilized after 10 consecutive batches and maintained for another 14 batches without further decrease, indicating the fermentation system could be operated reliably.

Improvement of stress tolerance to various adverse environmental conditions (such as toxic products, high temperature) of the industrial microorganisms is important for industrial applications. Ethanol produced by yeast fermentation is inhibitory to both yeast cell growth and metabolisms, and consequently is one of the key stress elements of brewer's yeast. Research on the biochemical and molecular mechanism of the tolerance of yeast can provide basis for breeding of yeast strain with improved ethanol tolerance. In recent years, employing global gene transcriptional analysis and functional analysis, new knowledge on the biochemical and molecular mechanisms of yeast ethanol tolerance has been accumulated, and novel genes and biochemical parameters related to ethanol tolerance have been revealed. Based on these studies, the overexpression and/or disruption of the related genes have successfully resulted in the breeding of new yeast strains with improved ethanol tolerance. This paper reviewed the recent research progress on the molecular mechanism of yeast ethanol tolerance, as well as the genetic engineering manipulations to improve yeast ethanol tolerance. The studies reviewed here not only deepened our knowledge on yeast ethanol tolerance, but also provided basis for more efficient bioconversion for bio-energy production.

A unique one-step ethanol fermentation process was developed with the inulinase-producing strain Kluyveromyces marxianus YX01. Firstly, the impact of temperature on ethanol fermentation was investigated through flask fermentation, and the temperature of 35 degrees C was observed to be the optimum to coordinate inulinase production, inulin saccharification and ethanol fermentation. And then, the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L fermentor, and the experimental data indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min, but higher ethanol yield was obtained under non-aeration conditions with more substrate directed to ethanol production. The ethanol concentration of 92.2 g/L was achieved with the substrate containing 235 g/L inulin, and the ethanol yield was calculated to be 0.436, equivalent to 85.5% of its theoretical value. Finally, Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment, 84.0 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal, and the ethanol yield was calculated to be 0.405, indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production.

Ethanol tolerance of self-flocculating yeast SPSC01 was studied in a 3-L bioreactor under fed-batch culture. Yeast floc populations with the average sizes around 100, 200, 300, and 400 microm were obtained by adjusting the mechanical stirring rates of the fermentation system. When subjected to 20% (V/V) ethanol shock for 6 h at 30 degrees C, the remained cell viability was 3.5%, 26.7%, 48.8% and 37.6% for the aforementioned four floc populations, respectively. The highest ethanol yield 85.5% was achieved for the 300 microm flocs, 7.2% higher than that of the 100 microm flocs. The amounts of trehalose and ergosterol (including free ergosterol and total ergosterol) were positively correlated with the average size distributions from 100 to 300 microm. However, in the 400 microm flocs, the content of trehalose and ergosterol decreased, which coincided with its reduced ethanol tolerance compared to that of the 300 microm flocs. Furthermore, when subjected to 15% (V/V) ethanol shock at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient(P') of the 300 microm flocs accounted for only 43% and 52% respectively of those of the 100 microm and 200 microm populations. The effect of floc size distribution on the ethanol tolerance of the self-flocculating yeast strain SPSC01 was closely related to plasma membrane permeability. An optimal floc size distribution with the highest ethanol tolerance and ethanol production level could be obtained by controlling mechanical stirring speed of the bioreactor, which provides basis for the process optimization of fuel ethanol production using this self-flocculating strain.