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4. PNSB AS BIOREMEDIATORS

4.1. INTRODUCTION

Shrimp farming is a common activity in coastal zones of many

tropical and subtropical nations (Boyd and Green, 2002). The

commercial scale shrimp farming using semi-intensive method started

only in late 1980s and early 1990s with the setting up several large

ventures in the coastal regions of Andhra Pradesh and Tamil Nadu.

India, a minor player in the total seafood trade, enjoys a fairly

prominent position in the world production of shrimps and commands

an 8 % share in the world trade of shrimp exports (Rajalakshmi, 2002).

Shrimp farming can be separated into extensive, semi intensive

and intensive culture systems (Macintosh and Phillips, 1992).

Extensive culture systems have large pond sizes (>5 ha), relatively low

stocking densities (<10 per m2), no aeration, and natural food sources

(through fertilisation). Intensive farming consists of smaller ponds (1

ha), very high stocking densities (>20 per m2), aeration, and

formulated high protein feed pellets. Intensive farming is becoming

more prominent, and at the same time the potential for environment

impact from shrimp farming is considerably high (Phillips et al.,

1993).

Semi-intensive cultivation of prawn is done in countries like

India, Bangladesh, Indonesia, the Philippines and Viet-Nam, in 1-5 ha

ponds that are commonly stocked with hatchery-produced seeds at the

rate of 5 to 20 PL/m². Water exchange is regularly carried out by tide

and supplemented by pumping. The shrimps feed on natural foods and

enhanced by pond fertilization, supplemented by artificial diets.

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Production yields range from 500 to 4000 kg/ha/yr. Intensive shrimp

farming is done in countries like Thailand, Philippines, Malaysia and

Australia. These ponds are generally small (0.1 to 1.0 ha) with a square

or rectangular shape. Stocking density ranges from 20 to 60 PL/m² and

production yields of 4000 to 15000 kg/ha/yr are achieved

(http://www.fao.org/fishery/culturedspecies/Penaeus_monodon/en).

Shrimp aquaculture brings with it the social and economic

benefits of rural employment, but it has damaging effects that may take

time to surface. Experiences in other shrimp producing countries have

shown that shrimp farming is likely to cause pollution to water and

land. There are three main negative effects on the environment. Firstly,

effluent discharge from the farms causes eutrophication. Secondly, soil

on which shrimp ponds are situated becomes polluted due to the

sediments accumulated during the farming period. Pond lands have

high concentrations of salt and cannot be used for cultivation (Patil et

al., 2002). Thirdly, salt water leaches from the pond to contaminate the

groundwater beneath (Páez-Osuna, 2001b). Negative impacts of

shrimp aquaculture include coastal ecosystem damages, decrease in

fisheries yields and environmental pollution by solids and nutrients

from shrimp ponds (Paez-Osuna et al., 2003; Primavera, 2006).

GESAMP (1991) an International advisory body on marine

pollution problems, defined marine pollution as,

"Marine pollution means introduction by man, directly or indirectly, of

substances or energy into the marine environment (including estuaries)

resulting in such deleterious effects as harm to living resources,

hazards to human health, hindrance to marine activities including

fishing, impairment of quality for use of sea water and reduction of

amenities."

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4.1.1 Shrimp farm effluents and their environmental impacts

Shrimp farms are constructed near sources of brackish water or

seawater, and ponds for shrimp culture are filled and maintained by

pumping water from these sources. Fertilizers and feeds are applied to

ponds to promote shrimp growth. Nitrogen and phosphorus in

fertilizers enhance phytoplankton production, enlarging the base of the

food chain for shrimp. Feed is consumed directly by shrimp, often

creating much greater production than with fertilizers alone. However,

uneaten feed, faeces, and other metabolic wastes increase nutrient

concentrations in pond water, also stimulating phytoplankton growth.

