A deep-sea hydrogen peroxide-stable alkaline serine protease from Aspergillus flavus.

We report here the production of an alkaline serine protease by Aspergillus flavus isolated at 5600-m depth from deep-sea sediments of the Central Indian Basin. When grown on defatted groundnut oil meal at 30 °C for 48-72 h, this fungal isolate produced 2000-2500 ACU mL-1 of alkaline protease. The purified protease had activity optima at pH 10.0 and 45 °C. It was a thiol-independent serine protease, identified as an alkaline serine protease ALP1 with a molecular mass of 42.57 kDa. The thermostability and activity of the enzyme increased at 60 °C, in the presence of additives such as sucrose, Tween 20, sorbitol, Ca2+ and glycerol and was not adversely affected by H2O2 indicating its potential as a detergent additive.

Heavy metal tolerance in the psychrotolerant Cryptococcus sp. isolated from deep-sea sediments of the Central Indian Basin.

A deep-sea isolate of the psychrotolerant yeast Cryptococcus sp. (NIOCC#PY13) obtained from polymetallic nodule-bearing sediments of the Central Indian Basin was examined for its capacity to grow in the presence of various concentrations of the heavy metal salts i.e., ZnSO4 , CuSO4 , Pb(CH3 COO)2 and CdCl2 . It demonstrated considerable growth in the presence of 100 mg/l concentrations of the above-mentioned four heavy metal salts both at 30°C and 15°C. This strain tolerated comparatively higher levels of these four metal salts than other deep-sea and terrestrial yeast isolates belonging to Cryptococcus, Rhodotorula, Rhodosporidium and Sporidiobolus spp. Optimum pH for growth of this isolate was in the range of 6-8 in the presence of heavy metal salts at these two temperatures. Scanning electron microscopic (SEM) studies exhibited altered cell surface morphology of the cells under the influence of heavy metals compared to that with control. The adsorption of heavy metals to the cells was demonstrated by FTIR and EDAX analysis. As evidenced by atomic absorption spectrophotometric (AAS) analysis, about 30-90% of the heavy metals were removed from the culture supernatant after 4 days of growth at 30°C. This deep-sea yeast isolate appears to be a potential candidate for bioremediation of metal-contaminated sites. Moreover, its metal tolerance properties provide a significant insight into its ecological role and adaptations to growth in such extreme conditions.

Fungal diversity from various marine habitats deduced through culture-independent studies.

Studies on the molecular diversity of the micro-eukaryotic community have shown that fungi occupy a central position in a large number of marine habitats. Environmental surveys using molecular tools have shown the presence of fungi from a large number of marine habitats such as deep-sea habitats, pelagic waters, coastal regions, hydrothermal vent ecosystem, anoxic habitats, and ice-cold regions. This is of interest to a variety of research disciplines like ecology, evolution, biogeochemistry, and biotechnology. In this review, we have summarized how molecular tools have helped to broaden our understanding of the fungal diversity in various marine habitats. Majority of the environmental phylotypes could be grouped as novel clades within Ascomycota, Basidiomycota, and Chytridiomycota or as basal fungal lineages. Deep-branching novel environmental clusters could be grouped within Ascomycota as the Pezizomycotina clone group, deep-sea fungal group-I, and soil clone group-I, within Basidiomycota as the hydrothermal and/or anaerobic fungal group, and within Chytridiomycota as Cryptomycota or the Rozella clade. However, a basal true marine environmental cluster is still to be identified as most of the clusters include representatives from terrestrial regions. The challenge for future research is to explore the true marine fungi using molecular techniques.

Differentially expressed genes under simulated deep-sea conditions in the psychrotolerant yeast Cryptococcus sp. NIOCC#PY13.

