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Phototropic bacteria is a group of bacteria that carry out their own photosynthesis by deriving energy from sunlight in order to produce phosphate bond energy (ATP) and reductants [e.g., NAD(P)Hand reduced ferredoxin] through photosynthetic electron transport. Anoxygenic and oxygenic phototrophs have different electron transport system. Oxygenic phototrophic bacteria (cyanobacteria) have similar electron transport system like plants, algae and uses water as electron donor while in anoxygenic (non-oxygen evolving) the photosynthetic electron transport pathways are different which contain either a type II (quinine type) or a type I (Fe-S type) reaction centre (RC) and uses reduced sulphur compounds, elemental sulphur, or elemental hydrogen for electron donor. Anoxygenic phototrophic bacterial group includes anaerobic anoxygenic phototrophs (AnAPs), aerobic anoxygenic phototrophic (AAPs), green sulphur bacteria (GSBs), filamentous anoxygenic phototrophs (FAPs; or green non-sulfur / gliding bacteria). AnAPs, AAPs, GSBs, FAPs; or green non-sulfur / gliding bacteria contain a type II RC, and green Sulfur bacteria, heliobacteria have a type I RC. (Tang et al, 2011).
These Anoxygenic phototrophic bacteria found in almost all types of habitats for instance aquatic and marine habitats, lake, lagoons, oceans, marine sediments, shallow water of costal line, freshwater bodies, intertidal sand flats, hypersaline ponds, microbial mats, lake water and almost all the extreme environments like soda lakes, alkaline as well as acidic hot springs with or without high sulfur content, yellow stone in US, solar lakes, marine sponges, dead sea and so on. Hence these bacteria are thermophiles, halophiles, akaliphiles, haloalkaliphiles, acidophiles, and psychrophiles as well (Imhoff, 2001; Madigan, 2003; YaÅŸa, 2006; Hubas et al, 2011; Hirose et al, 2012).
The culturing of anoxygenic phototrophic bacteria in laboratory conditions is tedious and extensively laborious and usually takes six months to a year to culture and to get a single purified colony. It's a slow grower with a bit of stringent conditions like reduced compounds, anaerobic conditions, pH, temperature, saline conditions, plays a very crucial role for the growth and culture of anoxygenic phototrophic bacteria. Due to these reasons, the diversity of phototrophic bacteria is still not yet fully explored (Madigan and Jung, 2008).
Photosynthetic system of anoxygenic phototrophic bacteria consists of bacteriocholophylls and carotenoids. These bacteriochlorophylls are the primarily light harvesting system which differed chemically from chlorophyll a in their substituted side chain and absorbs at 800-1000 nm in the far red region of light spectrum while carotenoids play crucially important physiological and structural roles in phototrophic bacteria. They are accessory pigments or secondary light harvesting pigments which help in transferring of light (400-600 nm) to the reaction centre almost with 100% efficiency and simultaneously, protect the photosynthetic reaction centre from reactive oxygen species by quenching chlorophyll triplet states and dissipating the energy as heat (YaÅŸa, 2006; Zhu et al, 2010).
Carotenoids are principally C-40 isoprenoid pigments having nine or more conjugated double bonds can inactivate certain reactive oxygen species (Chandi and Gill, 2011). Carotenoids consist of polyisoprenoid structure, a long conjugated chain of double bond and a near bilateral symmetry around the central double bond, as common chemical features (Britton, 1995).The absorbance of light by carotenoids depend upon its number of double conjugated bonds and functional group, hence imparts colours to the bacterial colony ranging from light yellow to orange-red (Zhu et al, 2010; Vachali et al, 2012). Due to its anti-oxidant (anti-radical) activity, are highly used in pharmaceutical industries for the production of drugs. It is also taken as supplement in order to increase the immunity. Experimental data has already suggested that oxygen radicals play a very crucial role in onset of many diseases. Lung cancer, age related problems, arteriosclerosis, cataracts, age-related macular degeneration, multiple sclerosis, and, perhaps most importantly, cancer , Parkinson disease, and cardiovascular disease, for skin related problems, eye diseases has already been proved that carotenoids can play a significant role in reducing infection by quenching radicals only if taken at threshold level (Carotenoids and human health). Studies has also shown that, apart from its antioxidant activity, carotenoids may mediate their effects via mechanisms such as gap junction communication, cell growth regulation, modulating gene expression, immune response and as modulators of Phase I and II drug metabolizing enzymes (Paiva and Russell 1999; Ong and Tee, 1992; Jewell and O'Brien, 1999; Bertram, 1999)
Table 1: Structures and commercial utility of important carotenoids produced by microorganisms
Against cardiovascular disease, prostate cancer, and cosmetic preparations
Anti-cancer agent, , food colorants, photo protectant, vitamin A source and cosmetic preparations
Feed additive mainly in aqua culture and as food colorant
Prevention of age related Muscular degeneration (AMD). Cosmetic preparation
Prevention of age related Muscular degeneration (AMD). Cosmetic preparation
Despite the availability of a variety of natural and synthetic carotenoids, there is currently renewed interest in microbial sources (Nelis and DeLeenheer, 1991; Johnson and Schroeder, 1996; Ausich, 1997; Lee and Schmidt- Dannert, 2002). As a part of their response to various environmental stresses microorganisms tend to accumulate several types of carotenoids and thereby protect themselves from radicals and play an important role in photo protection. Several types of microbes, such as algae, fungi, and bacteria, have been reported to produce carotenoids; but only a few of them have been exploited commercially (Liaaen-Jensen and Andrewes 1972; Nelis and DeLeenheer 1991; Johnson and Schroeder 1996). It is interesting to know that a variety of organisms produce carotenoids which can counteract the effect of harmful food additives. Hence the project is to isolate, purify and structural determination of carotenoids from the anoxygenic phototrophic bacteria and subsequently studying its anti-oxidant activity through in-vitro assays.