Effluents from shrimp ponds typically are enriched with nutrients,

especially nitrogen and phosphorus, and they have high concentrations

of particulate organic matter resulting from live plankton and decaying

plankton. Waters in shrimp ponds usually are eutrophic, and the degree

of eutrophication increases as shrimp production levels increase. In

semi-intensive shrimp farming, water is flushed through ponds to

reduce concentrations of nutrients, phytoplankton, ammonia, and other

potentially toxic metabolites, as well as organic matter. In intensive

shrimp farming, mechanical aeration is used to prevent low dissolved

oxygen concentrations, but water exchange (flushing) is also

commonly used. Water flushed from ponds enters coastal ecosystems,

where it can cause eutrophication (Jones et al., 2000; Boyd and Green,

2002).

The shrimp pond water quality tends to deteriorate through the

grow-out period, as feeding rate increases with shrimp size and

biomass. Thus, the highest quantity and poorest quality of waste water

(in terms of nutrient load, total ammonia and ionized ammonia and

total suspended solid) are found just before harvest time, when shrimp

biomass is at the maximum. Waste water discharge during harvest

(especially the last 5 cms drainage) is usually the most important

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contributor to overall waste water loading, comprising over 75% of the

total load (Aquaculture authority, 2001). Due to intensive/semi-

intensive farming methods the effluent quality is extremely poor with

high concentrations of nitrogenous compounds (ammonia) due to the

excretion of faeces, organic matter, nitrogen, phosphorus and addition

to this loss of feeds are the main sources of pollution loads. These

feeds are not fully utilized by the shrimps. These feeds are rich in

nutrients like phosphorus and nitrogenous compounds. These

compounds are limiting nutrients for primary producers in marine

environment, and their excessive release can contribute to hyper-

nutrification and in extreme cases, to eutrophication, and ultimately

leading to coastal or marine pollution (Chien, 1992; Boyd, 2003; Biao,

et al., 2004).

Discharge at harvest, includes sediment and nutrients that have

accumulated during the culture period. Pond waters include a

flocculent layer of organic matter that forms on the pond bottom from

dead plankton, uneaten feed, and culture animal waste (Boyd, 1995).

Biochemical oxygen demand of discharge water is important to the

ecology of receiving waters because large quantities of high BOD

water may result in critically low oxygen conditions (Teichert-

Coddington et al., 1999). Effluent from aquaculture ponds are enriched

in suspended organic solids, carbon, nitrogen and phosphorus. This

may contribute significantly to elevated nutrient loadings in coastal

environments (Chua et al., 1989; Dierberg and Kiattisimkul, 1996;

Paez- Osuna et al., 1997; 1998). In India, the development of shrimp

farming has generated many public debates over environmental

impact, such as, the use of mangrove ecosystems for pond

construction, salinization of groundwater and agricultural land,

pollution of coastal waters due to pond effluents, biodiversity issues

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arising from collection of wild seed and broodstock, social conflicts

with other users of resources (Rajalakshmi, 2002 ) .

4.1.2 Regulations, Acts and rules regarding shrimp farm effluents

in India

The Hon‟ble Supreme Court in it‟s judgement dated 11

December, 1996 in WPC No.561/94 (Shri S. Jagannathan vs Union of

India and others) directed the Central Government to set up an

Aquaculture Authority under section 3 (3) of the Environment

Protection Act, 1986 and confer on the Authority all the powers

necessary to protect the ecologically fragile coastal areas, sea shore,

water front areas, other coastal areas and specially deal with the

situation created by the shrimp culture industry in the coastal

States/Union Territories. The Hon‟ble Court also directed that any

aquaculture activity including intensive and semi-intensive which has

the effect of causing salinity of soil; or the drinking water of wells and/

or by the use of chemical feeds increases shrimp or prawn production

with consequent increase in sedimentation which on putrefaction is a

potential health hazard, apart from causing siltation/ turbidity of water

courses and estuaries with detrimental implication on local fauna and

flora shall not be allowed by the aforesaid authority (Aquaculture

authority, 2001). The aquaculture authority of India has put forth

standards for the discharge of effluent from marine shrimp ponds and

given in the table 39.

The Indian Parliament in 2005 passed the Coastal Aquaculture

Authority Act (Act 24 of 2005) and amended the act Act 24 of 2005

with ref. to G.S.R.302(E) dated 30.4.2009 and Central Government of

India, has framed the Rules and Guidelines to improve the productivity

under sustained conditions. The Central Government has established

the Coastal Aquaculture Authority, with its head quarters at Chennai.

