Use of <Emphasis Type="Italic">Cupriavidus basilensis</Emphasis>-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC^®BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.     

<Emphasis Type="Italic">Talaromyces heiheensis</Emphasis> and <Emphasis Type="Italic">T. mangshanicus</Emphasis>, two new species from China

Two new species, Talaromyces heiheensis from rotten wood and T. mangshanicus isolated from soil, are illustrated and described as new to science in sections Trachyspermi and Talaromyces. The phylogenetic positions of the two new species inferred from the internal transcribed spacer, beta-tubulin, calmodulin and RNA polymerase II second largest subunit regions were carried out. Talaromyces heiheensis is phylogenetically closely related to T. albobiverticillius, T. rubrifaciens, T. solicola and T. erythromellis, and characterised by slow growth on Czapek yeast autolysate agar at 25 °C, orange conidia en masse on malt extract agar at 25 °C, biverticillate and terverticillate conidiophores, acerose phialides and subglobose to ellipsoidal, smooth-walled conidia. Talaromyces mangshanicus is related to T. kendrickii, T. qii and T. thailandensis, and characterised by slow-growing colonies with absent or sparse sporulation on CYA agar at 25 °C, conidia en masse greyish purple, purplish red soluble pigment on yeast extract agar (YES) at 25 °C, biverticillate conidiophores, ampulliform phialides and subglobose to ellipsoidal conidia with echinulate walls. They are distinguished from the known species in culture characteristics on four standard media, microscopic features and sequence data.     

Low temperature and <Emphasis Type="Italic">Daphnia</Emphasis>-associated infochemicals promote colony formation of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> and its harvesting

Objective

To explore the combined effects of temperature and Daphnia-associated infochemicals on colony formation of Scenedesmus obliquus to faciliate harvesting the algal biomass.

Results

A three-parameter modified Gaussian model fitted the changes of the number of cells per particle in S. obliquus induced by Daphnia culture filtrate well under any temperature. Decreases in temperature enhanced the induced–colony formation of Scenedesmus. The maximum colony size at 15–25 °C was significantly larger than those at 30–35 °C. An additional 1 or 2 days at low temperature was needed to reach the maximum colony size, which indicates the best harvest time for algal biomass.

Conclusion

Induced-colony formation of Scenedesmus by Daphnia culture filtrate at 15–25 °C is recommended to settle algal cells. This condition facilitates harvesting the biomass.

     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Sequence analysis of <Emphasis Type="Italic">KmXYL1</Emphasis> genes and verification of thermotolerant enzymatic activities of xylose reductase from four <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

Expression and Characterization of Glucose Oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Precultivation of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during <Emphasis Type="SmallCaps">l</Emphasis>(+)-lactic acid fermentation

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in l(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

<Emphasis Type="Italic">Metschnikowia</Emphasis> mating genomics

Genes involved in mating type determination and recognition were examined in Metschnikowia and related species, to gather insights on factors affecting mating compatibility patterns among haplontic, heterothallic yeast species of the genus. We confirmed the universality of the special mating locus organisation found in Clavispora lusitaniae across and exclusive to the family Metschnikowiaceae (i.e., Metschnikowia and Clavispora). Timing of the divergence between idiomorphs was confirmed to coincide with the origin of the larger (CUG-ser) clade comprising the Debaryomycetaceae and the Metschnikowiaceae, exclusive of Cephaloascus fragrans. The sequence of the a mating pheromone is highly conserved within the large-spored Metschnikowia species, including Metschnikowia orientalis and Metschnikowia hawaiiana, but not Metschnikowia drosophilae or Metschnikowia torresii, which have a pattern of their own, as do other clades in the genus. In contrast, variation in α pheromones shows a more continuous, although imperfect correlation with phylogenetic distance as well as with in vivo mating compatibility.     

Comparison of bioethanol production from cultivated versus wild <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> and <Emphasis Type="Italic">Gracilaria gigas</Emphasis>

The seaweed genus Gracilaria is a potential candidate for the production of bioethanol due to its high carbohydrate content. Gracilaria is abundant throughout the world and can be found in both wild and cultivated forms. Differences in the ecological factors such as temperature, salinity, and light intensity affecting wild and cultivated specimens may influence the biochemical content of seaweeds, including the carbohydrate content. This study aimed to investigate the proximate composition and potential bioethanol production of wild and cultivated G. gigas and G. verrucosa. Bioethanol was produced using separate hydrolysis fermentation (SHF), employing a combination of enzymatic and acid hydrolysis, followed by fermentation with Saccharomyces cerevisiae ATCC 200062. The highest carbohydrate content was found in wild G. gigas. The highest galactose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L^?1, respectively), as well as the highest production of bioethanol (3.56 ± 0.02 g L^?1), were also found in wild G. gigas. Thus, we conclude that wild G. gigas is the most promising candidate for bioethanol production. Further research is needed to optimize bioethanol production from wild G. gigas. Domestication of wild G. gigas is a promising challenge for aquaculture to avoid overexploitation of this wild seaweed resource.     

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in <Emphasis Type="Italic">Thermoanaerobacter pseudethanolicus</Emphasis> 39E

Background

Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results

Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions

T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.