Prospects for the use of new basidiomycete strains for the direct conversion of lignocellulose into ethanol

Sixty-six isolates of basidiomycete fungi were screened for the ability to synthesize cellulase. The effect of temperature on cellulase activity was studied for eight basidiomycete strains as perspective producers of ethanol. The temperature optima of enzyme activity ranged between 26 and 32°C. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and rye straw were studied for seven basidiomycetes strains: Fomitopsis pinicola MT-5.09, F. pinicola MT-5.21, Piptoporus betulinus MT-30.04, Fomes fomentarius MT-4.05, F. fomentarius MT-4.23, Trametes hirsuta MT-24.24, Flammulina velutipes MT-3.03 Maximum ethanol production from Na-carboxymethyl cellulose (1.3 g/dm³) was achieved by strain F. velutipes MT-3.03. Strain F. fomentarius MT-4.05 more effectively converted rye straw to ethanol with yield of 1.1 g/dm³.     

The key role of lignin decomposing fungi in the decay of roofs thatched with water reed

We investigated the involvement of microorganisms in the rapid reed decay of roofs thatched with water reed. Numerous bacteria and fungi were isolated by enrichment cultures from reed samples and from fungal fruit bodies on roofs. All strains were characterised in respect to their abilities to degrade cellulose, hemicelluloses and the lignin model substance Poly-R-478. Only 15 of the 92 isolated bacterial strains were capable of degrading cellulose and hemicelluloses. However, nearly all 61 of the identified fungal isolates had these abilities. Nevertheless, only 14 of the isolated fungal strains as well as a reference isolate of Trametes versicolor were capable of degrading Poly-R-478. The ability of the microorganisms to attack complete reed was assessed using a newly developed test system which measures the loss of dry weight during the incubation. A significant loss of dry weight was apparent only in tests using the ligninolytic fungi Pycnoporus cinnabarinus, Trametes versicolor, Phlebia tremellosa and some Mycena species, but not in the case of the majority of cellulolytic bacteria and fungi. From these results, we conclude that ligninolytic fungi are capable of destroying complete reed structure and that they play the key role in the process of the rapid decay of roofs thatched with reed. Directly after the initial lignin attack, cellulose and hemicellulose were degraded to a great extent, evidenced by the large loss of dry weight (up to 72 %), which significantly exceeds the lignin content of reed (ca. 15 %). However, after the initial attack by ligninolytic fungi, bacteria or other fungi capable of degrading cellulose and hemicelluloses may contribute to the progressive decay of reed under natural conditions. Furthermore, we show that the rate of decay depends on the source of the reed and on the reed quality.     

A novel ethanol-tolerant laccase,Tvlac, from <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Objectives

To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source.

Results

Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml^?1 within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 °C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V ₀.

Conclusions

The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.

     

<Emphasis Type="Italic">Cachaça</Emphasis> yeast strains: alternative starters to produce beer and bioethanol

This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H₂S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.     

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

Background

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

Results

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

Conclusions

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

     

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

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

Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Bioethanol, as a form of renewable and clean energy, has become increasingly important to the energy supply. One major obstacle in ethanol production is developing a high-capacity system. Existing approaches for regulating the ethanol production pathway are relatively insufficient, with nonspecific genetic manipulation. Here, we used CRISPR/Cas9 technology to disrupt the alcohol dehydrogenase (ADH) 2 gene via complete deletion of the gene and introduction of a frameshift mutation in the ADH2 locus. Sequencing demonstrated the accurate knockout of the target gene with 91.4% and near 100% targeting efficiency. We also utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single-guide RNAs. This extensive analysis indicated the mutations in the CRISPR/Cas9-engineered strains were homozygous. We applied the engineered Saccharomyces cerevisiae strains for bioethanol production. Results showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain. This work illustrates the applicability of this highly efficient and specific genome engineering approach to promote the improvement of bioethanol production in S. cerevisiae via metabolic engineering. Importantly, this study is the first report of the disruption of a target gene, ADH2, in S. cerevisiae using CRISPR/Cas9 technology to improve bioethanol yield.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.     

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach

Production of bioethanol from brewers spent grains (BSG) using consolidated bioprocessing (CBP) is reported. Each CBP system consists of a primary filamentous fungal species, which secretes the enzymes required to deconstruct biomass, paired with a secondary yeast species to ferment liberated sugars to ethanol. Interestingly, although several pairings of fungi were investigated, the sake fermentation system (A. oryzae and S. cerevisiae NCYC479) was found to yield the highest concentrations of ethanol (37 g/L of ethanol within 10 days). On this basis, 1 t of BSG (dry weight) would yield 94 kg of ethanol using 36 hL of water in the process. QRT-PCR analysis of selected carbohydrate degrading (CAZy) genes expressed by A. oryzae in the BSG sake system showed that hemicellulose was deconstructed first, followed by cellulose. One drawback of the CBP approach is lower ethanol productivity rates; however, it requires low energy and water inputs, and hence is worthy of further investigation and optimisation.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

Bioethanol production from steam-exploded rice husk by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> as a biocatalyst for biofuel production

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.     

Metabolic engineering for high glycerol production by the anaerobic cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.