Low Culture Temperature Research Articles

Temperature Down-Shift Modifies Expression of UPR-/ERAD-Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells.

Low culture temperature enhances the cell-specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r-) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)-resident proteins, appear to be involved in the improvements of r-protein production under low temperature conditions. The transcriptional regulation of UPR-specific targets is studied in response to decreased culture temperature in relation to production of a difficult-to-express protein. A clonally-derived CHO cell line expressing a chimeric fusion protein (human erythropoietin [hEPO] linked to a murine Fc region, hEPO-Fc) is evaluated in terms of growth, metabolism, r-protein production and UPR-/ER associated degradation (ERAD)-specific gene expression at standard (37°C) and low (32°C) temperature in batch and fed-batch systems. Low temperature decreased peak cell density, improved viability, generated cell cycle arrest in the G1 phase and enhanced hEPO-Fc expression in both batch and fed-batch cultures. A low culture temperature significantly upregulated genes encoding UPR-specific transcriptional activators (xbp1s, ddit3, and atf5) and ER-resident proteins (grp78, grp94, trib3, and ero1α), that are associated with folding and processing of proteins within the ER. Further, low culture temperature decreased expression of genes involved in ERAD (edem3, sels, herpud1, and syvn1) indicating a decreased potential for protein degradation.

The role of temperature, pH and nutrition in process development of the unique oleaginous yeast Metschnikowia pulcherrima

AbstractBACKGROUNDLipids produced from oleaginous yeasts are a promising alternative to terrestrial oils. Despite promising cellular yields of lipid however, an industrial process remains elusive. One key processing bottleneck is the need to provide nutrient‐rich conditions for cellular growth and then extended nutrient‐depleted conditions for lipid accumulation. Surprisingly, investigations detailing process development, particularly with a focus on kinetics, are rare in this field.RESULTSIn this investigation we report on the unique oleaginous yeast Metschnikowia pulcherrima, where lipid accumulation ≥ 29.8% (w/w) was achieved without apparent nutrient limitation. The process was developed in stirred tank reactors through determining the influence of temperature, pH and nutrition on lipid production. A temperature of up to 25 °C and initial pH 5 could be applied to enhance initial reaction kinetics. Through the increased supply of yeast extract of up to 5% (w/w) of glucose, a maximum lipid production rate of 0.60 g L−1 h−1 (4 h‐average), productivity of 0.29 g L−1 h−1, and yield of 0.17 g g−1 glucose were achieved – the highest yet recorded with this yeast. Suitable to combat the excessive secretion of polyols of up to 0.11 g g−1 glucose was an excess nutrient supply as well as a low cultivation temperature of 15 °C and moderate pH 5.CONCLUSIONSThis study demonstrates that the lack of a starvation stage, coupled with effective process development is required for oleaginous yeasts to achieve the yields and productivities required for commercial lipid production. © 2019 Society of Chemical Industry

Reduced culture temperature attenuates oxidative stress and inflammatory response facilitating expansion and differentiation of adipose-derived stem cells

BackgroundAdipose-derived stem cell (ASC) expansion under atmospheric oxygen levels (21%) was previously shown to cause increased reactive oxygen species (ROS) accumulation and genetic instability compared to cells cultured under physiological oxygen levels (2–8%). However, since culture under physiological oxygen levels is costly and complicated, a simpler method to reduce ROS accumulation is desirable. The current study aimed to determine whether lower culture temperature can reduce ROS production in ASCs without impairing their culture expansion.MethodsProliferation, differentiation, ROS accumulation, and gene expression were compared between ASC cultures at 35 °C and 37 °C. ASCs isolated either from rat fat depots or from human lipoaspirates were examined in the study.ResultsRat visceral ASCs (vASCs) cultured at 35 °C demonstrated reduced ROS production and apoptosis and enhanced expansion and adipogenic differentiation compared to vASCs cultured at 37 °C. Similarly, the culture of human ASCs (hASCs) at 35 °C led to reduced ROS accumulation and apoptosis, with no effect on the proliferation rate, compared to hASCs cultured at 37 °C. Comparison of gene expression profiles of 35 °C versus 37 °C vASCs uncovered the development of a pro-inflammatory phenotype in 37 °C vASCs in correlation with culture temperature and ROS overproduction. This correlation was reaffirmed in both hASCs and subcutaneous rat ASCs.ConclusionsThis is the first evidence of the effect of culture temperature on ASC growth and differentiation properties. Reduced temperatures may result in superior ASC cultures with enhanced expansion capacities in vitro and effectiveness in vivo.

Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells.

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.