Microorganisms exhibit varying degrees of tolerance to extreme conditions of pressure and temperature. In the present study, the psychrotolerant deep-sea yeast, Cryptococcus sp. NIOCC#PY13, was exposed to elevated hydrostatic pressure and low temperature to explore the differentially expressed genes responsible for its survival at such extreme conditions. The suppression subtractive hybridization technique was employed for identification of expressed upregulated genes at extreme conditions of pressure and temperature. The effect of elevated pressure was found to be different than that of combined pressure and temperature exposures. Altogether, 17 and 20 upregulated genes were identified at 50 MPa and 50 MPa/5 °C, respectively. These differentially expressed genes were similar to the NCBI database ESTs (expressed sequence tags), coding for proteins such as arachidonic acid metabolism, amino acid transport and unsaturation of membrane fatty acids, which have been previously demonstrated to assist in the survival of microorganisms under stress conditions. Interestingly, about 50 % of the upregulated genes matched with hypothetical proteins at a percentage similarity of ≤96, suggesting their probability of being novel. Detailed studies of the above genes/proteins from deep-sea microorganisms are suggested for future investigations, which may shed more light on the existence and adaptation mechanisms adopted by these for their survival under such extreme conditions.

Assessment of fungal diversity in deep-sea sediments by multiple primer approach.

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79-97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.

Fungi and their role in corals and coral reef ecosystems.

Fungi in coral reefs exist as endoliths, endobionts, saprotrophs and as pathogens. Although algal and fungal endoliths in corals were described way back in 1973, their role in microboring, carbonate alteration, discoloration, density banding, symbiotic or parasitic association was postulated almost 25 years later. Fungi, as pathogens in corals, have become a much discussed topic in the last 10 years. It is either due to the availability of better tools for investigations or greater awareness among the research communities. Fungi which are exclusive as endoliths (endemic) in corals or ubiquitous forms seem to play a role in coral reef system. Fungi associated with sponges and their role in production or induction of secondary metabolites in their host is of primary interest to various pharmaceutical industries and funding agencies. Fungal enzymes in degradation of coral mucus, and plant detritus hold great promise in biotechnological applications. Unravelling fungal diversity in corals and associated reef organisms using culture and culture-independent approaches is a subject gaining attention from research community world over.

Monitoring the sedimentary carbon in an artificially disturbed deep-sea sedimentary environment.

An area of 0.6 km(2) in the manganese nodule field of the Central Indian Basin was physically disturbed and sediments discharged in the near bottom waters to simulate seabed mining and study its impact on benthic ecosystem. An estimated 2 to 3 tonnes of sedimentary organic carbon (C(org)) was resuspended into the water column during a 9-day experiment. The majority of the sediment cores from within the disturbed area and areas towards the south showed a ~30% increase in C(org) content as well as an increase in carbon burial rates after disturbance, though with a reduction in carbon/phosphorus ratios. High specific surface area (SSA~25 m(2) g(-1)) and low C(org)/SSA ratios (mostly <0.5) are typical of deep-sea sediments. The increased C(org) values were probably due to the organic matter from dead biota and the migration and redeposition of fine-grained, organic-rich particles. Spatial distribution patterns of C(org) contents of cores taken before and after disturbance were used to infer the direction of plume migration and re-sedimentation. A positive relationship was observed between total and labile C(org) and macrobenthos density and total bacterial numbers prior to disturbance, whereas a negative relationship was seen after disturbance owing to drastic reduction in the density of macrofauna and bacteria. Overall decrease in labile organic matter, benthic biota and redistribution of organic matter suggest that the commercial mining of manganese nodules may have a significant immediate negative effect on the benthic ecosystem inducing changes in benthic community structure.

A novel hybrid technology for remediation of molasses-based raw effluents.

A novel three-step technology for treatment of four molasses-based raw industrial effluents, varying in their COD, color and turbidity is reported here. Sequential steps involved in this treatment are; (1) sonication of the effluents, (2) whole-fungal treatment of these by a ligninolytic marine fungus and (3) biosorption of the residual color with heat-inactivated biomass of the same fungus. Sonication reduced the foul odor and turbidity of the effluents. It increased biodegradability of the effluents in the second stage of treatment. Laccase production in the presence of all the four effluents was directly correlated with their decolorization. After the third step, a reduction of 60-80% in color, 50-70% in COD and 60-70% in total phenolics were achieved. Comparative mass and nuclear magnetic resonance spectra indicated increasing degradation of the effluent components after each stage. Toxicity (LC(50) values) against Artemia larvae was reduced by two to five folds.

