Biotechnological Applications of Cultivated and Uncultivated Marine Microorganism - Assignment Example

The life under water is very diverse. Tremendous biotechnological potentials possessed by the diverse microorganisms benefit every field of life. To date, a large number of cultivable and non-cultivable microorganisms find diverse biotechnological applications in medicine and many other fields. The screening and investigative studies have attributed the marine microorganisms for the production of novel bioactive substance, in the aquatic ecosystem (Yves Le Gal et al, 2005). Study of the application of a particular marine microbe is succeeded by a number of identification and characterisation studies. Marine microbes are studied and identified based on identification, enumeration, activity, colony structure. The study of microorganisms is based on the identification techniques. Identification is essential for the classification of microorganisms. The various identifications methods for the cultivated microorganisms include morphological identification, differential staining, differential media, serological methods and flow cytometry (Abedon, 1998). Identification of uncultivated microorganisms is based on the protein analysis and the comparison of the nucleotide sequences. Enumeration is a methodology used to estimate the number of different microorganism in a given sample. It is either achieved through the direct enumeration technique and other modern filter techniques. Direct enumeration method utilises microscope to count the number of microbes in a given sample (O’Leary, 1989). There are many other methods for enumeration, including minimum dilution, MPN method, most probable number methods, and plate count methods using agar media or silicic acid gels. The selection of any of the method is based on the specie and nature of the microorganism being studied. For instance, the MPN method is used for the enumeration of the hydrocarbon degrading bacteria in marine environment (Sato Higashihara et al, 1978) In the marine environment, various microorganisms are related to diverse activities. For instance, biosurfactant producing marine bacteria can be studied through haemolytic assay (HA), modified drop collapse (MDC), tilted glass slide, oil spread method (OSM), blue agar plate(BAP), emulsification index(EI) and emulsification assays (S K Satpute, 2008). Munn (2004) showed that the study of the community structure and the allocation of function to different groups of microorganisms could be achieved through microelectrodes and biosensor methods. Microelectrodes are sensitive to change in pH, oxygen, carbon dioxide, hydrogen, and hydrogen sulphide. Any change in the concentrations is detected by the microelectrodes. The environmental change helps to infer the type and function of microorganism based on the change in composition of sample. Every living cell needs to synthesise energy to carry out cell functions. They store energy in form of ATP molecules. The ATP synthesis and successful growth of microorganisms requires water, mineral elements, nitrogen, phosphorus, growth factors, and gases such oxygen, carbon dioxide etc. Different growth factors such as vitamins, amino acids, purines, and pyrimidines are essential for the microbial growth. Carbon, nitrogen, and phosphorus are essential components of proteins, carbohydrates, DNA and RNA (CliffNotes, n.d.). Munn (2004) argues that microorganisms have been classified based on carbon source as well as the energy source for ATP conservation. One the basis of carbon source the microorganisms are classified into autotroph and heterotroph. In autotroph, the sole source of carbon is carbon dioxide whereas heterotrophs extract carbon from other organic compounds. Classification based n the energy source classifies microorganisms into two major categories. Microorganisms that utilise light as a source of ATP synthesis are called phototrophs whereas some microorganisms that generate ATP by the oxidation of organic and inorganic compounds are called chemotrophs. Chemotrophs are further classified into two main categories. Chemotroph that solely depends upon inorganic compounds for ATP generation are called chemolithotroph whereas the chemotrophs that can synthesise ATP only from organic compounds are called chemo-organotroph. The ATP generation is carried out through redox reactions. Redox reactions or reduction and oxidation reactions are important for the conversion of chemical energy into biological energy carrier ATP. This reaction involves a series of electron acceptors and donors. The mechanism is based on the transport of electron from the donor to the electron acceptor. Organic and inorganic compounds are the electron donors while oxygen and other charged compounds are the electron acceptor for carrying out energy conservation through the redox reaction. The energy released as a result, of this electron transfer is utilised for various cell functions. The process of energy conservation or ATP generation is carried out through the biochemical pathway. The biochemical pathway is classified into three major