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Background Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses. Results It was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. Conclusions The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Inference of gene regulatory networks (GRNs) is one of the major challenges in molecular biology, understanding of which can reveal the regulatory relationship between transcription factors (TFs) and target genes. Although in the past decades many methods were developed to reconstruct GRNs, the accuracy of traditional methods can be further improved. In this work, we proposed a new method, GRN-LightGBM (Light Gradient Boosting Machine), to reconstruct GRNs. GRN-LightGBM is a non-linear. Ordinary differential equations (ODEs) model established by LightGBM, which is considering regulatory and target genes for a specific gene. Furthermore, GRN-LightGBM utilizes time-series data, steady-state data, and temporal time-delay data together to evaluate the features of regulatory genes important for target genes. GRN-LightGBM is evaluated both in the DREAM4 simulated datasets and Escherichia coli real datasets. The results show that the proposed method outperforms other popular inference algorithms in terms of area under the receiver operating characteristic curve (AUROC) and area under the precision-recall curve (AUPR).

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate

High DHA production cost caused by low DHA titer and productivity of the current Schizochytrium strains is a bottleneck for its application in competition with traditional fish-oil based approach. In this study, atmospheric and room-temperature plasma with iodoacetic acid and dehydroepiandrosterone screening led to three mutants, 6–8, 6–16 and 6–23 all with increased growth and DHA accumulations. A LC/MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle might be relevant to the increased growth and DHA biosynthesis in the mutants. Finally, the mutant 6–23, which achieved the highest growth and DHA accumulation among all mutants, was evaluated in a 5 L fermentor. The results showed that the DHA concentration and productivity in mutant 6–23 were 41.4 g/L and 430.7 mg/L/h in fermentation for 96 h, respectively, which is the highest reported so far in literature. The study provides a novel strain improvement strategy for DHA-producing Schizochytrium.

The Biosecurity Law has laid down a regulatory framework on bioethics in China, from raising awareness through education, requiring researchers to conform to ethical principles and conduct ethical reviews on biomedical research, to giving special attention to human genetic resources. The law constructively leaves a wide range of discretion to medical institutions and professionals in ethical decision-making, adaptive to the biotechnology-ethics-regulation dynamics. This regulatory strategy poses crucial institutional challenges in its implementation, particularly on how to safeguard institutional review boards (IRB), a core mechanism in the governance, to effectively protect human subjects but not unnecessarily hinder the progress of biomedical research. Further measures need to clarify important issues on the IRB-based governance, including legal status of the IRB review decision, potential liabilities, and protections of the IRB members.

Synthetic biology is a new interdisciplinary research area that uses engineering principles as guidelines for biological investigation. With research goals to modify existing biological systems or to create new ones, the recent applications of synthetic biology have expanded approaches and tools for conventional biological research. In this article, we first briefly review the development and progress of synthetic biology over the past decade. Although the contributions of synthetic biology to basic life science research, human health, environmental protection, and even economic growth have been widely observed, potential biosafety, biosecurity, and ethical risks related to synthetic biology have also emerged in recent years as technology becomes less expensive, more mature, and more accessible. We provide a brief assessment of the risks associated with the possible misuse or abuse of this technology in various areas and discuss concerns from three points of view: biosafety, biosecurity risks, and ethics. Finally, to address challenges arising from the rapid progress of synthetic biology, technical, ethical, and regulatory measures were developed or discussed in recent years, including laboratory level precautionary measures for biosafety and biosecurity related to synthetic biology (such as genetic safeguards and firewalls), ethical codes of conduct for biological scientists, and regulations or oversight rules from personal, national, and international perspectives. A brief summary of these efforts is provided. Keywords: Synthetic biology, Biosafety, Biosecurity, Ethics, Genetic firewall, Genetic safeguard, Code of conduct, Governance regulation

Dietary omega-3 long-chain polyunsaturated fatty acids docosahexaenoic acid (DHA, C22:6) can be synthesized in microalgae Crypthecodinium cohnii; however, its productivity is still low. Here, we established a new protocol termed as “chemical modulator based adaptive laboratory evolution” (CM-ALE) to enhance lipid and DHA productivity in C. cohnii. First, ACCase inhibitor sethoxydim based CM-ALE was applied to redirect carbon equivalents from starch to lipid. Second, CM-ALE using growth modulator sesamol as selection pressure was conducted to relive negative effects of sesamol on lipid biosynthesis in C. cohnii, which allows enhancement of biomass productivity by 30% without decreasing lipid content when sesamol was added. After two-step CM-ALE, the lipid and DHA productivity in C. cohnii was respectively doubled to a level of 0.046 g/L/h and 0.025 g/L/h in culture with addition of 1 mM sesamol, demonstrating that this two-step CM-ALE could be a valuable approach to maximize the properties of microalgae.