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The Act enables the Central Government to regulate coastal

aquaculture and to ensure sustained increase in the aquaculture

products. The act mandates every shrimp farm to have effluent

treatment system and according to the act, effluent treatment system

(ETS) is mandatory for farms above 5 ha. At least 10 per cent of the

total pond area should be earmarked for the ETS which may be used

for secondary aquaculture projects, particularly for culture of mussels,

oysters, seaweed, other fin fishes, etc. Such integrated projects would

help improving the wastewater quality, reducing the organic and

nutrient loads and producing an additional cash crop (Coastal

Aquaculture Authority, 2006).

4.1.3 Bioremidiation of shrimp farm effluents using heterotrophic

bacteria

The current approach to improving water quality in aquaculture

as well as detoxifying the fish and shrimp culture wastes is by

bioremediation. Bioremidiation is a general concept that includes all

those processes and actions that take place in order to bio-transform an

environment, already altered by contaminants, to its original status.

Although the processes that can be used in order to achieve the

desirable results vary, they still have the same principles; the use of

microorganisms or their enzymes, that are either indigenous and are

stimulated by the addition of nutrients or optimization of condition, or

are seeded into the medium (Thassitou and Arvanitoyannis, 2001).

Bioremidiation is recognized as an inexpensive, effective and

environmentally safe technology, which offers new and innovative

ways to clean up hazardous wastes. When microorganisms and or their

products are used as additives to improve water quality, they are

referred to as bio (microbial)-remediators or microbial remidiating

agents (Moriarty, 1998). They result in a lower accumulation of slime

or organic matter at the bottom of the pond, better penetration of

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oxygen into the sediment and a generally better environment for the

farmed stock (Rao and Karunasagar, 2000).

The isolation and development of indigenous bacteria are

required for successful microbial remediation (Austin and Brunt,

2009). A successful microbial remediation process in the aquaculture

system involves: maximizing carbon mineralization to carbon dioxide

to minimize sludge accumulation; maximizing primary productivity

that stimulates shrimp production and also secondary crops.

Optimizing nitrification rates to keep ammonia concentration low;

Optimizing denitrification rates to eliminate excess nitrogen from

ponds as nitrogen gas; maximizing sulphide oxidation to reduce

accumulation of hydrogen sulphide; and maintaining a diverse and

stable pond community where undesirable species do not become

dominant (Bratvold et al., 1997). The dissolved and suspended organic

matter contains mainly carbon chains and available in plenty to

microbes and algae. A good bioremediator must contain microbes that

are capable of effectively clearing carbonaceous wastes from water,

capable of multiplying rapidly and have good enzymatic activity.

Members of the genus Bacillus, like B. subtilis, B. licheniformis,

B. cereus, B. coagulans, and of the genus Phenibacillus spp., like P.

polymyxa, are good examples of bacteria suitable for bioremediation of

organic detritus. However, these are not normally present in the

required amounts in the water column, their natural habitat being the

sediment. When certain Bacillus strains are added to the water in

sufficient quantities, they can make an impact. They compete with the

bacterial flora naturally present for the available organic matter like

leached or excess feed and shrimp faeces (Sharma, 1999).

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As a part of bio-augmentation, the Bacillus can be produced,

mixed with sand or clay and broadcasted, to be deposited in the pond

bottom (Singh et al., 2001). Lactobacillus spp., is also used along with

Bacillus to break down the organic detritus. These bacteria produce a

variety of enzymes that break down proteins and starch to small

molecules, which are then taken up as energy sources by other

organisms. The removal of large organic compounds reduces water

turbidity (Haung, 2003). Nitrogen applications in excess of pond

assimilatory capacity can lead to deterioration of water quality through

the accumulation of nitrogenous compounds (e.g., ammonia and

nitrite) with toxicity to fish and shrimp. The principal sources of

ammonia are fish/shrimp excretion and sediment flux derived from the

mineralization of organic matter and molecular diffusion from reduced

sediment, although cyanobacterial nitrogen fixation and atmospheric

deposition are occasionally important (Ayyappan and Mishra, 2003).

