CRISPR-based bioengineering in microalgae for production of industrially important biomolecules.

Microalgae, as photosynthetic organisms, have the potential to produce biomolecules for use in food, feed, cosmetics, nutraceuticals, fuel, and other applications. Faster growth rates and higher protein and lipid content make microalgae a popular chassis for many industrial applications. However, challenges such as low productivity and high production costs have limited their commercialization. To overcome these challenges, bioengineering approaches such as genetic engineering, metabolic engineering, and synthetic biology have been employed to improve the productivity and quality of microalgae-based products. Genetic engineering employing genome editing tools like CRISPR/Cas allows precise and targeted genetic modifications. CRISPR/Cas systems are presently used to modify the genetic makeup of microalgae for enhanced production of specific biomolecules. However, these tools are yet to be explored explicitly in microalgae owing to some limitations. Despite the progress made in CRISPR-based bioengineering approaches, there is still a need for further research to optimize the production of microalgae-based products. This includes improving the efficiency of genome editing tools, understanding the regulatory mechanisms of microalgal metabolism, and optimizing growth conditions and cultivation strategies. Additionally, addressing the ethical, social, and environmental concerns associated with genetic modification of microalgae is crucial for the responsible development and commercialization of microalgae-based products. This review summarizes the advancements of CRISPR-based bioengineering for production of industrially important biomolecules and provides key considerations to use CRISPR/Cas systems in microalgae. The review will help researchers to understand the progress and to initiate genome editing experiments in microalgae.

Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide.

Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes.

CRISPR-based assays for rapid detection of SARS-CoV-2.

COVID-19 pandemic posed an unprecedented threat to global public health and economies. There is no effective treatment of the disease, hence, scaling up testing for rapid diagnosis of SARS-CoV-2 infected patients and quarantine them from healthy individuals is one the best strategies to curb the pandemic. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid based tests are considered to be more sensitive with respect to serological tests but present gold standard qRT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. CRISPR based novel techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here, we discuss and present compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology which has tremendous potential to transform diagnostics and epidemiology.

Optimization and validation of RT-LAMP assay for diagnosis of SARS-CoV2 including the globally dominant Delta variant.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.

Characterization of nanocellulose production by strains of Komagataeibacter sp. isolated from organic waste and Kombucha.

Bacterial nanocellulose production is gaining popularity owing to its applications in food, cosmetics and medical industry. Three Acetobacter strains isolated from organic waste and fermented tea were identified using 16S rDNA sequencing and their ability to produce nanocellulose was studied. Strain isolated from Kombucha has 99% homology with Komagataeibacter rhaeticus DSM 16663 T. This is the first report where nanocellulose productivity of this strain with different carbon sources such as glucose, glycerol, fructose and sucrose has been studied. 1% glycerol was found to be optimal concentration, with up to 69% of the utilized carbon converted to nanocellulose. Maximum productivity of 4.5 g/L of bacterial nanocellulose was obtained. Average nitrogen and phosphorus consumption rate was 45 mg/L/day each. Physical properties such as crystallinity, fibril dimensions, and glass transition temperature were studied. Bacterial cellulose was 80% crystalline when glycerol and glucose were used as carbon source and 73% for fructose and sucrose. Renewable materials such as bacterial cellulose with their unique properties are the future for applications in the field of cosmetics, composite and wound care.

CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.

The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase ß-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.

Potential of natural astaxanthin in alleviating the risk of cytokine storm in COVID-19.