Origin of the proposal
The diversity of phototrophic bacteria has not been yet explored fully and still taxa of this group are yet to be identified. The major problem while dealing with phototrophs as culture based method is determining the exact conditions for its survival like the electron donor source, maintaining the anaerobic conditions, the light intensity, temperature, pH, saline conditions. If all the conditions given for its growth are more or less optimum, even then the initial growth from the actual sample like soil, sediment, water bodies etc may take a month or so and later, streaking on agar media plate. Once, active culture is acquired on the media plate after optimising all conditions like temperature, pH, salt concentration, carbon source requirement, reduced sulfur compound etc the purification might take another one- two months. Due to so much of extensive and laborious work the diversity of these phototrophs has not yet been fully explored.
The potential of these phototrophic bacteria probably as the carotenoids source for its anti-oxidant activity, food additives, food colorant, in cosmetic industry is highly anticipated. Epidemiological and experimental data has already shown that most of the onset of the diseases like cancer is due to the free radicals present inside the body which initiates the chemical chain reaction which lead to death or damage to the cell. Anti-oxidant molecules like carotenoids present in the vegetables are the source to combat these radicals. Anti-oxidant molecule basically terminates the chain reaction by quenching the free radicals intermediates, and inhibits the other oxidation reactions. Human body could not synthesise on its own hence rely on the daily basis of its consumption of vegetables or fruits. But, present life style of human beings, pollution, deforestation, industrialization all has led to the deterioration of health and increase in reactive species or free radicals inside the body which on later stage of life result into the onset of diseases like cancer, cardiovascular diseases, Parkinson and so on.
The extensively studied and known carotenoids which is already being consumed by the human population and taken as supplement as well is β-carotene, lycopene, zeaxanthin , astaxanthin . Our interest is to study carotenoids extracted from the phototrophic bacteria. Hence the objectives are:-
To obtain pure culture of purple sulfur and green sulfur bacteria (Anoxygenic Phototrophic Bacteria).
16S rRNA gene sequencing of phototrophic bacteria for its identification and the absorption spectra of whole cells for its carotenoids determination.
Purification and structural determination of the carotenoids extracted from anoxygenic phototrophic bacteria.
In-vitro assays of the carotenoids for its anti-oxidant activity.
Phototrophic bacteria required specific conditions for its growth and survival like anaerobic/aerobic conditions, electron source, temperature, pH, light intensity, saline conditions as well as carbon source. Standardisation of optimum conditions for each above mentioned factor has to be carried out. Once the growth observed in broth, purification of culture is done by 0.8% (w/v) agar dilution and then subsequently on its respective media agar plates maintaining all the standardised conditions.16SrRNA sequencing is done with the help of universal primers 27F and 1492R to identify the phototrophic bacteria in the Eztaxon database.
Extraction of carotenoids is done in 100% acetone keeping in the dim light, at low temperature and N2 purging is done to avoid the oxidation of carotenoids. Then the sample will be run in HPLC with C18/C30 analytical column to isolate as well as to see its absorption spectra of the carotenoids. Pure fractions of carotenoids can further identified with the help of LC-MS and NMR.
Carotenoids anti-oxidant activity
The sample (soil, water or sediment) is usually collected which has pungent smell (due to presence of reduced sulphur compounds) or green/red/pink etc coloured sample (as pigments impart the colour to the bacteria) are likely to found anoxygenic phototrophs. These samples are inoculated in PSB (enrichment broth for Purple Sulfur bacteria) or GSB (enrichment broth for Green Sulfur bacteria) broth.