Bacteriological nitrification is the most practical method for the

removal of ammonia from closed aquaculture systems and it is

commonly achieved by setting of sand and gravel bio-filter through

which water is allowed to circulate. The ammonia oxidisers are placed

under five genera, Nitrosomonas spp., Nitrosovibrio spp.,

Nitrosococcus spp., Nitrolobus spp., and Nitrospira spp., and nitrite

oxidisers under three genera, Nitrobacter spp., Nitrococcus spp., and

Nitrospira spp. There are also some heterotrophic nitrifiers that

produce only low levels of nitrite and nitrate and often use organic

sources of nitrogen rather than ammonia or nitrite. Nitrifiers in

contaminated cultures have been demonstrated to nitrify more

efficiently. Nitrification not only produces nitrate but also alters the pH

slightly towards the acidic range, facilitating the availability of soluble

materials (Ayyappan and Mishra, 2003). The vast majority of

aquaculture ponds accumulate nitrate, as they do not contain a

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denitrifying filter. Denitrifying filters helps to convert nitrate to

nitrogen. It creates an anaerobic region where anaerobic bacteria can

grow and reduce nitrate to nitrogen gas (Rao, 2002). Unlike the limited

species diversity of bacteria mediating nitrification, at least 14 genera

of bacteria can reduce nitrate. Among these, Pseudomonas, Bacillus

and Alkaligenes are the most prominent numerically (Focht and

Verstraete, 1977). Sequencing batch reactors have been used to treat

shrimp farm waste water where the indigenous heterotrophic bacterial

flora viz., Nitrosomonas, Nitrobacter, and Pseudomonas spp., carried

out the metabolism of nitrogen in the wastewater and as a result there

was a tremendous decrease in chemical oxygen demand (COD) and the

levels of nitrogenous substances like nitrate, ammonia and nitrite in the

shrimp farm waste water (Boopathy et al., 2007; Lyles et al., 2008;

Boopathy, 2009).

4.1.4 Utilization of anoxygenic Photosynthetic bacteria to maintain

water quality in shrimp ponds

The photosynthetic benthic bacteria that break H2S at pond

bottom have been widely used in aquaculture to maintain a favourable

environment (Singh and Radhika, 2001). These bacteria contain

bacterio-chlorophyll that absorb light (blue to infrared spectrum,

depending on type of bacterio-chlorophyll) and perform photosynthesis

under anaerobic conditions (Haung, 2003).

They are purple and green sulphur bacteria that grow at the

anaerobic portion of the sediment-water interface. Photosynthetic

purple non-sulphur bacteria can decompose organic matter, H2S, NO2

and harmful wastes of ponds. The green and purple sulphur bacteria

split H2S to utilize the wavelength of light not absorbed by the

overlying phytoplankton. The purple and green sulphur bacteria obtain

reducing electrons from H2S at a lower energy cost than H2O splitting

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photoautotrophs and thus require lower light intensities for carrying

out photosynthesis (Antony and Philip, 2006). Purple non sulfur

bacteria are good mineralizers at shrimp pond bottom as they grow in

both aerobic and anaerobic conditions as heterotrophic bacteria even in

the dark without utilizing solar energy (Singh and Radhika, 2001).

The purple non sulfur bacteria of importance in aquaculture are

the following Rhodospirillum spp., Rhodopseudomonas spp., and

Rhodomicrobium spp., (Haung, 2003). In Chinese aquaculture, many

commercial photosynthetic bacterial products are labeled as either

single or multiple species at concentrations higher than 109 mL

-1, and

are often combined with growth promoters or conditioners, and are

claimed to have multifunctional effects such as improvement of water

quality, enhancement of growth rate and prevention of disease (Qi et

al., 2009). Purple non sulfur bacterial members are efficient in

flocculating heavy metals like, cadmium, lead, copper and zinc from

shrimp ponds (Watanabe et al., 2003; Panwichian et al., 2010; 2011).

In spite of the diversified prevalence of PNSB in shrimp ponds

and their profound applications in shrimp farming, very few reports are

available on the utilization of purple non sulfur bacteria as potential

bioremidiators of shrimp pond harvest discharges. In the light of the

above, the present study was carried out with the following objectives.