Host excessive inflammatory immune response to SARS-CoV-2 infection is thought to underpin the pathogenesis of COVID-19 associated severe pneumonitis and acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Once an immunological complication like cytokine storm occurs, anti-viral based monotherapy alone is not enough. Additional anti-inflammatory treatment is recommended. It must be noted that anti-inflammatory drugs such as JAK inhibitors, IL-6 inhibitors, TNF-α inhibitors, colchicine, etc., have been either suggested or are under trials for managing cytokine storm in COVID-19 infections. Natural astaxanthin (ASX) has a clinically proven safety profile and has antioxidant, anti-inflammatory, and immunomodulatory properties. There is evidence from preclinical studies that supports its preventive actions against ALI/ARDS. Moreover, ASX has a potent PPARs activity. Therefore, it is plausible to speculate that ASX could be considered as a potential adjunctive supplement. Here, we summarize the mounting evidence where ASX is shown to exert protective effect by regulating the expression of pro-inflammatory factors IL-1ß, IL-6, IL-8 and TNF-α. We present reports where ASX is shown to prevent against oxidative damage and attenuate exacerbation of the inflammatory responses by regulating signaling pathways like NF-ĸB, NLRP3 and JAK/STAT. These evidences provide a rationale for considering natural astaxanthin as a therapeutic agent against inflammatory cytokine storm and associated risks in COVID-19 infection and this suggestion requires further validation with clinical studies.

CRISPR-Cas9 System for Genome Engineering of Photosynthetic Microalgae.

Targeted genome editing using RNA-guided endonucleases is an emerging tool in algal biotechnology. Recently, CRISPR-Cas systems have been widely used to manipulate the genome of some freshwater and marine microalgae. Among two different classes, and six distinct types of CRISPR systems, Cas9-driven type II system has been widely used in most of the studies for targeted knock-in, knock-out and knock-down of desired genes in algae. CRISPR technology has been demonstrated in microalgae including diatoms to manifest the function of the particular gene (s) and developing industrial traits, such as improving lipid content and biomass productivity. Instead of these, there are a lot of gears to be defined about improving efficiency and specificity of targeted genome engineering of microalgae using CRISPR-Cas system. Optimization of tools and methods of CRISPR technology can undoubtedly transform the research toward the industrial-scale production of commodity chemicals, food and biofuels using photosynthetic cell factories. This review has been focused on the efforts made so far to targeted genome engineering of microalgae, identified scopes about the hurdles related to construction and delivery of CRISPR-Cas components, algae transformation toolbox, and outlined the future prospect toward developing the CRISPR platform for high-throughput genome-editing of microalgae.

Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites.

Diversity of yeasts from puddles in the vicinity of midre lovénbreen glacier, arctic and bioprospecting for enzymes and fatty acids.

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C(18:1 (w9C)) and C(18:2 (w9,12C)) were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8 degrees C compared to 22 degrees C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.

Thiomonas bhubaneswarensis sp. nov., an obligately mixotrophic, moderately thermophilic, thiosulfate-oxidizing bacterium.

A novel aerobic, obligately mixotrophic, moderately thermophilic, thiosulfate-oxidizing bacterium, S10T, was isolated from hot-spring sediment samples collected from Atri, Bhubaneswar, India. The cells of this isolate stained Gram-negative and were strictly aerobic, non-sporulating, rod-shaped and motile with a single polar flagellum. Strain S10T was positive for oxidase and catalase activities. It was capable of utilizing thiosulfate under mixotrophic growth conditions. Mixotrophic growth was observed at pH 6.0-8.5 and 25-45 degrees C; optimum growth occurred at pH 7.5-8.0 and 30-37 degrees C. The major cellular fatty acids were C12:0 3-OH, C16:1omega7c, C16:0, C17:0 cyclo, C18:1omega7c and C19:0 cyclo omega8c. The DNA G+C content of strain S10T was 64.8 mol%. 16S rRNA gene sequence analysis indicated that the bacterium clustered within the radiation of the genus Thiomonas and showed 98.0% similarity with Thiomonas perometabolis ATCC 23370T and Thiomonas intermedia ATCC 15466T. However, DNA-DNA reassociation values of strain S10T with Thiomonas perometabolis JCM 20426T and Thiomonas intermedia JCM 20425T, its nearest phylogenetic relatives, were 46 and 39%, respectively. On the basis of phenotypic, physiological and chemotaxonomic properties, 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, it is proposed that strain S10T represents a novel species of the genus Thiomonas, Thiomonas bhubaneswarensis sp. nov.; the type strain is S10T (=DSM 18181T=JCM 14806T).

Pichia garciniae sp. nov., isolated from a rotten mangosteen fruit (Garcinia mangostana L., Clusiaceae).