categories based on electron acceptor. The redox reaction involved in the fermentation does not involve any added electron acceptor. The aerobic pathway utilises oxygen as an electron acceptor whereas the last type, anaerobic pathway utilises any non-oxygen electron acceptor (George, 2000). According to Madigan (2000), microorganism having fermentative biochemical pathway synthesise ATP by substrate level phosphorylation. It transforms the high-energy phosphate bond to ADP from any of the ten high-energy phosphorylated intermediates with the help of some other metabolic products as electron acceptors. Carbon and Electron Flow in Fermentation1 Source: Computational subsurface hydrology: reactions, transport, and fate by Yeh The respiratory mechanism in microorganisms is carried out through a sequential transfer of electrons through a series of membrane bound proteins. This transfer mechanism is called electron transport chain. The electrons are transferred to reducing terminal electron acceptor, through the membrane carriers (Terry, 2004). Gale (2005) explains the electron transport system and shows that phosphorylation pumps hydrogen ions to enzymes that bring about synthesis of ATP. The enzyme utilises the energy of ions to make ATP. The system works by transferring the hydrogen ions from the gel like cytoplasm to NADH. With the gain of proton or H ion, NADH shuttles an electron and is converted into NAD. This released electron is accepted by the flavoproteins and cytochromes in the membrane. The release of hydrogen outside the cell results in the increase in hydroxyl ion concentration in interior of cell. The sequential transfer of electrons ends up at a protein ATP synthase. This enzyme takes in hydrogen ion to manage equilibrium which was previously disturbed. The energy released by the downhill movement of hydrogen ions result in the addition of a phosphate bond to ADP. Source: http://www.microbialfuelcell.org/MFC/images/etf.jpg Microorganisms use various reduction reactions to use the electrons, generated by the oxidation of the energy yielding substrate. The electron receptors of the electron transport chain are different for aerobic and anaerobic respiration. In aerobic respiratory pathway, oxygen acts as the terminal electron acceptor and yield water. O2 + 4H+ + 4e- ------- 2H2O Under anoxic conditions other electron acceptors such as NO3-, SO42-, CO2 are utilised. It reduces NO3- to NH2OH and then finally into ammonia. This type of anaerobic respiration is carried out in denitrifying bacteria. Source: Computational subsurface hydrology: reactions, transport, and fate by Yeh2 Similarly, the electron transport mechanism utilising SO42- as an electron acceptor yields four molecules of water and reduces sulphate into hydrogen sulphide. The process is called sulphate reduction. On the other hand, when carbon dioxide is used as an electron acceptor, the final product is always methane. This process is found in methanogenic bacteria and the process is called methane fermentation. All such processes result in the generation of energy. The amount of energy, released as a result of oxidation and reduction reactions depends upon the nature of different electron acceptors. It varies directly with the difference in the reduction potential between the electron acceptors and the donors such that greater the difference, greater will be the energy yielded. For a particular substrate or donor, energy yield will be greater for the acceptor having higher reduction potential. Moreover this energy can be mathematically calculated by the formula ΔG = - n x F x Eemf    Where n is the number of electrons, F is the Faraday’s constant. Its value is 96485 C/mol and Eemf   or ΔV is the equilibrium cell potential. The ecological niche is the place where specie lives (Sharov, 1997). According to Gray (2001), microorganisms are divided into diverse ecological niches depending upon the gradients of light, redox, temperature, and salinity. It is also characterised based on mechanism of respiration through different donors. The culture independent analysis including 16s rRNA and fluorescence in situ hybridisation reveals the microbial activity of aerobic, anoxic niches at different concentration gradients. The microorganisms have the ability to adapt with the changing salt concentration. The microorganism such as eubacteria undergoes haloadaption by producing certain compatible products. It is the property of every cell to maintain the inner environment with respect to the external environment. A microorganism adapts to high salt concentration by through strategies. The first process is the accumulation of the molar concentrations of potassium chloride. It modifies the enzymatic machinery to the high concentration of the ions, as it is essential for protein enzymes to maintain their conformation. The second concept related to adaptation is the accumulation of organic solutes including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The microorganisms that have undergone such adaptation include halophilic methanogenic Archaea, Halanaerobiales (Firmicutes), halophilic