The heterotrophic microalga Crypthecodinium cohnii accumulates lipids with a high fraction of docosahexaenoic acid (DHA). However, lack of genomic information limits the understanding of its physiological metabolism for better DHA production. In this study, de novo assembly of the C. cohnii transcriptome from three growth stages (i.e., fast growth, fatty acid accumulation and DHA conversion stages) was conducted, leading to identification of a total of 82,106 unigenes with an N50 of 1822 bp, among which 64.7% were annotated based on sequence similarity to known genes in the database. In addition, pathway enrichment analysis showed that transcripts related to fatty acid biosynthesis, starch and sucrose metabolism as well as biosynthesis of unsaturated fatty acids were significantly up-regulated during late-stage fermentation. Interestingly, several polyketide synthases (PKSs) and multiple fatty acid desaturases potentially involved in DHA biosynthesis were identified in the C. cohnii transcriptome, most of which were significantly up-regulated during lipid and DHA accumulation, implying that C. cohnii might utilize a combination of PKS systems and desaturase steps for DHA biosynthesis. The results were further confirmed by qRT-PCR and GC–MS-based metabolomic analyses. Overall, integrative analysis of de novo transcriptomic and metabolomic data provided important functional genomic information necessary for not only a better understanding of C. cohnii growth and DHA biosynthesis but also future genetic engineering of industry-important C. cohnii.

Background Docosahexaenoic acid (DHA, C22:6) and odd-chain fatty acids (OCFAs, C15:0 and C17:0) have attracted great interest, since they have been widely used in food and therapeutic industries, as well as chemical industry, such as biodiesel production and improvement. The oil-producing heterotrophic microalgae Schizochytrium sp. 31 is one of main DHA-producing strains. Recently, it was found that Schizochytrium can also synthesize OCFAs; however, contents and titers of DHA and OCFAs in Schizochytrium are still low, which limit its practical application. Results In this study, we found that acetyl-CoA carboxylase suffered from a feedback inhibition by C16-CoA in Schizochytrium, and relief of the inhibition resulted in improved both lipid content and the ratio of OCFAs in total fatty acids. Based on this finding, a novel strategy for elevating both DHA and OCFAs contents was established. First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from Mortierella alpina, which encodes an elongase enzyme responsible for converting C16 into C18 fatty acids. After the above two-step engineering, contents of DHA and OCFAs were increased by 1.39- and 3.30-fold, reaching a level of 26.70 and 25.08% of dry cell weight, respectively, which are the highest contents reported so far for Schizochytrium. The titers of DHA and OCFAs were elevated by 1.08- and 2.57-fold, reaching a level of 3.54 and 3.32 g/L, respectively. Notably, the OCFAs titer achieved was 2.66-fold higher than the highest reported in Escherichia coli (1.25 g/L), implying potential value for industry application. To reveal the potential metabolic mechanism for the enhanced biosynthesis of both DHA and OCFAs, LC–MS metabolomic analysis was employed and the results showed that the pentose phosphate pathway and the glycolysis pathway were strengthened and intracellular propionyl-CoA concentration were also significantly increased in the engineered Schizochytrium, suggesting an increased supply of NADPH, acetyl-CoA, and propionyl-CoA for DHA and OCFAs accumulation. Conclusions The discovery provides a new source of OCFAs production, and proposes a new strategy to improve contents and titers of both DHA and OCFAs in Schizochytrium. These will be valuable for improving commercial potential of Schizochytrium and guiding the engineering strategy in other fatty acids producing heterotrophic microalga. Electronic supplementary material The online version of this article (10.1186/s13068-019-1484-x) contains supplementary material, which is available to authorized users.