Ascogenous yeasts were isolated from a decaying mangosteen fruit (Garcinia mangostana L., Clusiaceae). Based on colony morphology and RAPD analysis, the strains were grouped into four groups (G-I, G-II, G-III and G-IV). Phenotypic properties and analysis of the D1/D2 domain sequence of the 26S rRNA gene identified representative members of groups G-II, G-III and G-IV as Pichia manshurica (=Pichia galeiformis), Pichia fermentans and Pichia anomala, respectively. Representatives of group G-I, YS110(T) and YS111, showed similar phenotypic traits, 99.9 % similarity in the sequence of the D1/D2 domain of the 26S rRNA gene and ITS1-5.8S rRNA gene-ITS2 sequence and 92 % DNA-DNA relatedness. Hence, YS110(T) and YS111 are regarded as members of the same species. Based on D1/D2 domain and ITS region sequences, the nearest phylogenetic neighbours of YS110(T) and YS111 were identified as Pichia membranifaciens and P. manshurica. However, YS110(T) differs from these two nearest phylogenetic neighbours by >3 % in the D1/D2 domain sequence and by >18 % with respect to the ITS region sequence. In addition, YS110(T) and YS111 differ from P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T) with respect to a number of phenotypic traits. The strains show 52-57 % DNA-DNA relatedness with P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T). Strains YS110(T) and YS111 are proposed as two strains of a novel species, for which the name Pichia garciniae sp. nov. is proposed. The type strain is YS110(T) (=NRRL Y-48422(T) =CBS 10758(T)).

Exiguobacterium soli sp. nov., a psychrophilic bacterium from the McMurdo Dry Valleys, Antarctica.

A novel psychrophilic bacterium, designated strain DVS 3Y(T), was isolated from a moraine sample from the McMurdo Dry Valleys, Antarctica. Phenotypic and chemotaxonomic characteristics and data from a phylogenetic analysis based on 16S rRNA gene sequences indicated that strain DVS 3Y(T) was related to the genus Exiguobacterium. Strain DVS 3Y(T) exhibited >97 % gene sequence similarity with respect to Exiguobacterium acetylicum DSM 20416(T) (97.4 %), Exiguobacterium oxidotolerans DSM 17272(T) (97.4 %), Exiguobacterium indicum IAM 15368(T) (97.6 %), Exiguobacterium undae DSM 14481(T) (98.2 %), Exiguobacterium sibiricum DSM 17290(T) (98.6 %) and Exiguobacterium antarcticum DSM 14480(T) (99.6 %). However, the levels of DNA-DNA relatedness between strain DVS 3Y(T) and E. acetylicum DSM 20416(T), E. oxidotolerans JCM 12280(T), E. indicum IAM 15368(T), E. undae DSM 14481(T), E. sibiricum DSM 17290(T) and E. antarcticum DSM 14480(T) were less than 70 % (52, 25, 16, 33, 16 and 38 %, respectively). Strain DVS 3Y(T) also differed from these six closely related species in a number of phenotypic traits. Phenotypic, chemotaxonomic and phylogenetic data suggest that strain DVS 3Y(T) represents a novel species of the genus Exiguobacterium, for which the name Exiguobacterium soli sp. nov. is proposed. The type strain is DVS 3Y(T) (=MTCC 4816(T)=JCM 14376(T)).

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.

Yeasts and yeast-like fungi associated with tree bark: diversity and identification of yeasts producing extracellular endoxylanases.

A total of 239 yeast strains was isolated from 52 tree bark samples of the Medaram and Srisailam forest areas of Andhra Pradesh, India. Based on analysis of D1/D2 domain sequence of 26S rRNA gene, 114 strains were identified as ascomycetous; 107 strains were identified as basidiomycetous yeasts; and 18 strains were identified as yeast-like fungi. Among the ascomycetous yeasts, 51% were identified as members of the genus Pichia, and the remaining 49% included species belonging to the genera Clavispora, Debaryomyces, Kluyveromyces, Hanseniaspora, Issatchenkia, Lodderomyces, Kodamaea, Metschnikowia, and Torulaspora. The predominant genera in the basidiomycetous yeasts were Cryptococcus (48.6%), Rhodotorula (29%), and Rhodosporidium (12.1%). The yeast-like fungi were represented by Aureobasidium pullulans (6.7%) and Lecythophora hoffmanii (0.8%). Of the 239 yeast strains tested for Xylanase, only five strains of Aureobasidium sp. produced xylanase on xylan-agar medium. Matrix-assisted laser desorption ionization-time of flight analysis and N-terminal amino-acid sequence of the xylanase of isolate YS67 showed high similarity with endo-1-4-beta-xylanase (EC 3.2.1.8) of Aureobasidium pullulans var. melanigenum.