Salinibacter (Bacteroidetes) (Gray, 2001). Costantino Vetriani et al. (2005) argues that the microorganism adapt to high temperature by producing certain changes in the membrane lipid composition, enhancing thermostabilities of the (membrane) proteins and increasing turnover rates of the energy transducing enzymes. At higher temperature, the cytoplasmic membrane becomes more permeable to the transport of proton. This change limits the growth of microorganism. The thermophiles adapt to the change by using the less permeable sodium ions. Microorganisms such as bacteria can adapt to the low temperature. The microorganisms respond to the hypothermic conditions by synthesising certain cold shock proteins. They are able to grow at low temperatures, even below the freezing temperature. They maintain the growth by managing certain parameters. Hebraud and Potier (1999) argue that one of the most requisite for the growth is the membrane fluidity. It copes with the low temperature by increasing, unsaturated fatty-acyl content in the membrane, cis double bonds and by undergoing methyl branching. The target is accomplished through enzyme activation responsible for pre-existed lipid modification and de novo synthesis of certain enzymes or by inserting des gene. This gene is responsible for producing membrane phospholipid desasturase. Moreover, microbial cell produces Casp-A proteins. It is the immediate response produced due to a decrease in temperature. It is constantly synthesised except the lag phase of microbial growth. Sea ice is a remarkable feature of polar seas. There are microbes that occupy the sea ice. The microbes inhabiting it belong to the microbial sea ice community. The microorganisms of sea ice community contribute to the food webs. They are responsible for decreasing the light in sea ice and thus change the spectral quality of the light reaching the base of sea ice. It is also involved in the increased absorption of heat and influences the physical structure and break up of sea ice. They are classified into two major types. The land-fast ice community are found along the coastal lines whereas the rest is occupied by the pack ice community. The former is dominated by the diatoms and the later is dominated by diatoms as well as some autotrophic and phototrophic protists (Stoecker, Kurt & Putt, 1993). According to CRRC & NOAA (2007) report, the microbial sea ice community contributes to the food web by recycling the nutrients present in its semi-conserved environment. The microbial sea ice community consist of heterotrophic bacteria, unicellular algae, and protists. The micro algae and bacteria at the sea ice surface photosynthesise, and contribute to the biomass. These algae are grazed by the zooplanktons. These zooplanktons reach the underside of the sea ice interface after being eaten up by the deep-water fish. Later on, it becomes the source for the heterotrophic bacteria and the cycle continues. When we talk about the properties of microorganisms, pathogencity is the most important feature. There are many pathogens associated with the marine environment. The main pathogens of the marine environment include acinetobacter colcoaceticus, Aeromonas hydrophila , A. Caviae, A.sobria, Brucella maris, Edwardsiella tarda, Enterobacter, Erysipelothris, Francisella, Legionella pneumophilla, , Legionella bozemanii, Halomonas , Klebsiella, Lactococcus, Shigella, Staphylococcus, Serratia, Salmonella, Streptococcus and many more (Thompson, Luisa & Polz, 2006). Apart from the harmful pathogenic effects, the marine microbes are involved in many beneficial activities. They help in monitoring the faecal contamination in the costal and marine areas. The most common methods are the most probable number and membrane filtration. These methods are simple but very time consuming. The latest methods include various enzyme assays. The enzyme assays for 4-methylumbelliferyl-β-D-glactopyranosidase and 4-methylumbelliferyl-β-D- glucuronidase is the fastest process for the fecal contaminant detection. The results of the assays depend on the sensitivity limit corresponded to the bacterial concentration (Liv Fiksdal, 1994). The quantification of the fecal pollution is carried out either by the enumeration of the colony forming units or through quantitative polymerase chain reaction. The qPCR utilises genus and specie based primer sets. These are based on the indicator microorganism used during the study (Clayton R. Morrison et al, 2008). Microorganisms are very important in the sewage treatment. The sewage treatment is done through the activated sludge process. The process consists of the primary, secondary, and tertiary steps. The primary step is the simple sedimentation of organic matter. In this process the sewage is subjected to forced aeration for eight hours Secondary treatment is the biological treatment involving microorganisms. As a result, the fungi and bacteria present in the sewage multiply and oxidise the organic matter. The oxidation products are carbon dioxide and water. Tertiary treatment is carried out to get the highest quality of effluent. It is carried