Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Using a Taguchi DOE to investigate factors and interactions affecting germination in Miscanthus sinensis.

The Miscanthus genus of perennial grasses is grown for bioenergy and biorenewable feedstocks. Most Miscanthus crop is M × giganteus which is rhizome propagated and therefore difficult to multiply at large scale. Seed-based propagation of new hybrids is being developed, but Miscanthus is difficult to establish from seed especially in the field. Miscanthus is often grown on marginal land adding to the challenge of successfully establishing the crop. Improved understanding of the limits and biology of germination in Miscanthus species is needed. Seed germination is affected by physical and chemical factors that impact germination differently depending on level of exposure. In this investigation of Miscanthus germination, four hormones plus water stress were investigated and the range over which these factors affect germination was determined. An efficient Taguchi experimental design was used to assess the five factors in combination with the effects of light and seed priming. This determined an example of a set of optimum conditions for Miscanthus germination and demonstrated how this could change based on fixing one condition. The experiment showed how environmental stress impacted germination and how treatments such as gibberellic acid could be used to mitigate stress.

Draft Genome Assemblies of Xylose-Utilizing Candida tropicalis and Candida boidinii with Potential Application in Biochemical and Biofuel Production.

Non-albicans Candida species are growing in prominence in industrial biotechnology due to their ability to utilize hemicellulose. Here, we present the draft genome sequences of an inhibitor-tolerant Candida tropicalis strain (Y6604) and Candida boidinii NCAIM Y01308T.

Apiotrichum terrigenum sp. nov., a soil-associated yeast found in both the UK and mainland Europe.

Five arthroconidium-producing yeast strains representing a novel Trichosporon-like species were independently isolated from the UK, Hungary and Norway. Two strains (Bio4T and Bio21) were isolated from biogas reactors used for processing grass silage, with a third strain (S8) was isolated from soil collected at the same UK site. Two additional strains were isolated in mainland Europe, one from soil in Norway (NCAIM Y.02175) and the other from sewage in Hungary (NCAIM Y.02176). Sequence analyses of the D1/D2 domains of the LSU rRNA gene and internal transcribed spacer (ITS) region indicated that the novel species belongs to the recently reinstated genus Apiotrichum and is most closely related to Apiotrichum scarabaeorum, a beetle-associated species first found in South Africa. Despite having similar physiological characteristics, the two species can be readily distinguished from one another by ITS sequencing. The species name Apiotrichum terrigenum sp. nov. is proposed to accommodate these strains, with Bio4T (=CBS 11373T=NCYC 3540T) designated as the type strain. The Mycobank deposit number is MB817431.

New aspects and strategies for methane mitigation from ruminants.

The growing demand for sustainable animal production is compelling researchers to explore the potential approaches to reduce emissions of greenhouse gases from livestock that are mainly produced by enteric fermentation. Some potential solutions, for instance, the use of chemical inhibitors to reduce methanogenesis, are not feasible in routine use due to their toxicity to ruminants, inhibition of efficient rumen function or other transitory effects. Strategies, such as use of plant secondary metabolites and dietary manipulations have emerged to reduce the methane emission, but these still require extensive research before these can be recommended and deployed in the livestock industry sector. Furthermore, immunization vaccines for methanogens and phages are also under investigation for mitigation of enteric methanogenesis. The increasing knowledge of methanogenic diversity in rumen, DNA sequencing technologies and bioinformatics have paved the way for chemogenomic strategies by targeting methane producers. Chemogenomics will help in finding target enzymes and proteins, which will further assist in the screening of natural as well chemical inhibitors. The construction of a methanogenic gene catalogue through these approaches is an attainable objective. This will lead to understand the microbiome function, its relation with the host and feeds, and therefore, will form the basis of practically viable and eco-friendly methane mitigation approaches, while improving the ruminant productivity.

Laccase production by Coriolopsis caperata RCK2011: optimization under solid state fermentation by Taguchi DOE methodology.