Brevibacterium oceani sp. nov., isolated from deep-sea sediment of the Chagos Trench, Indian Ocean.

Two bacterial strains, designated BBH5 and BBH7(T), were isolated from a deep-sea sediment sample collected from the Chagos Trench of the Indian Ocean (1 degrees 06' S 7 degrees 31' E). Based on their 16S rRNA gene sequence similarity (99.9%), level of DNA-DNA relatedness (93%) and a number of similar phenotypic characteristics, the two strains are identified as representing the same species. Their phylogenetically nearest neighbours, based on 16S rRNA gene sequence similarity values (97.9-98.4%), were identified as Brevibacterium iodinum, Brevibacterium epidermidis, Brevibacterium linens and Brevibacterium permense. However, strains BBH5 and BBH7(T) could be distinguished from the above four species by a number of phenotypic characteristics, and levels of DNA-DNA relatedness between the two new isolates and these Brevibacterium species were 35-42%. Therefore, strains BBH5 and BBH7(T) are considered to represent a novel species of the genus Brevibacterium, for which the name Brevibacterium oceani sp. nov. is proposed. The type strain is BBH7(T) (=LMG 23457(T) =IAM 15353(T)).

Microbacterium indicum sp. nov., isolated from a deep-sea sediment sample from the Chagos Trench, Indian Ocean.

Two bacterial strains, BBH6(T) and BBH9, were isolated from a deep-sea sediment sample collected from the Chagos Trench, Indian Ocean, at a depth of 5904 m. The two strains were closely related in their 16S rRNA gene sequences (99.7 %), belonged to one genomic species and were virtually identical at the phenotypic level. Microbacterium barkeri DSM 20145(T) was the nearest phylogenetic neighbour to the new isolates, with 16S rRNA gene sequence similarity levels of 97.2-97.4 %. The new isolates exhibited levels of DNA-DNA relatedness of 32-34 % to M. barkeri and differed from it in a number of phenotypic characteristics. Therefore, it is suggested that strains BBH6(T) and BBH9 represent a novel species of the genus Microbacterium, for which the name Microbacterium indicum sp. nov. is proposed. The type strain is BBH6(T) (=LMG 23459(T)=IAM 15355(T)).

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months.

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0-691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn(2+), comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5-10 microM Zn(2+ )(325-650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 microM Zn(2+ )(260 ppb). The lag phase was reduced by 6-8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.

Pichia cecembensis sp. nov. isolated from a papaya fruit (Carica papaya L., Caricaceae).

The ascogenous yeast YS16T was isolated from a decaying papaya fruit. Phenotypic traits such as multilateral budding, spheroidal or elongate shape, pseudohyphae formation, asci with one or more ascospores, ability to ferment d-glucose, inability to assimilate nitrate and the presence of Q7 ubiquinone suggest its affiliation to the genus Pichia. The nearest phylogenetic neighbor, based on D1/D2 domain sequence of the 26S rRNA gene and ITS region sequence, was identified as Issatchenkia orientalis (NRRL Y-5396T, a synonym of Pichia kudriavzevii) with similarities of 98.2% and 97% respectively. In addition to the difference in the D1/D2 and ITS region sequence, YS16T differs from I. orientalis with respect to a number of phenotypic traits. However, in the phylogenetic analysis, YS16T showed close relatedness to the P. membranifaciens clade. Thus, it is proposed to assign the status of a new species to YS16T, for which the name P. cecembensis sp. nov. is proposed. The type strain of P. cecembensis sp. nov. is YS16T (=NRRL Y-27985T=JCM 13873T=CBS 10445T).