Natural antifouling compound production by microbes associated with marine macroorganisms — A review

In the marine environment, all hard surfaces including marine macroorganims are colonized by microorganisms mainly from the surrounding environment. The microorganisms associated with marine macroorganisms offer tremendous potential for exploitation of bioactive metabolites. Biofouling is a continuous problem in marine sectors which needs huge economy for control and cleaning processes. Biotechnological way for searching natural product antifouling compounds gained momentum in recent years because of the environmental pollution associated with the use of toxic chemicals to control biofouling. While, natural product based antifoulants from marine organisms particularly sponges and corals attained significance due to their activities in field assays, collection of larger amount of organisms from the sea is not a viable one. The microorganisms associated with sponges, corals, ascidians, seaweeds and seagrasses showed strong antimicrobial and also antifouling activities. This review highlights the advances in natural product antifoulants research from microbes associated with marine organisms.

Molecular cloning of alkaline protease gene from marine fungus Engyodontium album

Department of Biotechnology, Cochin University of Science and Technology

A marine actinomycete nocardiopsis mccb 110 as source of novel drugs to manage vibriosis

Aquaculture is a form of agriculture that involves the propagation, cultivation and marketing of aquatic plants and animals in a controlled environment (Swann, 1992). After growing steadily, particularly in the last four decades, aquaculture is for the first time set to contribute half of the fish consumed by the human population worldwide. Given the projected population growth over the next two decades, it is estimated that at least an additional 40 million tonnes of aquatic food will be required by 2030 to maintain the current per capita consumption (FAO, 2006). Capture fisheries and aquaculture supplied the world with about 110 million tonnes of food fish in 2006. Of this total, aquaculture accounted for 47 percent (FAO, 2009). Globally, penaeid shrimp culture ranks sixth in terms of quantity and second in terms of value amongst all taxonomic groups of aquatic animals cultivated (FAO, 2006). In places where warm-water aquaculture was possible black tiger shrimp, Penaeus monodon became the preferred variety of shrimp cultivar owing to its fast growth, seed availability and importantly due to high prices it fetches (Pechmanee, 1997). World shrimp production is dominated by P.monodon, which accounted for more than 50 % of the production in 1999 (FAO, 2000). In the last few years the whiteleg shrimp, Litopenaeus vannamei, has replaced P.monodon in many countries. Indian shrimp culture is dominated by P.monodon with the East Coast accounting for 70% of the production (Hein, 2002). Intensive culture, apart from other problems, results in enhanced susceptibility of the cultured species to diseases (Jory, 1997), which in fact have become the biggest constraint in shrimp aquaculture (FAO, 2003).

Atmospheric deposition of methanol over the Atlantic Ocean

In the troposphere, methanol (CH3OH) is present ubiquitously and second in abundance among organic gases after methane. In the surface ocean, methanol represents a supply of energy and carbon for marine microbes. Here we report direct measurements of air-sea methanol transfer along a similar to 10,000-km north-south transect of the Atlantic. The flux of methanol was consistently from the atmosphere to the ocean. Constrained by the aerodynamic limit and measured rate of air-sea sensible heat exchange, methanol transfer resembles a one-way depositional process, which suggests dissolved methanol concentrations near the water surface that are lower than what were measured at similar to 5 m depth, for reasons currently unknown. We estimate the global oceanic uptake of methanol and examine the lifetimes of this compound in the lower atmosphere and upper ocean with respect to gas exchange. We also constrain the molecular diffusional resistance above the ocean surface-an important term for improving air-sea gas exchange models.