Quality Assessment of Aflatoxins Contamination in Red Chillies

In this work, have been presented results of bio productivity study and quantity of formed MDA in Dunaliella cells at high salinity in optimal and low-temperature cultivation conditions. It became clear that, in conditions of low temperature stress, bioproductivity of cells decreases 25% in regard to optimal cultivating regime. The cell modification by 2,6 di-tret-butyl cresol (ionol) leads to growth stimulation of Dunaliella culture in optimal regime: (25-350mkM) 2-7% respectively, and in conditions of low temperature stress in range of concentrations (25-350mkM) 5-8%. It was established that, the concentrations 25-50mkm 2,6 di-tret-butyl cresol in mineral medium cause 30-70% increase of MDA content in cells and then 35-60% decrease of its content at concentrations 15-300mkm in optimal regime. In low temperature regime concentrations 25-50mkm of BHT, MDA quantity remains in control, but at concentrations 15-300mkm decreases 4-20%.

Lipid Peroxidation in Dunaliella Salina IPPAS-294 Cells Modified by Ionol at High Salinity in Optimal and Low-Temperature Cultivation Conditions

Lipid Peroxidation in Dunaliella Salina IPPAS-294 Cells Modified by Ionol at High Salinity in Optimal and Low-Temperature Cultivation Conditions

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

There are several factors influencing the protein expression. The study was aimed to increase the yield of protein recombinant JSU-pET by applying combination of temperature and medium culture composition. This research was composed in a 2x2 factorial design with two level temperatures of 37<sup>o</sup>C and 25<sup>o</sup>C and 2 medium compositions of LB and mLB (modified). <em>Escherichia coli</em> bearing JSU-pET plasmid were grown in two applied media and incubated in two applied temperatures. The cells were harvested by centrifugation the pellets were then lysed. The lysed cells were centrifuged, the supernatant then purified by Ni-NTA Resin Agarose. The purified JSU protein recombinant was characterized and quantified by spectrophotometer. The Result showed that the JSU protein recombinant was successfully expressed on the right size (37kDa) in all treatments even though the JSU-pET showed sharper band in m LB at 37<sup>o</sup>C. Statistically the protein yield was not significant different (P>0.05), however culture temperature of 25<sup>o</sup>C showed higher yield (0.618±0.095 vs 0.704±0.094) than cultured at 37<sup>o</sup>C (0.598±0.137 vs 0.553±0.041), respectively either cultured in LB or mLB. This finding suggests that a lower culture temperature could increase protein yield both either in LB or modified LB.

Enzymatic preparation and identification of 5′-adenosyl-methylthiopropylamine for the impurity control in SAM fermentation

Abstract S-adenosyl-L-methionine (SAM) is an important biosynthetic drug used for various diseases such as depression, arthritis syndromes and cirrhosis in clinical therapeutics. In order to ensure the high quality of SAM as a clinical drug, the key impurity, 5′-adenosyl-methylthiopropylamine (decarboxylated SAM, dC-SAM) should be monitored during the whole production process and in end tablet product. In the present work, the SPE2 gene encoding SAM decarboxylase was inserted into pET28a to obtain the expression plasmid pET28a-SPE2, which was transferred into Escherichia coli BL21 to construct the recombinant strain. With low temperature (26 °C) cultivation and 0.1 mM IPTG induction, the recombinant cells were collected to prepare cell lysates, which can be utilized to effectively prepare dC-SAM in vitro with the addition of SAM as the substrate. Then dC-SAM sample with high-purity (99.7%) was isolated from this biotransformation mixture by semi-preparative high performance liquid chromatography. Further, the molecular structure of the purified dC-SAM sample was confirmed by liquid chromatography-mass spectrometry. Finally, prepared dC-SAM was employed to detect the content of the impurity during one-month industrial SAM fermentation processes. Our work will be very helpful to the control of impurities during the whole production process of SAM and its end products.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody.

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

The Adhesiveness of the OmpF and OmpC Porins from Yersinia pseudotuberculosis to J774 Macrophages

Abstract—The significance of the Yersinia pseudotuberculosis porins OmpF and OmpC for adhesion to macrophages J774 was assessed using optical trapping. The passive sensitization of polystyrene microspheres with the preparations of these porins was verified. Using a set of differently functionalized microspheres, it was shown that OmpF produced at a lower culture temperature contributes to the adhesiveness of the bacteria Y. pseudotuberculosis to macrophages, while OmpC synthesized at a temperature of 37°C does not have this property. These results can be explained by thermoinducible differences in the primary structure and conformational features of the outer loops of porin molecules. These differences are suggested to be necessary for effective circulation in the environment and manifestation of the invasive properties of Y. pseudotuberculosis as an agent of saprozoonotic infection.