Fungal community analysis in the deep-sea sediments of the Central Indian Basin by culture-independent approach.

Few studies have addressed the occurrence of fungi in deep-sea sediments, characterized by elevated hydrostatic pressure, low temperature, and fluctuating nutrient conditions. We evaluated the diversity of fungi at three locations of the Central Indian Basin (CIB) at a depth of ~5,000 m using culture-independent approach. Community DNA isolated from these sediments was amplified using universal and fungal-specific internal transcribed spacers and universal 18S rDNA primer pairs. A total of 39 fungal operational taxonomic units, with 32 distinct fungal taxa were recovered from 768 clones generated from 16 environmental clone libraries. The application of multiple primers enabled the recovery of eight sequences that appeared to be new. The majority of the recovered sequences belonged to diverse phylotypes of Ascomycota and Basidiomycota. Our results suggested the existence of cosmopolitan marine fungi in the sediments of CIB. This study further demonstrated that diversity of fungi varied spatially in the CIB. Individual primer set appeared to amplify different fungal taxa occasionally. This is the first report on culture-independent diversity of fungi from the Indian Ocean.

Four marine-derived fungi for bioremediation of raw textile mill effluents.

Textile dye effluents pose environmental hazards because of color and toxicity. Bioremediation of these has been widely attempted. However, their widely differing characteristics and high salt contents have required application of different microorganisms and high dilutions. We report here decolorization and detoxification of two raw textile effluents, with extreme variations in their pH and dye composition, used at 20-90% concentrations by each of the four marine-derived fungi. Textile effluent A (TEA) contained an azo dye and had a pH of 8.9 and textile effluent B (TEB) with a pH of 2.5 contained a mixture of eight reactive dyes. The fungi isolated from mangroves and identified by 18S and ITS sequencing corresponded to two ascomycetes and two basidiomycetes. Each of these fungi decolorized TEA by 30-60% and TEB by 33-80% used at 20-90% concentrations and salinity of 15 ppt within 6 days. This was accompanied by two to threefold reduction in toxicity as measured by LC(50) values against Artemia larvae and 70-80% reduction in chemical oxygen demand and total phenolics. Mass spectrometric scan of effluents after fungal treatment revealed degradation of most of the components. The ascomycetes appeared to remove color primarily by adsorption, whereas laccase played a major role in decolorization by basidiomycetes. A process consisting of a combination of sorption by fungal biomass of an ascomycete and biodegradation by laccase from a basidiomycete was used in two separate steps or simultaneously for bioremediation of these two effluents.

Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation.

In order to study fungal diversity in oxygen minimum zones of the Arabian Sea, we analyzed 1440 cloned small subunit rRNA gene (18S rRNA gene) sequences obtained from environmental samples using three different PCR primer sets. Restriction fragment length polymorphism (RFLP) analyses yielded 549 distinct RFLP patterns, 268 of which could be assigned to fungi (Dikarya and zygomycetes) after sequence analyses. The remaining 281 RFLP patterns represented a variety of nonfungal taxa, even when using putatively fungal-specific primers. A substantial number of fungal sequences were closely related to environmental sequences from a range of other anoxic marine habitats, but distantly related to known sequences of described fungi. Community similarity analyses suggested distinctively different structures of fungal communities from normoxic sites, seasonally anoxic sites and permanently anoxic sites, suggesting different adaptation strategies of fungal communities to prevailing oxygen conditions. Additionally, we obtained 26 fungal cultures from the study sites, most of which were closely related (>97% sequence similarity) to well-described Dikarya. This indicates that standard cultivation mainly produces more of what is already known. However, two of these cultures were highly divergent to known sequences and seem to represent novel fungal groups on high taxonomic levels. Interestingly, none of the cultured isolates is identical to any of the environmental sequences obtained. Our study demonstrates the importance of a multiple-primer approach combined with cultivation to obtain deeper insights into the true fungal diversity in environmental samples and to enable adequate intersample comparisons of fungal communities.

A thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus.