1. Collection of harvest discharges from shrimp ponds (Brackish

and direct sea water) and analyzing for their physico-chemical

properties.

2. Bioremidiation of shrimp pond harvest discharges using native

PNSB strains and post bioremidial analysis of physico-chemical

parameters.

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4.2 MATERIALS AND METHODS

4.2.1 Bioremidiation studies of shrimp pond harvest discharge

using PNSB strains

Bioremediation of shrimp pond harvest discharge using purple

non sulfur bacteria (PNSB) was done by growing the strains in

bioreactor with shrimp pond harvest discharge supplemented with cane

molasses serving as medium, the following factors were optimized

with reference to the bioreactor.

Effect of inoculum size

Volume of natural raw carbon source (Cane molases)

Purified PNSB strains were mass cultured anaerobically in 5

litre glass jars, in modified Biebl and Pfennig‟s medium with succinate

(0.2%) as carbon source and cultured for 48- 72 hours and the active

log phase cultures were used in the study. Screw capped bottles (100

ml) served as culture vessels, for determining the optimal inoculum

size and amount of natural carbon source. Cane black strap molasses

obtained from Kothari sugars Ltd., served as the natural carbon source

for the growing PNSB, Borosil Glass stoppered bottles (1000 ml) were

used as bioreactors for bioremediation experiments.

4.2.1.1 TREATMENT OF CANE MOLASSES (Panda et al., 1984

modified.)

The cane molasses was autoclaved at121°C at 15 lbs pressure

for 10 min, and cooled. The thick molasses was made into syrup by

measuring 100 ml of molasses and dispensing into 100 ml of sterile

distilled water and mixing the contents aseptically using a magnetic

stirrer. To 100 ml of molasses syrup 2ml of 1N H2SO4 was added and

boiled for 30 minutes, cooled and neutralized with sufficient amount of

saturated solution of calcium hydroxide and was left to stand overnight

for clarification and re-filtered with sterile filter paper and the pH

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adjusted to 7.5±1, using 1N NaoH. The clarified molasses syrup was

filtered using a 0.45µm porosity membrane filter and refrigerated.

4.2.1.2 Optimization studies

Effect of inoculum size and volume of natural carbon source on the

growth of Purple non sulfur bacterial strains in shrimp farm

harvest discharge

100 ml screw capped bottles were filled with shrimp harvest

discharge (pH adjusted to 7.4 ±1). The clarified molasses syrup was

used as the natural carbon source. Clarified molasses syrup at different

concentrations (0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5 %) was

added seperately into different screw capped bottles containing the

harvest discharge. Among the PNSB strains some were isolated

exclusively from brackish water, some exclusively from direct sea

water (marine) and a few others from both brackish and direct sea

water. For optimization studies one strain each representing a

particular habitat viz., brackish (BRP1), marine (BRP9) (or) both

(BRP5) was chosen as representative strains. The inoculum of the

individual PNSB strains was prepared by picking a colony from the

modified Biebl and Pfennig agar slants and inoculated individually into

glass stoppered bottles (100ml) containing sterile modified Biebl and

Pfennigs‟s broth and incubated under constant illumination (2,400 lux)

at 30 + 2 C for 72 hours.

After 72 hours of growth, the cultures of PNSB strains were

used as inoculum and the cell suspension of each PNSB strain was

adjusted to an optical density (OD) of 0.3 at 660nm, using sterile

modified Biebl and Pfennig‟s broth as a diluent. The sterile modified

Biebl and Pfennig broth was used as the blank. In order to assess the

optimal inoculum percentage, 10ml, 15ml and 20 ml of the cell

suspensions of the PNSB strains (standardized to an optical density at

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660nm (OD660) to 0.3) were taken separately in sterile centrifuge tubes

and was centrifuged at 4000rpm for 10minutes and the cell pellet was

collected. The cell pellet of each PNSB strain was washed twice with

double denionized distilled water and inoculated separately into the

screw capped bottles (100ml) containing harvest discharge and

incubated under incandescent illumination at 2400 lux at 30±2°C, for

12 days.