Microbial acetone oxidation in coastal seawater

Acetone is an important oxygenated volatile organic compound (OVOC) in the troposphere where it influences the oxidizing capacity of the atmosphere. However, the air-sea flux is not well quantified, in part due to a lack of knowledge regarding which processes control oceanic concentrations, and, specifically whether microbial oxidation to CO2 represents a significant loss process. We demonstrate that 14C labeled acetone can be used to determine microbial oxidation to 14CO2. Linear microbial rates of acetone oxidation to CO2 were observed for between 0.75-3.5 h at a seasonally eutrophic coastal station located in the western English Channel (L4). A kinetic experiment in summer at station L4 gave a Vmax of 4.1 pmol L-1 h-1, with a Km constant of 54 pM. We then used this technique to obtain microbial acetone loss rates ranging between 1.2 and 42 pmol L-1 h-1.(monthly averages) over an annual cycle at L4, with maximum rates observed during winter months. The biological turnover time of acetone (in situ concentration divided by microbial oxidation rate) in surface waters varied from ~3 days in February 2011, when in situ concentrations were 3 ± 1 nM, to >240 days in June 2011, when concentrations were more than twofold higher at 7.5 ± 0.7 nM. These relatively low marine microbial acetone oxidation rates, when normalized to in situ concentrations, suggest that marine microbes preferentially utilize other OVOCs such as methanol and acetaldehyde.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

In this thesis, the production and characterization of ligninolytic enzymes using the fungi isolated from mangrove area are studied. The objective of the present work are isolation and screening of dye decolorizing micro-organisms from mangrove area, screening of the selected microorganisms for the production of lignin degrading enzymes, identification of the potent micro-organisms, characterization of the crude enzyme, lignin peroxidase, of the selected fungi—Aspergillus sp. SIP 11 and Penicillium sp. SIP 10 etc. This included the determination of the optimum pH, temperature, veratryl alcohol and H2O2 concentration. Besides the stability of crude LiP at different pHs and temperatures were studied. The immense applications, particularly in bioremediation, to which the lignin degrading micro-organisms could be used make this study important, the ascomycetes and deuteromycetes fungi, especially form the marine environment were studied with respect to their ligninolytic enzyme system making this study an initial step in unraveling the vast hidden potential of these microbes in bioremediation, the marine microbes are halophilic in nature which make them better suited to cope with the high salinity of industrial effluents thereby giving them added advantage in the filed of bioremediation. The thesis deals with the isolation and screening of lignin degrading enzyme-producing microbes from mangrove area. The identification of the most potent fungal isolates and characterization of LiP from these are also done.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

The continually growing worldwide hazardous waste problem is receiving much attention lately. The development of cost effective, yet efficient methods of decontamination are vital to our success in solving this problem.Bioremediation using white rot fungi, a group of basidiomycetes characterized by their ability to degrade lignin by producing extracellular LiP, MnP and laccase have come to be recognized globally which is described in detail in Chapter 1.These features provide them with tremendous advantages over other micro-organisms.Chapter 2 deals with the isolation and screening of lignin degrading enzyme producing micoro-organisms from mangrove area. Marine microbes of mangrove area has great capacity to tolerate wide fluctuations of salinitie.Primary and secondary screening for lignin degrading enzyme producing halophilic microbes from mangrove area resulted in the selection of two fungal strains from among 75 bacteria and 26 fungi. The two fungi, SIP 10 and SIP ll, were identified as penicillium sp and Aspergillus sp respectively belonging to the class Ascomycetes .Specific activity of the purified LiP was 7923 U/mg protein. The purification fold was 24.07 while the yield was 18.7%. SDS PAGE of LiP showed that it was a low molecular weight protein of 29 kDa.Zymogram analysis using crystal violet dye as substrate confirmed the peroxidase nature of the purified LiP.The studies on the ability of purified LiP to decolorize different synthetic dyes was done. Among the dyes studied, crystal violet, a triphenyl methane dye was decolorized to the greatest extent.