Laccase production by Coriolopsis caperata RCK2011 under solid state fermentation was optimized following Taguchi design of experiment. An orthogonal array layout of L18 (2(1) × 3(7)) was constructed using Qualitek-4 software with eight most influensive factors on laccase production. At individual level pH contributed higher influence, whereas, corn steep liquor (CSL) accounted for more than 50% of the severity index with biotin and KH2PO4 at the interactive level. The optimum conditions derived were; temperature 30°C, pH 5.0, wheat bran 5.0 g, inoculum size 0.5 ml (fungal cell mass = 0.015 g dry wt.), biotin 0.5% w/v, KH2PO4 0.013% w/v, CSL 0.1% v/v and 0.5 mM xylidine as an inducer. The validation experiments using optimized conditions confirmed an improvement in enzyme production by 58.01%. The laccase production to the level of 1623.55 Ugds(-1) indicates that the fungus C. caperata RCK2011 has the commercial potential for laccase.

Optimizing anaerobic digestion by selection of the immobilizing surface for enhanced methane production.

Maximizing methane production while maintaining an appreciable level of process stability is a crucial challenge in the anaerobic digestion industry. In this study, the role of six parameters: the type of immobilizing supports, loading rate, inoculum levels, C:N ratio, trace nutrients concentrations and mixing rate, on methane production were investigated under thermophilic conditions (55 ± 1°C) with synthetic substrate medium. The immobilizing supports were Silica gel, Sand, Molecular Sieve and Dowex Marathon beads. A Taguchi Design of Experiment (DOE) methodology was employed to determine the effects of different parameters using an L(16) orthogonal array. Overall, immobilizing supports influenced methane production substantially (contributing 61.3% of the observed variation in methane yield) followed by loading rate and inoculum which had comparable influence (17.9% and 17.7% respectively). Optimization improved methane production by 153% (from 183 to 463 ml CH(4)l(-1)d(-1)).

Candida northwykensis sp. nov., a novel yeast isolated from the gut of the click beetle Melanotus villosus.

Two yeast morphotypes, BET 4(T) and BET 7, were isolated from the gut of click beetle Melanotus villosus. Click beetles were collected from the decaying timber within the woodlands of North Wyke Research, South West England, UK (latitude, 50°46'29″N; longitude, 3°55'23″W). Morphotype BET 7 was identified as Debaryomyces hansenii, and the other morphotype, BET 4(T), was found to differ from Priceomyces castillae and Priceomyces haplophilus, its closest phylogenetic neighbours, by 5.0% with respect to the nucleotide sequence of the D1/D2 domain of the large-subunit (LSU) rRNA gene, and by 8.0% with respect to the ribosomal internal-transcribed spacer (ITS) region. BET 4(T) also differ from P. castillae and P. haplophilus in a number of different phenotypic characteristics. Thus, based on the unique nucleotide sequences of its D1/D2 domain and ITS region, its physiological characteristics and an inability to sporulate, strain BET 4(T) is assigned the status of a new species of Candida, for which the name Candida northwykensis sp. nov., is proposed. The type strain is BET 4(T) (NCYC 3525(T) = CBS 11370(T)).

Cryptococcus shivajii sp. nov.: a novel basidiomycetous yeast isolated from biogas reactor.

Five yeast morphotypes were isolated from biogas reactors at North Wyke Research, Okehampton, UK. Out of the five morphotypes, four were identified as known species. In contrast, the fifth morphotype strain, Bio10(T), was found to differ from Bullera dendrophila and Kwoniella mangroviensis, its closest phylogenetic neighbours, by 2.6-2.9% with respect to the nucleotide sequence of the D1/D2 domain of the 26S rRNA gene and by 5.6-6.2% with respect to the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-ITS2 region. Bio10(T) also differs from these two species by a number of phenotypic characteristics. Thus, based on the phenotypic differences and phylogenetic analysis, strain Bio10(T) is assigned the status of a new species of Cryptococcus, for which the name Cryptococcus shivajii sp. nov. is proposed. The type strain is Bio10(T) (NCYC 3541(T) = CBS 11374(T)).

The Taguchi methodology as a statistical tool for biotechnological applications: a critical appraisal.