Laccase, an oxidoreductive enzyme, is important in bioremediation. Although marine fungi are potential sources of enzymes for industrial applications, they have been inadequately explored. The fungus MTCC 5159, isolated from decaying mangrove wood and identified as Cerrena unicolor based on the D1/D2 region of 28S and the 18S ribosomal DNA sequence, decolorized several synthetic dyes. Partially purified laccase reduced lignin content from sugarcane bagasse pulp by 36% within 24 h at 30 degrees C. Laccase was the major lignin-degrading enzyme (approximately 24,000 U L(-1)) produced when grown in low-nitrogen medium with half-strength seawater. Three laccases, Lac I, Lac II, and Lac III, of differing molecular masses were produced. Each of these, further resolved into four isozymes by anion exchange chromatography. The N-terminal amino acid sequence of the major isozyme, Lac IId showed 70-85% homology to laccases from basidiomycetes. It contained an N-linked glycan content of 17%. The optimum pH and temperature for Lac IId were 3 and 70 degrees C, respectively, the half-life at 70 degrees C being 90 min. The enzyme was most stable at pH 9 and retained >60% of its activity up to 180 min at 50 degrees C and 60 degrees C. The enzyme was not inhibited by Pb, Fe, Ni, Li, Co, and Cd at 1 mmol. This is the first report on the characterization of thermostable metal-tolerant laccase from a marine-derived fungus with a potential for industrial application.

Anaerobic denitrification in fungi from the coastal marine sediments off Goa, India.

Denitrification is a microbial process during which nitrate or nitrite is reduced under anaerobic condition to gaseous nitrogen. The Arabian Sea contains one of the major pelagic denitrification zones and in addition to this, denitrification also takes places along the continental shelf. Prokaryotic microorganisms were considered to be the only players in this process. However recent studies have shown that higher microeukaryotes such as fungi can also adapt to anaerobic mode of respiration and reduce nitrate to harmful green house gases such as NO and N2O. In this study we examined the distribution and biomass of fungi in the sediments of the seasonal anoxic region off Goa from two stations. The sampling was carried out in five different periods from October 2005, when dissolved oxygen levels were near zero in bottom waters to March 2006. We isolated mycelial fungi, thraustochytrids and yeasts. Species of Aspergillus and thraustochytrids were dominant. Fungi were isolated under aerobic, as well as anaerobic conditions from different seasons. Four isolates were examined for their denitrification activity. Two cultures obtained from the anoxic sediments showed better growth under anaerobic condition than the other two cultures that were isolated from oxic sediments. Our preliminary results suggest that several species of fungi can grow under oxygen deficient conditions and participate in denitrification processes.

Effects and interactions of medium components on laccase from a marine-derived fungus using response surface methodology.

The effects of various synthetic medium components and their interactions with each other ultimately impact laccase production in fungi. This was studied using a laccase-hyper-producing marine-derived basidiomycete, Cerrena unicolor MTCC 5159. Inducible laccases were produced in the idiophase only after addition of an inducer such as CuSO(4). Concentration of carbon and nitrogen acted antagonistically with respect to laccase production. A combination of low nitrogen and high carbon concentration favored both biomass and laccase production. The most favorable combination resulted in 917 U L(-1) of laccase. After sufficient growth had occurred, addition of a surfactant such as Tween 80 positively impacted biomass and increased the laccase activity to around 1,300 U L(-1). Increasing the surface to volume ratio of the culture vessel further increased its activity to almost 2,000 U L(-1).

Treatment of colored effluents with lignin-degrading enzymes: an emerging role of marine-derived fungi.

Some of the industries that discharge highly colored effluents are paper and pulp mills, textiles and dye-making industries, alcohol distilleries, and leather industries. Terrestrial white-rot basidiomycetous fungi and their lignin-degrading enzymes laccase, manganese-peroxidase and lignin peroxidases are useful in the treatment of colored industrial effluents and other xenobiotics. Free mycelia, mycelial pellets, immobilized fungi or their lignin-degrading enzymes from terrestrial fungi have been reported in treatment of several effluents. Marine obligate or facultative (marine-derived) fungi may have unique properties but have not been explored sufficiently for this purpose. This article presents a critical review of bioremediation potential of such fungi and their lignin-degrading enzymes in comparison with the state-of-the-art in terrestrial white-rot fungi.

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)).

Fungi and macroaggregation in deep-sea sediments.