Seawater carbonate chemistry, biomass and metabolic rates (leucine incorporation, CO2 fixation and respiration) of Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217) in a laboratory experiment

Experimental results related to the effects of ocean acidification on planktonic marine microbes are still rather inconsistent and occasionally contradictory. Moreover, laboratory or field experiments that address the effects of changes in CO2 concentrations on heterotrophic microbes are very scarce, despite the major role of these organisms in the marine carbon cycle. We tested the direct effect of an elevated CO2 concentration (1000 ppmv) on the biomass and metabolic rates (leucine incorporation, CO2 fixation and respiration) of 2 isolates belonging to 2 relevant marine bacterial families, Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217). Our results demonstrate that, contrary to some expectations, high pCO2 did not negatively affect bacterial growth but increased growth efficiency in the case of MED217. The elevated partial pressure of CO2 (pCO2) caused, in both cases, higher rates of CO2 fixation in the dissolved fraction and, in the case of MED217, lower respiration rates. Both responses would tend to increase the pH of seawater acting as a negative feedback between elevated atmospheric CO2 concentrations and ocean acidification.

Biomineralization of manganese by Bacillus spp isolated from a marine biofilm

Biomineralization of manganese on titanium condenser material exposed to seawater has been illustrated. Biomineralization occurs when the fouling components, namely, the microbes, are able to oxidize minerals present in water and deposit them as insoluble oxides on biofilm surfaces. Extensive biofilm characterization studies Showed that an alarmingly large number of bacteria in these biofilms are capable of oxidizing manganese and are, thereby, capable of causing biomineralization on the condenser material exposed to seawater. This paper addresses studies on understanding the exact role of the microbes in bringing about oxidation of manganese. The kinetics of manganese oxidation by marine Gram-positive manganese oxidizing bacterium Bacillus spp. that was isolated front the titanium surface was studied in detail. Manganese oxidation in the presence of Bacillus cells, by cell free extract (CFE) and heat-treated cell free extract was also studied. The study confirmed that bacteria mediate manganese oxidation and lead to the formation of biogenic oxides of MnO2 eventually leading to biomineralization on titanium surface exposed to seawater.

Exploiting the diverse microbial ecology of marine sponges

Marine sponges (phylum Porifera) are the oldest extant metazoan animals on earth and host large populations of symbiotic microbes: Bacteria, Archaea and unicellular Eukaryota. Those microbes play ecological functions which are essential to the health of the host including carbon, nitrogen and sulfur cycling as well as host defence through the production of bioactive secondary metabolites which protect against infection and predation. The diversity of sponge-associated microbes is remarkable with thousands of OTUs reported from individual sponge species. Amongst those populations are sponge-specific microbes which may be specific to sponges or specific to sponge species. While marine natural product discovery concerns many animal phyla, Porifera account for the largest proportion of novel compounds. Evidence suggests that many of these compounds are the products of symbiotic microbes. Descriptions of sponge-associated microbial community structures have been advanced by the development of next-generation sequencing technologies while the discovery and exploitation of sponge derived bioactive compounds has increased due to developments in sequence-based and function-based metagenomics. Here, we use pyrosequencing to describe the bacterial communities associated with two shallow, temperate water sponges (Raspailia ramosa and Stelligera stuposa) from Irish coastal waters and to describe the bacterial and archaeal communities of a single sponge species (Inflatella pellicula) from two different depths in deep waters in the Atlantic Ocean, including at a depth of 2900m, a depth far greater than that of any previous sequence-based sponge-microbe investigation. We identified diverse microbial communities in all sponges and the presence of sponge-specific taxa recruiting to previously described and novel spongespecific clusters. We also identified archaeal communities which dominated sponge-microbe communities. We demonstrate that sponge-associated microbial communities differ from seawater communities indicating host selection processes. We used sequence-based metagenomic techniques to identify genes of potential industrial and pharmacological interest in the metagenomes of various sponge species and functionbased metagenomic screening in an attempt to identify lipolytic and antibacterial activities from metagenomic clones from the metagenome of the marine sponge Stelletta normani. In addition we have cultured diverse bacterial species from sponge tissues, many of which display antimicrobial activities against clinically relevant bacterial and yeast test strains. Other isolates represent novel species in the genus Maribacter and require emendments to the description of that genus.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.