out through sand filters, mechanical filtration or by passing the effluent through a constructed wetland such as a reed bed or grass plot. Diagram: Sewage Treatment Source: EUWFD The discharge of poorly treated effluents into the river, lakes, and other water resources pose serious dangers to the wildlife as well as human beings. The contaminants may contain estrogens. These xenoestrogens impair the endocrine functioning, structure, and function of the reproductive system in wildlife. The poorly treated water may contain viruses that cause infectious hepatitis, polio, and gastroenteritus (Hurst 1991). The cultivable as well as uncultivable marine microorganisms can significantly act the indicators of pollution. According to McLemore and Keeler (1995), rapid bioassesment protocols utilises a number of biological indicators to study the changes in the communities of certain microorganisms. The indicators are the biomarkers that indicate the change through adaptive biochemical and immunological responses, response of stress proteins and change in DNA due to exposure to the pollutants. The bioindicators of quality of aquatic sources include algae and micro-invertebrates. The biological indicators give the more refined knowledge of quality of water. Microbes are used as biosensors. Biosensor can be defined as biological entity that reacts with the target and produces readily quantifiable signals. The traditional biosensors work on basis of specificity of enzyme to substrates, antibodies to antigens or that of nucleic acids to their complementary sequences. The latest biosensors utilises the genetically engineered cells that respond to the predetermined group of chemicals. The biosensors are constructed to check toxicity caused by one or more pollutants by, measuring light, fluorescence, colour, and electric current. The electrochemical biosensors can be employed for measuring general toxicity as well as hydrocarbons and heavy metals through the in situ measurement protocols (Paitan et al, 2003). There are a large number of hydrocarbon degrading microorganisms, which play a significant role in biological removal of oil hydrocarbons. According to Yakimov et al. (2007), the release of oil into seawater results in a bloom of indigenous marine genera- Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and others. This bloom causes the degradation of oil constituents. They have a modified hydrocarbon utilisation metabolic route which helps them to mitigate oil pollution. In this regard, the most active role is played by the obligate hydrocarbonoclastic bacteria (OHCB). There are many approaches used to investigate the strain of microorganism responsible for oil degradation. A specie –specific PCR technique, with the help of 16s rRNA based primers, helps to investigate the oil degrading activity of strains like cyanobacterium. The genomic sequencing of the cultivated and uncultivated microorganism provides remarkable information about the action of a particular strain. The marine environment presents resource for novel products and processes across a very wide range of sectors. For instance anti-viral compounds, TAQ polymerase enzyme for DNA sequencing and coral-derived materials are used as bone replacements (BIS, n.d.).  According to NRC (2002), the marine natural products are used as potential pharmaceuticals, biopolymers, and biocatalysts. Various efforts are still being made for the discovery of the new and improved drug therapies, through marine microorganisms. Many useful products were discovered at the high latitudes an under the Antarctic sea. The molecular approaches such as isolation cloning, heterogonous expression of genes have played a remarkable role in the identification of the cultivated microbes. The discovery of the uncultivated microbes entirely depends upon the gene sequencing and biochemical pathway identification. A broad range of biological assays has been designed for such biologically active producers. Metagenomics involve the construction of the gene libraries followed by the marine DNA recovery and sequence analysis. The approach is important for the discovery of novel genes and their regulated pathways. It enables biotechnologists to generate datasets for the marine ecosystem (Osborn & Smith, 2005). The metagenomic approach has been aided with the latest techniques for the identification of uncultivated microorganisms. Friedrich (2006) explains that DNA sequence is identified with the help of stable-isotopic probing of the DNA. It is then followed by the fractional centrifugation to identify the active community. In short, Marine biotechnology is such an advanced field of biological science that benefits every field including health, environment, and agriculture. The rich physiological and genetic diversity in the marine ecosystem is the strength of all the biotechnological applications. The wide scope of biotechnology has stirred up the scientists to use the microscopic world for the benefit of the macroscopic world. References 1. Abedon. T. S. (1998) Identification of Bacteria. 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