Sphingolipidomics of Thermotolerant Yeasts.

Mass spectrometry-based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes-phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)2 Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long-chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)2 Cer decreased. Molecular species having C18-LCB were associated with low cultivation temperature, and a higher proportion of C20-LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long-chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2-hydrocylo-lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.

Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets

Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20–33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.

The role of temperature and bivalent ions in preparing competent Escherichia coli.

Several factors including the culture temperature, bivalent ion, and osmotic stress were gradually optimized for preparing efficient Escherichia coli competent cells. The effect of culture temperature on the transformation efficiency (TrE) of E. coli DH5α was tested with 100mM CaCl2. The lower culture temperature at 18°C resulted in higher TrE of 2.5 × 106 cfu/μg, which was about 3.5 times of that obtained at 37°C. Bivalent ions including Ca2+, Mn2+, Mg2+, and Ni2+ were tested independently or combinatorially at a total concentration of 100mM. Ni2+ showed a significantly inhibition on the TrE, and various concentration combinations of Ca2+, Mg2+, and Mn2+ were tested. The TrE was improved up to 1.8 ± 0.4 × 108 cfu/μg, when a combination of 25mM Ca2+, 50mM Mg2+, and 25mM Mn2+ was applied. Further supplement of 0.8% (w/v) PEG6000 lead to a slight decrease in the TrE, whereas supplement of 25mM sucrose contributed to another increase in the TrE by 17% up to 2.1 ± 0.3 × 108 cfu/μg. These results indicated that the culture temperature and bivalent ion were important factors affecting the TrE of E. coli. A chemical method for preparing efficient competent cells of E. coli was provided.

All-trans retinoic acid in combination with sodium butyrate enhances specific monoclonal antibody productivity in recombinant CHO cell line.

The effects of all-trans retinoic acid (RA) and sodium butyrate (NaBu) on growth, viability and antibody production of two types of transfected Chinese hamster ovary cell lines (CHO-K1 and CHO-S) were investigated using a batch mode cell culture. By adding 0.5mM NaBu in the CHO-K1 cell culture, the cell specific productivity (Qp) and antibody concentration increased by five- and threefold, respectively. The optimal concentration of RA was 100nM which resulted in twofold increase in antibody production. In a combination model, RA applied at early growth phase of CHO-K1 cells followed by addition of NaBu with lowering culture temperature at the end of stationary phase resulted in two- and threefold increase in Qp and final antibody concentration, respectively. The latter strategy was also applied on suspended CHO-S cells with enhanced Qp and antibody concentration, but to a lesser extent than the CHO-K1 cells. In conclusion, our results demonstrate that the addition of RA and NaBu along with lowering the culture temperature can increase cell culture period as well as Qp and the final concentration of recombinant monoclonal antibody in both CHO-K1 and CHO-S cells without any significant change in binding affinity of the mAb.

High-throughput screening of Mucoromycota fungi for production of low- and high-value lipids

BackgroundMucoromycota fungi are important producers of low- and high-value lipids. Mortierella alpina is used for arachidonic acid production at industrial scale. In addition, oleaginous Mucoromycota fungi are promising candidates for biodiesel production. A critical step in the development of such biotechnological applications is the selection of suitable strains for lipid production. The aim of the present study was to use the Duetz-microtiter plate system combined with Fourier transform infrared (FTIR) spectroscopy for high-throughput screening of the potential of 100 Mucoromycota strains to produce low- and high-value lipids.ResultsWith this reproducible, high-throughput method, we found several promising strains for high-value omega-6 polyunsaturated fatty acid (PUFA) and biodiesel production purposes. Gamma-linolenic acid content was the highest in Mucor fragilis UBOCC-A-109196 (24.5% of total fatty acids), and Cunninghamella echinulata VKM F-470 (24.0%). For the first time, we observed concomitant gamma-linolenic acid and alpha-linolenic acid (up to 13.0%) production in psychrophilic Mucor flavus strains. Arachidonic acid was present the highest amount in M. alpina ATCC 32222 (41.1% of total fatty acids). Low cultivation temperature (15 °C) activated the temperature sensitive ∆17 desaturase enzyme in Mortierella spp., resulting in eicosapentaenoic acid production with up to 11.0% of total fatty acids in M. humilis VKM F-1494. Cunninghamella blakesleeana CCM-705, Umbelopsis vinacea CCM F-539 and UBOCC-A-101347 showed very good growth (23–26 g/L) and lipid production (7.0–8.3 g/L) with high palmitic and oleic acid, and low PUFA content, which makes them attractive candidates for biodiesel production. Absidia glauca CCM 451 had the highest total lipid content (47.2% of biomass) of all tested strains. We also demonstrated the potential of FTIR spectroscopy for high-throughput screening of total lipid content of oleaginous fungi.ConclusionsThe use of Duetz-microtiter plate system combined with FTIR spectroscopy and multivariate analysis, is a feasible approach for high-throughput screening of lipid production in Mucoromycota fungi. Several promising strains have been identified by this method for the production of high-value PUFA and biodiesel.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BackgroundThe increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.ObjectivesHere, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and MethodsThe encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.ResultsIn fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.ConclusionsOur founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

