Mechanisms of weak acid-induced stress tolerance in yeasts: Prospects for improved bioethanol production from lignocellulosic biomass

Weak acids are known to have a negative impact on yeast performance, restraining production efficiency during the production of bioethanol and other fermentative yeast-derived products. These acids, which might be hydrophilic or lipophilic exert negative effects on yeasts when they diffuse into the cell in their unionized state as a result of their pH being lower than the pka of yeast growth medium. Consequently, the unionized acids dissociate into their respective cations and anions, as intracellular pH is typically neutral. Further, proton accumulation tends to reduce intracellular pH. As a result, the anions destabilize the internal cell machinery, thus affecting cellular metabolism on various levels. Overcoming this acid-mediated stress in budding yeast would in part, harness the potential of using lignocellulosic biomass hydrolysate – which is typically acetic acid-rich – as a cheaper feedstock for large-scale bioethanol production. Since organic acids are key intermediates in ethanol fermentation, this review focuses on the prospects of bioethanol production from lignocellulosic biomass using weak acid-tolerant strains of yeasts derived by metabolic engineering.     

Production of bioethanol from four species of duckweeds (Landoltia punctata,Lemna aequinoctialis,Spirodela polyrrhiza,and Wolffia arrhiza) through optimization of saccharification process and fermentation with Saccharomyces cerevisiae

Duckweeds are promising potential sources for bioethanol production due to their high starch content and fast growth rate. We assessed the potential for four species, Landoltia punctata, Lemna aequinoctialis, Spirodela polyrrhiza, and Wolffia arrhiza, for bioethanol production. We also optimized a possible production procedure, which must include saccharification to convert starch to soluble sugars that can serve as a substrate for fermentation. Duckweeds were cultivated on 10% Hoagland solution for 12 days, harvested, dried, homogenized, and dissolved in solutions that were tested as substrates for bioethanol production by the yeast Saccharomyces cerevisiae. First, we optimized the saccharification process, including the ideal ratio of the enzyme used to convert starch into simple sugars. The greatest starch-to-sugar conversion was obtained when the α-amylase and amyloglucosidase was 2:1 (v/v) and with a 24 h incubation period at 50 °C. After saccharification, the solutions were incubated with the yeast, S. cerevisiae. The fermentation process was carried out for 48 h with 10% (v/v) yeast inoculum. The ethanol content was maximal approximately 24 h after the start of incubation, and the sugars and protein were minimal, with little change over the next 24 h. The final ethanol concentration obtained were 0.19, 0.17, 0.19, and 0.16 g ethanol/g dry biomass for L. punctata, L. aequinoctialis, S. polyrrhiza, and W. arrhiza respectively. We suggest that these four species of duckweed have the potential to serve sources of bioethanol and hope that the procedure we have optimized proves useful in the endeavour.     

一株高产淀粉绿藻——标志链带藻在废水中的培养及对氮磷的去除

为了研究标志链带藻(Desmodesmus insignis strain JNU24)在不同Na NO3浓度和不同废水浓度培养下的生长与代谢产物积累状况,评估标志链带藻对废水的处理能力,本研究利用柱状光反应器对标志链带藻进行培养,分别对4种Na NO3浓度的BG-11培养基和4种废水浓度培养下藻细胞的生物量、蛋白质含量、碳水化合物含量和淀粉含量进行了测定。结果显示,在BG-11培养基中,氮浓度为9.0 mmol/L时,藻细胞生物质浓度最高,达6.23 g/L;在废水培养下,未稀释的原废水实验组,其藻细胞生物质浓度最高,达10.31 g/L;75%废水培养下,藻细胞的淀粉含量最高(达50.9%),单位体积藻细胞淀粉含量和产率分别为4.86 g/L和405 mg·L~(-1)·d~(-1),且显著高于不同浓度Na NO3的BG-11培养基组。本研究还测定了标志链带藻对废水中氮、磷的去除效率,结果显示不同浓度废水培养下,氮、磷的去除效率最高可达90.8%和98.7%。基于柱状反应器中的最佳培养效果,以9.0 mmol/L Na NO3的BG-11培养基和75%废水于3.0 cm光径平板光生物反应器中进行室内扩大培养,结果显示在75%废水培养下,藻细胞生物质浓度达9.75 g/L,淀粉单位体积含量和产率分别达到4.75 g/L和230 mg·L~(-1)·d~(-1),且为9.0 mmol/L Na NO3的BG-11培养基培养的3倍。通过沉降特性分析发现,藻细胞收获90 min后均完全沉降,具有较强的沉降性能。本研究标志链带藻能够耐受废水中较高的氮、磷浓度,且对废水中氮、磷有显著的去除作用;该藻能利用废水中的营养成分积累较高的生物量和淀粉含量并且藻细胞能快速沉降,具有极高的经济价值和应用价值,是一株淀粉生产能力较高和废水处理能力较强的极具开发潜力的藻株。     

Purification and characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.     

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co²⁺. Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co²⁺ stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Improving the remaining activity of lignocellulolytic enzymes by membrane entrapment

The production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years because of its low cost and great potential availability. However, the high cost of the enzyme required for this conversion is often considered to be the major bottleneck in the commercial lignocellulosic ethanol industry. In this work, the hydrolysis of rice straw by free and entrapped lignocellulolytic enzymes (cellulase, xylanase and laccase) was carried out at pH 5.5 and 37 °C. The hydrolysis of rice straw by enzymes entrapped in a membrane produced a higher monosaccharide content: 601.05 mg/g rice straw for entrapped enzymes vs. 465.46 mg/g rice straw for free enzymes. This study has shown that enzyme entrapment is an important technique for the efficient use and reuse of enzymes in industrial applications and also for the rapid separation of saccharide products from the reaction medium, thus improving the remaining enzymatic activities.     

The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal-analytical and complementary studies

Differential Scanning Calorimetry, Dynamic Mechanical Thermal Analysis, gravimetric and chemical techniques have been used to study hydrothermal reactions of straw biomass. Exothermic degradation initiates above 195 °C, due to breakdown of the xylose ring from hemicellulose, which may be similar to reactions occurring during the early stage pyrolysis of dry biomass, though activated at lower temperature through water mediation. The temperature and magnitude of the exotherm reduce with increasing acid concentration, suggesting a reduction in activation energy and a change in the balance of reaction pathways. The presence of xylan oligomers in auto-catalytic hydrolysates is believed to be due to a low rate constant rather than a specific reaction mechanism. The loss of the lignin glass transition indicates that the lignin phase is reorganised under high temperature auto-catalytic conditions, but remains partially intact under lower temperature acid-catalytic conditions. This shows that lignin degradation reactions are activated thermally but are not effectively catalysed by aqueous acid.     

The influence of ferrous sulfate utilization on the sugar yields from dilute-acid pretreatment of softwood for bioethanol production

By employing metal salts in dilute-acid pretreatment the severity can be reduced due to reduced activation energy. This study reports on a dilute-acid steam pretreatment of spruce chips by addition of a small amount of ferrous sulfate to the acid catalyst, i.e., either SO₂, H₂SO₃ or H₂SO₄. The utilization of ferrous sulfate resulted in a slightly increased overall glucose yield (from 74% to 78% of the theoretical value) in pretreatment with SO₂ and H₂SO₃. Impregnation with ferrous sulfate and sulfuric acid did not give any improvement compared with pretreatment based solely on H₂SO₄.