Additional file 8: Table S1. T. reesei strains used in this study.

Additional file 3: File S1. The sequence of codon-optimized ELO3 gene from Mortierella alpina.

Docosahexaenoic acid (DHA, C22: 6), which can be synthesized by the heterotrophic microalga Crypthecodinium cohnii, has received significant interest recently, due to its important roles in human health. However, the high production cost caused by low productivity and yield is still a major hurdle for industrial production of DHA using C. cohnii. The reduction of starch content has been found to be a promising mean to drive more carbon source to lipid biosynthesis in C. cohnii; however, it usually affects cell growth negatively, which limits its practical application. In this study, atmospheric and room temperature plasma mutagenesis associated with high-throughput screening based on iodine vapour was developed to identify a starch-deficient C. cohnii mutant 16D. The mutant 16D accumulated more biomass, lipid and DHA, with less starch and extracellular polysaccharide. The productivity and the yield of DHA were elevated 1.7 and 1.3 fold, reaching levels of 57.7 mg/L-h and 52.0 mg/g, respectively, in fed-batch fermentation, which are the highest reported so far for DHA production using C. cohnii. LC- and GC–MS based metabolomic analyses revealed multiple possible mechanisms relevant to increased growth and lipid content in mutant 16D, including strengthened Embden-Meyerhof, Krebs cycle and pentose phosphate pathways to provide more CoA, ATP and NADPH for supporting growth and lipid accumulation; and a significant increase in the intracellular abundance of tagatose, which was demonstrated to be involved in lipid content enhancement possibly by activating the triacylglycerol biosynthesis pathway in mutant 16D. The study presents a new strain improvement strategy for DHA-producing C. cohnii and new insights for switching metabolism from starch to growth and lipid in C. cohnii. In addition, the study also demonstrates the inductive effects of tagatose on lipogenesis in heterotrophic microalgae.

It is well-known that high-nitrogen content inhibits cell growth and docosahexaenoic acid (DHA) biosynthesis in heterotrophic microalgae Crypthecodinium cohnii. In this study, two nitrogen feeding strategies, pulse-feeding and continuous-feeding, were evaluated to alleviate high-nitrogen inhibition effects on C. cohnii. The results showed that continuous-feeding with a medium solution containing 50% ( w/v) yeast extract at 2.1 mL/h during 12-96 h was the optimal nitrogen feeding strategy for the fermentation process, when glucose concentration was maintained at 15-27 g/L during the same period. With the optimized strategy, 71.2 g/L of dry cell weight and DHA productivity of 57.1 mg/L/h were achieved. In addition, metabolomic analysis was applied to determine the metabolic changes during different nitrogen feeding conditions, and the changes in amino acids, polysaccharides, purines, and pentose phosphate pathway were observed, providing valuable metabolite-level information for exploring the mechanism of the high-nitrogen inhibition effect and further improving DHA productivity in C. cohnii.

Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression.

Metallic Pd and PdO electrodes were prepared by using Pd and PdCl2 slurries, respectively, and their electrochemical performance as a cathode for oxygen reduction reaction in intermediate temperature solid oxide fuel cells was evaluated by electrochemical impedance spectroscopy (EIS) and direct current polarization (DC polarization). The electrochemical activity of metallic Pd was much higher than that of PdO for the reaction of oxygen reduction; below the decomposition temperature, a thin layer of PdO formed on the surface of metallic Pd electrode, which increased its polarization resistance. The decomposition temperature of PdO decreased from 810 to 750 °C as oxygen partial pressure decreased from 20 to 5 kPa, and was further lowered under the influence of the applied current during DC polarization test. The charge transfer resistance of PdO increased by decreasing oxygen partial pressure, while that of metallic Pd was less sensitive to it.