Success in experiments and/or technology mainly depends on a properly designed process or product. The traditional method of process optimization involves the study of one variable at a time, which requires a number of combinations of experiments that are time, cost and labor intensive. The Taguchi method of design of experiments is a simple statistical tool involving a system of tabulated designs (arrays) that allows a maximum number of main effects to be estimated in an unbiased (orthogonal) fashion with a minimum number of experimental runs. It has been applied to predict the significant contribution of the design variable(s) and the optimum combination of each variable by conducting experiments on a real-time basis. The modeling that is performed essentially relates signal-to-noise ratio to the control variables in a 'main effect only' approach. This approach enables both multiple response and dynamic problems to be studied by handling noise factors. Taguchi principles and concepts have made extensive contributions to industry by bringing focused awareness to robustness, noise and quality. This methodology has been widely applied in many industrial sectors; however, its application in biological sciences has been limited. In the present review, the application and comparison of the Taguchi methodology has been emphasized with specific case studies in the field of biotechnology, particularly in diverse areas like fermentation, food processing, molecular biology, wastewater treatment and bioremediation.

Rhodotorula himalayensis sp. nov., a novel psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India.

Twenty-five psychrophilic yeasts were isolated from the soil of Roopkund Lake, Himalayas, India. Two colony morphotypes were identified and representatives of 'morphotype 1' were identified as Cryptococcus gastricus. Representatives of 'morphotype 2', namely 3AT, 4A, 4B and Rup4B, showed similar phenotypic properties and are identical with respect to the nucleotide sequence of the ITS1-5.8S rRNA gene-ITS2 region and D1/D2 domain of the 26S rRNA gene. The sequence of D1/D2 domain of 3AT shows 97.6-98.8% similarity with Rhodotorula psychrophila CBS10440T, Rhodotorula glacialis CBS10437T and Rhodotorula psychrophenolica CBS10438T and in the neighbour-joining phylogenetic tree strains; 3AT, 4A, 4B and Rup4B form a cluster with Rhodotorula glacialis and Rhodotorula psychrophila. Strains 3AT, 4A, 4B and Rup4B also differ from their nearest phylogenetic relatives in several biochemical characteristics such as in assimilation of D-galactose, L-sorbose, maltose, citrate, D-glucuronate and creatinine. Thus, based on the phylogenetic analysis and the phenotypic differences 3AT, 4A, 4B and Rup 4B are assigned the status of a new species of Rhodotorula for which the name Rhodotorula himalayensis sp. nov. is proposed with 3AT as the type strain (=CBS10539T =MTCC8336T).

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae).

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.

Candida hyderabadensis sp. nov., a novel ascomycetous yeast isolated from wine grapes.

Three ascomycetous yeast strains were isolated from decaying green wine grapes, collected from Hyderabad city in India. Two strains, YS9 and YS21, were identified as Kodamaea ohmeri and Candida fermentati, respectively. The third strain, YS12(T), differs from Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis, the nearest phylogenetic neighbours, by 1.6-1.9% with respect to the nucleotide sequence of the D1/D2 domain of the 26S rRNA gene and by 1.4-9.2% with respect to the nucleotide sequence of the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-ITS2 region. YS12(T) also differs from C. parapsilosis, C. metapsilosis and C. orthopsilosis by some phenotypic characteristics. Thus, based on the phenotypic differences and phylogenetic analysis, strain YS12(T) is assigned the status of a new species of Candida, for which the name C. hyderabadensis sp. nov. is proposed. The type strain is YS12(T) (NRRL Y-27953(T)=CBS10444(T)=IAM15334(T)).

Strain improvement of Candida tropicalis for the production of xylitol: biochemical and physiological characterization of wild-type and mutant strain CT-OMV5.

Candida tropicalis was treated with ultraviolet (UV) rays, and the mutants obtained were screened for xylitol production. One of the mutants, the UV1 produced 0.81 g of xylitol per gram of xylose. This was further mutated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the mutants obtained were screened for xylitol production. One of the mutants (CT-OMV5) produced 0.85 g/g of xylitol from xylose. Xylitol production improved to 0.87 g/g of xylose with this strain when the production medium was supplemented with urea. The CT-OMV5 mutant strain differs by 12 tests when compared to the wild-type Candida tropicalis strain. The XR activity was higher in mutant CT-OMV5. The distinct difference between the mutant and wild-type strain is the presence of numerous chlamydospores in the mutant. In this investigation, we have demonstrated that mutagenesis was successful in generating a superior xylitol-producing strain, CT-OMV5, and uncovered distinctive biochemical and physiological characteristics of the wild-type and mutant strain, CT-OMV5.

Optimization of lactic acid production in SSF by Lactobacillus amylovorus NRRL B-4542 using Taguchi methodology.

Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO4 . 7H20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO4 . 7H20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.