Over a 12 day period, samples were taken at 48 hrs. interval,

when the optical density (OD660) and dry cell mass were estimated in

the effluent (harvest discharge) medium. The uninoculated harvest

discharge (with natural carbon source) with a pH correction of 7.5,

served as the control. The lowest concentration of natural carbon

source with a high dry cell mass was chosen to be the optimal

concentration of natural carbon additive into the harvest discharge.

4.2.2 Physicochemical analysis (APHA, 1998)

Physicochemical analysis of shrimp harvest discharge was

undertaken before starting bioremediation studies. The following

physicochemical parameters like, pH, Temperature, Biological oxygen

demand (BOD),Chemical oxygen demand (COD), Total suspended

solids (TSS), Nitrite, Nitrate, Total ammonia nitrogen (TAN), and

dissolved phosphates, were analyzed. For TSS analysis the water

sample was centrifuged at 3000 rpm for 2 minutes and the cell pellet

was removed and suspended solids were estimated.

4.2.3 Bioremidiation studies using bioreactor

The shrimp harvest discharge with a pH correction of 7.5 was

supplemented with, the optimal percentage of clarified molasses syrup.

Optimized inoculum size of PNSB strains in their exponential phase of

growth with a cell density of (OD660) 0.3, were inoculated into, 1000ml

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glass stoppered bottles (Fig.21) which served as bioreactors. The

bioreactors were maintained at 29±2°C in a temperature controlled

environment and illumination was provided by placing the glass

stoppered bottles in an illuminated tissue culture rack fitted with three

60W florescent tubes and two 40 W tungsten bulbs. A constant light

intensity of 2400 ± 40 lux was maintained and measured using a

digital lux meter 401025( Extech Instruments , USA).

4.2.3.1 Post bioremidial analysis of harvest discharge

The physicochemical and microbiological analysis of harvest

discharge on course bioremediation was done over a 12 day period.

The samples from the bioreactors were subjected to physicochemical

analysis and PNSB enumeration, starting from the 0 to12 days at every

48 hour intervals.

4.2.3.2 Enumeration of PNSB strains post bioremediation

The enumeration of PNSB post bioremediation of harvest

discharge was done by Paraffin wax overlay of pour plate method

(Archana et al., 2004), starting from 0 day up to 12th

day, every 48

hour intervals.

4.2.4 Statistical analysis

The statistical significance (p=0.05) of the experimental data

was analyzed using one-way ANOVA using software SPSS 16.0. All

the experiments were performed in triplicate as described by Paniagua-

Michel and Garcia, (2003) for arriving at the experimental data.

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4.3 RESULTS

4.3.1 Optimization studies

Effect of inoculum size and volume of natural carbon source on the

growth of Purple non sulfur bacterial strains in shrimp farm

effluent

In order to assess the optimal natural carbon source and

inoculum percentage for optimization studies with regard to

bioremediation of shrimp farm harvest discharges, one strain each

representing a particular habitat viz., brackish (BRP1), marine (BRP9)

(or) both (BRP5) was chosen as representative strains. On inoculating

the above mentioned strains with various volumes inoculums viz.

10%,15%, and 20% and various concentrations of raw carbon source

(clarified molasses) viz. 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% its

influence on the optical density (OD660) and dry cell mass over a

period of 12 days was observed and given in fig (22a – 23c).

Based on the values obtained, inoculum size of 10% and the

raw carbon volume of 0.2% yielded good growth starting from the 6th

day onwards for all the representative strains and the growth was on

par with higher sizes of inoculum as well as higher concentration of

raw carbon source . Hence it was determined that a 10% inoculum and

0.2% of raw carbon source was found to be optimal, in order to carry

out bioremediation of harvest discharges.

4.3.2 Physicochemical analysis of harvest discharge (Pre

bioremediation)

The physico-chemical parameters viz., pH, Temperature,

Biological oxygen demand (BOD),Chemical oxygen demand (COD),

Total suspended solids (TSS), Nitrite, Nitrate, Total ammonia nitrogen

(TAN), and dissolved phosphates was analyzed and the values are

tabulated in the table 40.