The stability of La 0.6 Sr 0.4 Co 0.2 Fe 0.8 O 3− δ impregnated Y 2 O 3 stabilized ZrO 2 (LSCF–YSZ) cathodes was investigated under the condition of open circuit or current polarization at 750 °C in air. The electrochemical measurement and the microstructure characteristic show that the flattening of LSCF particles has great contribution to the increase of resistance of LSCF–YSZ cathodes after 500 h heat treatment at 750 °C. Microstructure coarsening and the damage of well-connected porous structure are main reasons of the performance degradation for LSCF–YSZ cathodes testing at 200 mA cm −2 and 750 °C in air. Higher current density of 500 mA cm −2 applying on cathodes accelerates degradation processes. X-ray photoelectron spectroscopy (XPS) shows that Sr concentration on the cathode surface decreases after current polarization, which plays a main role in performance activation processes observed at the beginning stage. The enhancement of cobalt activity in LSCF lattice by current polarization increases the conductivity and decreases the stability of LSCF–YSZ cathodes.

La 0.8 Sr 0.2 MnO 3 (LSM)-coated Ba 0.5 Sr 0.5 Co 0.8 Fe 0.2 O 3 (BSCF) composite powder (LSM-BSCF) was synthesized by a novel solid-solution method and investigated electrochemically as a cathode material for intermediate temperature solid oxide fuel cells. The cathode combined the merits of LSM and BSCF cathodes through an extended triple phase boundary and stabilized microstructure and demonstrated a polarization resistance between 0.61 and 0.09 Ω cm 2 at 600 to 750 °C. Compared with high performance cathodes prepared by solution impregnation, this LSM-BSCF cathode greatly improved performance stability.

The performance degradation of composite cathodes of La 0.6 Sr 0.4 Co 0.2 Fe 0.8 O 3− δ and Gd-doped ceria (LSCF–GDC), prepared by impregnating the porous GDC scaffold with a nitrate solution containing La, Sr, Co and Fe in desired composition, is investigated at 750 °C and open circuit in air for 500 h. The performance of the impregnated LSCF–GDC composite cathodes deteriorates after testing at 750 °C for 500 h; the electrode polarization resistance ( R p ) increases from 0.38 to 0.83 Ω cm 2 , and the electrode ohmic resistance ( R o ) increases from 1.79 to 2.14 Ω cm 2 . The grain growth and coarsening of impregnated LSCF nanoparticles are responsible for the performance degradation of the cathodes. XPS analysis shows the enrichment of cobalt on the surface of the infiltrated LSCF–GDC cathodes and such surface segregation could also contribute to the degradation of the electrocatalytic activity of the cathodes. Introducing MgO and LaNi 0.6 Fe 0.4 O 3 phases can effectively suppress the coarsening of LSCF nanoparticles and enhance the stability of the cathodes. However, the enhancing effect is related to the conductivity and electrocatalytic activity of the introduced phases.

Improvement of agitation is a commonly used approach for the optimization of fermentation processes. In this report, the response to improving agitation rate from 150 to 250 rpm on cellulase production from Penicillium decumbens JUA10-1 was investigated. It was shown that the production of all the major components of the cellulase mixture increased following improved agitation. Further investigations showed that at least three factors are involved in this improvement: the improved biomass accumulation, proportion of active/mature cellulases, and cellulase transcription level. The transcription levels of the cellulase repressing transcription factor ace1 and the cellulase activating transcription factor xlnR, however, both declined when a higher agitation was applied. These observations, along with our analysis of the carbon catabolite repressor CreA, lead to the suggestion that the molecular mechanism underlying improved cellulase transcription is the competition of two concurrent effects following improved agitation: CreA-mediated derepression induced by the downregulation of ace1, and CreA-mediated deactivation induced by the downregulation of xlnR.

LaCo0.6Ni0.4O3−δ (LCN64) was prepared through the polymeric steric entrapment precursor route with Polyvinyl alcohol (PVA) as the entrapment agent and was evaluated as a contact material between the metallic interconnect and the cathode in planar intermediate temperature solid oxide fuel cell stacks (IT-SOFC). The ratio of PVA to metal nitrates and the calcination temperature of the precursor were optimized for the process. The electrical conductivity and thermal expansion coefficient (TEC) of the synthesized LCN64 and its chemical compatibility with SUS 430 were also characterized. The results indicate that 1:4 is a proper ratio of PVA to metal nitrates for process control and safety management; and calcination of the precursor at temperatures above 650 °C leads to formation of single perovskite phase LCN64. The conductivity of fully sintered LCN64 is above 1150 S cm−1 in the temperature range between 100 °C and 800 °C, which is higher than those of conventional contact materials La1−xSrxMnO3 (LSM) and LaNiyFe1−yO3 (LNF). The average TEC is 17.22 × 10−6 K−1 at temperatures below 900 °C, which is higher than those of the metallic interconnect and cell components. Mn and Cr elements contained in SUS 430 migrated into the porous LCN64 layer at 800 °C without chemically forming resistive phases.