The composition of PSB broth is NaCl, 2% (w/v) for marine sample/ 0.4% (w/v) freshwater sample; KH2PO4 , 0.5 gL-1 ; CaCl2,0.25 gL-1; NH4Cl, 0.340 gL-1.; MgSO4.7H20, 1.0 gL-1; Na2S2O3, 0.74 gL-1; Ferric citrate(0.1% w/v), 5ml; Yeast extract,0.4 gL-1; Pyruvate,3 gL-1; adjust pH - 7 to 7.5 with the help of NaHCO3 (10% w/v) after autoclaving.
The composition of GSB broth is NaCl, 2% (w/v) for marine sample/ 0.4% (w/v) freshwater sample; KH2PO4 , 0.5 gL-1 ; CaCl2,0.15 gL-1; NH4Cl, 0.680 gL-1.; MgSO4.7H20, 3.0 gL-1; Na2S2O3, 0.74 gL-1; Na2S (1M), 3ml; B12 (2mg/100ml), 1ml; adjust pH - 7 to 7.5 with the help of NaHCO3 (10% w/v) after autoclaving.
The collected samples will be inoculated in GSB/PSB media respectively pH-7 to 7.5 in a screw capped tubes (10-100 mm). 0.8% agar dilution will be carried out in their respective media as soon as the colour growth observed (. Isolated colony in 0.8% agar tube was later on streaked on their respective agar media plates. Isolated colonies on agar tubes will be streaked on plates which may show growth within one- two weeks. Further, these were re-streaked until single and pure colony will be observed on plate. It usually takes six months to one year for sampling, enrichment, isolation and finally getting the pure culture. These isolated phototrophic bacteria will be examined under microscope for their cellular characteristics and then identified with the help of 16S rRNA gene sequencing and pure culture will be stored in 10% glycerol (v/v) in -70oC. The universal primers were used 27F and 1492R to get the 1400-1450 bp result. The identification of genera can be done with the help of the database of EZtaxon site.
For the next objective, the absorption spectra will be taken for whole cell culture in spectrophotometer within the range of 200-1000nm wavelength that will give the idea of the presence of bacteriochlorophylls and carotenoids. As these pigments have specific absorption for the given wavelength.
To confirm the presence of carotenoids, the cell culture is extracted in 100% acetone at least 2-3 times keeping the sample at low temperature (in ice), in dim light procedure and adding 0.01% BHT to avoid the oxidation of carotenoids during its extraction procedure. Then the sample will be run in HPLC with C18/C30 analytical column and mobile phase can be isocratic with 100% methanol or gradient with different percent concentrations of methanol, water, chloroform, acetone, diethyl ether (enzymology).
Once the carotenoids get purified with the help of HPLC, the collected sample can be used for the determination of mass and structural determination by LC-MS and NMR respectively.
In-vitro assays like ORAC, TRAP for anti-oxidation studies can be done which are kit based methods.
The samples were collected from the backwaters, Cochin and marine aquaculture ponds near Amalapuram, Andhra Pradesh and were inoculated in 2PSB broth with 2% NaCl (w/v). After three to five months, the growth was observed and streaked in 2PSB media in anaerobic jars which were flushed with 95% N2, 5% CO2 gas with the help of anoxomart anaerobic system. 16S rRNA sequencing was done for the isolated and pure culture as shown in table 1.
Table 1: 16SrRNA sequencing of the cultured and pure phototrophs
short curved rod
curved rods or vibroid
Vibroid to spiral shape
Once the pure culture of phototrophic bacteria was obtained on agar plate then carotenoids were extracted in 100% acetone and scanned it in spectrophotometer in the visible range. Few of the extracted carotenoids absorption spectra are shown below:-
Absporption spectra of carotenoid extracts in acetone
Carotenoid belongs to Spirilloxanthin series
Carotenoid belongs to chlorobactene series
Carotenoid belongs to spirilloxanthin series
Carotenoid belongs to spheroidene series
Carotenoid belong to spheroidene series
Carotenoid belongs to spirilloxanthin series
13 different genera and 20 different species of anoxygenic phototrophs has been cultured in our laboratory conditions from the samples marine aqua water Andhra Pradesh, backwaters of Kerala. The growth conditions are anaerobic condition, 0-2% NaCl (w/v), light intensity varies from 500-2000 lux, electron source is either 2mM sodium thiosulphate or 2mM sodium sulphide, carbon source is CO2 for the phototrophs.
One green sulfur bacteria has been successfully cultured and purified showing 99% similarity with Chlorobaculum parvum and its carotenoids belong to chlorobactene group.
Absorption spectra of acetone extracted carotenoids majorly belong to the series of either spheroidene or spirilloxanthin series.
To culture phototrophs in laboratory conditions by standardising the conditions
Identification of Pure culture by 16S rRNA gene sequencing
Extraction of carotenoids and its purification by HPLC
Structural identification of carotenoids by LC-MS and NMR method