Whereas fungi in terrestrial soils have been well studied, little is known of them in deep-sea sediments. Recent studies have demonstrated the presence of fungal hyphae in such sediments but in low abundance. We present evidence in this study that one of the apparent reasons for the poor detection of fungi in deep-sea sediments is their cryptic presence in macroaggregates. Fungal biomass carbon from different core sections of deep-sea sediments from approximately 5000 m depth in the Central Indian Ocean was estimated based on direct microscopic detection of fungal mycelia. Treatment of sediment samples with ethylenediamine tetra-acetic acid (EDTA) enabled more frequent detection and significantly higher biomass than in samples without such treatment. Treatment with EDTA resulted in detecting various stages of breakdown of aggregates in the sediments, gradually revealing the presence of fungal hyphae within them. Experimental studies of a deep-sea, as well as three terrestrial isolates of fungi, showed that all could grow at 200 bar and 5 degrees C in a nutrient medium and in deep-sea sediment extract. Hyphae of fungi grown in sediment extract under the above conditions showed various stages of accretion of particles around them, leading to the formation of aggregates. Such aggregates showed the presence of humic material, carbohydrate, and proteins. We suggest that fungi in deep-sea sediments may be involved in humic aggregate formation by processes very similar to those in terrestrial sediments. The importance of such a process in carbon sequestration and food web in the deep sea needs to be examined.

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)).

Molecular evidence of fungal signatures in the marine protist Corallochytrium limacisporum and its implications in the evolution of animals and fungi.

Fungi, animals, and single-celled organisms belonging to the choanozoans together constitute the supergroup Opisthokonta. The latter are considered crucial in understanding the evolutionary origin of animals and fungi. The choanozoan Corallochytrium limacisporum is an enigmatic marine protist of considerable interest in opisthokontan evolution. Several isolates of the organism were obtained from a coral reef lagoon in the Lakshadweep group of islands of the Arabian Sea. The capability of these cultures to grow on media containing inorganic nitrogen sources prompted us to examine the possible presence of fungal signatures, namely the enzyme alpha-aminoadipate reductase (alpha-AAR) involved in the alpha-aminoadipate (AAA) pathway for synthesizing lysine and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi, were detected in the organism. Phylogenetic trees based on the alpha-AAR gene suggested that Corallochytrium limacisporum is a sister clade to fungi, while those based on the C-14 reductase gene did not adequately resolve whether the organism was more closely related to fungi or animals. While many studies indicate that Corallochytrium is a sister clade to animals, we suggest that further studies are required to examine whether this protist is in fact more closely related to fungi rather than to animals.

Biological deinking of inkjet-printed paper using Vibrio alginolyticus and its enzymes.

Recycling of office waste paper (photocopy, inkjet, and laser prints) is a major problem due to difficulty in removal of nonimpact ink. Biological deinking of office waste paper is reported using several microorganisms and their enzymes. We report here deinking and decolorization of the dislodged ink particles from inkjet printed paper pulp by a marine bacterium, Vibrio alginolyticus isolate no. NIO/DI/32, obtained from marine sediments. Decolorization of this pulp was achieved within 72 h by growing the bacterium in the pulp of 3-6% consistency suspended in seawater. Immobilized bacterial cells in sodium alginate beads were also able to decolorize this pulp within 72 h. The cell-free culture supernatant of the bacterium grown in nutrient broth was not effective in deinking. However, when the culture was grown in nutrient broth supplemented with starch or Tween 80, the cell-free culture supernatant could effectively deink and decolorize inkjet-printed paper pulp within 72 h at 30 degrees C. The culture supernatant of V. alginolyticus grown in the presence of starch or Tween 80 showed 49 U ml(-1) and 33 U ml(-1) amylase and lipase activities, respectively. Dialysis of these culture supernatants through 10 kDa cut-off membrane resulted in a 35-40% reduction in their efficiency in decolorizing the pulp. It appears that amylase and lipase effectively help in dislodging the ink particles from the inkjet printed-paper pulp. We hypothesize that the bacterium might be inducing the formation of low molecular weight free radicals in the culture medium, which might be responsible for decolorization of the pulp.