Transarterial chemoembolization (TACE) has mostly been used in hypervascular tumours such as hepatocellular carcinoma, and may be an effective palliative treatment in patients with metastatic liver cancer. Our goal is to determine whether TACE increases overall survival (OS) of in patients with liver metastases. The retrospective cohort study included 171 patients with liver metastases diagnosed between 2001 and 2015. OS was compared between the TACE and non-TACE groups after propensity score matching to reduce the effects of selection bias and potential confounders. Multivariate analysis was conducted to confirm the confounding factors with OS. After excluding 43 patients, 128 patients were analysed and among thses 64 patients (50%) were included in the TACE group. In the propensity score matched cohort (42 pairs), the OS was non-significantly longer in the TACE group than in the non-TACE group (p = 0.789). Multivariate analysis revealed that international normalized ratio (INR) (HR 0.058, 95%CI: [0.005, 0.681]; p = 0.023) and Radiofrequency ablation (RFA) (HR 3.054, 95%CI: [1.418, 6.579]; p = 0.004) were independent risk factors for OS in patients with unresectable liver metastases. There were no significant differences in patients with unresectable liver metastases with or without TACE. INR and RFA can significantly affect OS in patients with unresectable liver metastases.

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4–12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.

Additional file 1. The change of glucan conversions of high-gravity SPRs when commercial pectinase solutions were added. All the reactions were performed at 45 °C and 200 rpm for 6 h with an initial pH of 4.8. The concentration of SPRs in reaction system was 36 % (w/v). Three independent replicates were carried out.

The contact resistance and chemical compatibility of LaCo 0.6 Ni 0.4 O 3−δ (LCN) coated Ni–Mo–Cr alloy are investigated at 750 °C in air for more than 530 h to simulate the contact situation of cathode/contact layer/interconnect in SOFC stacks. With La 0.72 Sr 0.18 MnO 3 (LSM) as the cathode, the area specific resistance (ASR) of LSM/LCN/Ni–Mo–Cr alloy assembly increases to a maximum of 240 mΩ cm 2 during the early stage of the test, and decreases after 55 h to a steady level of ∼220 mΩ cm 2 till the end of the test. The contribution of formed oxide scale on the alloy to the measured ASR is negligible, compared to that of big AgLaMo 2 O 8 particles sporadically distributed in LCN matrix. AgLaMo 2 O 8 is formed of evaporated Mo from the alloy, Ag from the testing lead and La from the LCN before the oxide scale on the alloy is well developed. This reaction is expected to cease once the oxide scale is fully established.

Background China is the largest sweet potato producer and exporter in the world. Sweet potato residues (SPRs) separated after extracting starch account for more than 10 % of the total dry matter of sweet potatoes. In China, more than 2 million tons of SPRs cannot be utilized, and the unutilized SPRs are perishable and result in environmental pollution. Thus, an environmentally friendly and highly efficient process for bioethanol production from SPRs should be developed. Results The swelling behaviour of cellulose causes high-gravity sweet potato residues to be recalcitrant to enzymatic hydrolysis. Cellulase plays a major role in viscosity reduction and glucose production. In contrast, pectinase has a minor role in viscosity reduction but acts as a “helper protein” to assist cellulase in liberating glucose, especially at low cellulase activity levels. In total, 153.46 and 168.13 g/L glucose were produced from high-gravity SPRs with cellulase and a mixture of cellulase and pectinase, respectively. These hydrolysates were fermented to form 73.37 and 79.00 g/L ethanol, respectively. Each kilogram of dry SPR was converted to form 209.62 and 225.71 g of ethanol, respectively. Conclusion The processes described in this study have an enormous potential for industrial production of bioethanol because they are environmentally friendly, highly productive, economic with low cost, and can be easily manipulated. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0464-7) contains supplementary material, which is available to authorized users.