Effect of Low Temperature Cultivation on the Phytochemical Profile and Bioactivity of Arctic Plants: A Case of Dracocephalum palmatum.

The influence of climatic factors, e.g., low temperature, on the phytochemical composition and bioactivity of the arctic plant Dracocephalum palmatum Steph. ax Willd. (palmate dragonhead), a traditional food and medical herb of Northern Siberia, was investigated. D. palmatum seedlings were grown in a greenhouse experiment at normal (20 °C, NT) and low (1 °C, LT) temperature levels and five groups of components that were lipophilic and hydrophilic in nature were characterized. The analyses indicated that D. palmatum under NT demonstrates high content of photosynthetic pigments, specific fatty acid (FA) profile with domination of saturated FA (53.3%) and the essential oil with trans-pinocamphone as a main component (37.9%). Phenolic compounds were identified using a combination of high performance liquid chromatography with diode array detection and electrospray ionization mass-spectrometric detection (HPLC-DAD-ESI-MS) techniques, as well as free carbohydrates and water soluble polysaccharides. For the first time, it was established that the cold acclimation of D. palmatum seedlings resulted in various changes in physiological and biochemical parameters such as membrane permeability, photosynthetic potential, membrane fluidity, leaf surface secretory function, reactive oxygen species–antioxidant balance, osmoregulator content and cell wall polymers. In brief, results showed that the adaptive strategy of D. palmatum under LT was realized on the accumulation of membrane or surface components with more fluid properties (unsaturated FA and essential oils), antioxidants (phenolic compounds and enzymes), osmoprotectants (free sugars) and cell wall components (polysaccharides). In addition, the occurrence of unusual flavonoids including two new isomeric malonyl esters of eriodictyol-7-O-glucoside was found in LT samples. Data thus obtained allow improving our understanding of ecophysiological mechanisms of cold adaptation of arctic plants.

Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent-A comparative study.

Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69-80, 2018.

Influence of initial anolyte pH and temperature on hydrogen production through simultaneous saccharification and fermentation of lignocellulose in microbial electrolysis cell

An investigation on the performance of hydrogen production by simultaneous saccharification and fermentation (SSF) in a dual-chamber microbial electrolysis cell (MEC) was carried out to consider different anolyte pH levels and culture temperatures, and the influences of anolyte pH value and culture temperature on changes of current, organic acid and pH value were also evaluated. The maximal hydrogen production rate (HPR) of 2.46 mmol/L/D (hydrogen energy recovery 219.02%) was obtained at the initial anolyte pH of 6.5. Within the range of the tested operation temperatures (30–50 °C), the optimal temperature for hydrogen production by SSF in the MEC systems was 35 °C. Moreover, the contents of organic acids and reducing sugar significantly changed with varying in initial anolyte pH and temperature levels. The result indicates that a low initial anolyte pH value and high culture temperature was beneficial to hydrolysis of cellulose, and a high initial anolyte pH value and a moderate culture temperature to hydrogen production.

Exploration of a mechanism for the production of highly unsaturated fatty acids in Scenedesmus sp. at low temperature grown on oil crop residue based medium

The ability of algae to produce lipids comprising of unsaturated fatty acids varies with strains and culture conditions. This study investigates the effect of temperature on the production of unsaturated fatty acids in Scenedesmus sp. grown on oil crop residue based medium. At low temperature (10°C), synthesis of lipids compromising of high contents of unsaturated fatty acids took place primarily in the early stage while protein accumulation mainly occurred in the late stage. This stepwise lipid-protein synthesis process was found to be associated with the contents of acetyl-CoA and α-KG in the algal cells. A mechanism was proposed and tested through simulation experiments which quantified the carbon flux allocation in algal cells at different cultivation stages. It is concluded that low culture temperature such as 10°C is suitable for the production of lipids comprising of unsaturated fatty acids.