Solution impregnated La 0.8 Sr 0.2 Co 0.8 Ni 0.2 O 3 + Gd-doped CeO 2 (LSCN + GDC) cathodes for intermediate temperature solid oxide fuel cells (IT-SOFC) are prepared and their electrochemical properties are evaluated and compared with the conventional LSCN cathodes. The results indicate that the cathode performance can be enhanced by the presence of the nanosized microstructure produced with the solution impregnation method. It is determined that the amount of LSCN loading in the LSCN + GDC composite cathode needs to be higher than 35 wt% in order to achieve a performance superior to that of the conventional LSCN cathode. The optimum amount of LSCN loading is in the range of 45–55 wt% with an activation energy near 1.32 eV for oxygen reduction. At temperatures between 600 and 750 °C, the polarization resistance of the 55 wt% LSCN loaded LSCN + GDC cathode is in the range of 1.07 and 0.08 Ω cm 2 , which is only about one half of that for the conventional cathode.

The lignocellulose degradation capacity of filamentous fungi has been widely studied because of their cellulase hypersecretion. The p24 proteins in eukaryotes serve important functions in this secretory pathway. However, little is known about the functions of the p24 proteins in filamentous fungi. In this study, four p24 proteins were identified in Penicillium oxalicum. Six p24 double-deletion strains were constructed and further studies were carried out with the ΔerpΔpδ strain. The experimental results suggested that Erp and Pδ form a p24 heterodimer in vivo. This p24 heterodimer participates in important morphogenetic events, including sporulation, hyphal growth and lateral branching. The results suggested that the p24 heterodimer mediates protein transport, particularly that of cellobiohydrolase. Analysis of the intracellular proteome revealed that the ΔerpΔpδ double mutant is under secretion stress due to attempts to remove proteins that are jammed in the endomembrane system. These results suggest that the p24 heterodimer participates in morphogenesis and protein transport. Compared with P. oxalicum Δerp, a greater number of cellular physiological pathways were impaired in ΔerpΔpδ. This finding may provide new insights into the secretory pathways of filamentous fungi.

Despite the important role of MAPKs in signal transduction, their functions in the cellulase hyper-producing filamentous fungus Hypocrea jecorina haven't been studied except for the Hog1-like Tmk3. In this work, we constructed and explored the features of H. jecorina Δtmk2 to identify the role of this Slt2-homologous Tmk2. It is suggested from the results that Tmk2 is involved in cell wall integrity, sporulation and cellulase production. Although bearing similar roles in cell wall integrity maintenance, Tmk2 and Tmk3 appear to also have distinct functions: Tmk3 participates in high osmolarity resistance while Tmk2 does not; Tmk2 participates in sporulation but not Tmk3; Tmk3 is involved in promoting cellulase production while Tmk2 is involved in repressing cellulase formation. These studies provide the first insight into the function of Tmk2 in H. jecorina and contribute to understanding the signal transduction processes leading to the regulation of cellulase production in this important cellulase hyper-producer.

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading.

The protein hyper-secreting filamentous fungi impact their surrounding environments by secreting cellulases and digesting plant cell wall via microbe-plant interspecies interaction. This process is of paramount importance in biofuel production from the renewable lignocellulosic biomass, because cellulase production is the key factor in cost determination. Despite the importance of protein secretion, p24 protein, a key factor in eukaryotic protein maturation and secretion, was never investigated in filamentous fungi. The erp genes encoding p24γ homologues were identified in Trichoderma reesei and Penicillium decumbens. The roles of Erp and their participated cellular pathways were investigated via disruption of erp, revealing significant differences: sporulation was hampered in T. reesei Δerp but not in P. decumbens Δerp; in both species Erp maintains membrane integrity; Erp is likely involved in hyphae polarity maintenance in T. reesei. Protein- and transcription-level investigations of Erp participation in cellulase production revealed distinct regulatory mechanisms. In T. reesei, cellulase encoding genes were repressed under secretion stress. In contrast, activation of the same genes under the same stress was identified in P. decumbens. These observations revealed a novel cellulase gene regulation mechanism, clearly suggested the different physiological roles of Erp, and further demonstrated the different physiology of T. reesei and P. decumbens, despite above 75% sequence identity between the proteins and the close evolutionary relationship between the two species.