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4.3.3 Post bioremedial analysis of harvest discharges

4.3.3.1 Post bioremedial analysis of BOD

The effects of 12 PNSB strains on the reduction of BOD level

of harvest discharge are given in the tables 41 to 44. There was an

overall reduction of BOD starting from the day-0 to day-12. The ability

of BRP strains to reduce the BOD were not consistent through day- 0

to day-12, as evident from post hoc analysis. However among the 12

PNSB strains BRP12 showed maximum reduction of BOD in the

harvest discharges of all stations under study. Post bioremedial

analysis of BOD from the station Vadakkupoygainallur , the BOD on

day-0 was 26.83±0.10 mg/L, and on the day-12 the level of BOD

reduced to 02.03±0.12 mg/L by the strain BRP12, likewise in other

stations viz., Pappakovil, Sethubavachatram, and Karankadu the level

of reduction of BOD by the strain BRP12 from day-0 to day -12 was

31.39±0.20 mg/L to 02.18±0.08 mg/L, 34.61±0.14 mg/L to 02.37±0.06

mg/L, and 36.73±0.12 mg/L to 03.19±0.12 mg/L, respectively.

4.3.3.1.1 Grouping of PNSB strains based on BOD reduction

Since there was significant difference among the strains across

the days, a post-Hoc (Duncan multiple range test) was performed and

the results are given in the tables 41 to 44. The variations among the

strains with regard to BOD reduction from samples collected from

various stations from day-0 and day-12 are given in the tables 41 to 44.

For the post bioremedial reduction of BOD from the harvest

discharge collected from the station Vadakkupoygainallur, on day-0

five different groups were identified with similar means. However on

day-12 many of the PNSB strains isolated from the groups (based on

day-0) and hence the number of the groups became ten.

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With regard to the post bioremedial analysis of BOD in the

sample collected from the stations viz., Pappakovil ,Sethubavachatram

and Karankadu, the groups identified with similar means on day-0 was

4 for Pappakovil and Sethubavachatram and 5 (Karankadu). On the

day-12 for the above mentioned stations, the number of groups

became, eleven (Pappakovil and Sethubavachatram) and seven

(Karankadu).

With regard to the BOD reduction from Vadakkupoygainallur

sample the strain BRP12 was most effective followed by BRP6, BRP5,

BRP3, BRP9, BRP4, BRP7, BRP1, BRP11, BRP8, BRP10, BRP2 and

control. The percentage of reduction of BOD by BRP12 was 92.43%,

while in control it was 54.12%. With regard to the BOD reduction

from Pappakovil sample the strain BRP12 was most effective followed

by BRP6, BRP5, BRP3, BRP7, BRP9, BRP11, BRP1, BRP4, BRP2,

BRP8, BRP10, and control. The percentage of reduction of BOD by

BRP12 was 93.05% while in control it was 50.92%.

With regard to the BOD reduction from Sethubavachatram

sample the strain BRP12 was most effective followed by BRP6,

BRP5, BRP11, BRP7, BRP3, BRP9, BRP4, BRP10, BRP8, BRP2,

BRP1, and control. The percentage of reduction of BOD by BRP12

was 93.15%, while in control it was 44.05%. With regard to the BOD

reduction from Karankadu sample the strain BRP12 was most effective

followed by BRP6, BRP5, BRP3, BRP9, BRP4, BRP7, BRP11, BRP8,

BRP10, BRP2, BRP1 and control. The percentage of reduction of

BOD by BRP12 was 91.31 %, while in control it was 47.55 %.

4.3.3.2 Post bioremedial analysis of COD

The effect of PNSB strains in reducing the COD level in the

harvest discharges, across different days by different strains are given

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in the tables 45 to 48. There was an overall reduction of COD starting

from the day-0 today-12. The ability of BRP strains to reduce the COD

were not constant through day- 0 to day-12, as evident from post Hoc

analysis.

However among the 12 PNSB strains BRP12 showed maximum

reduction of COD in the harvest discharges of all stations under study.

Post bioremedial analysis of COD from the station

Vadakkupoygainallur , the COD on day-0 was 76.58±0.03mg/L, and

on the day-12 the level of COD reduced to 18.36±0.03 mg/L by the

strain BRP12, likewise in other stations viz., Pappakovil,

Sethubavachatram, and Karankadu the level of COD reduction by the

strain BRP12 from day-0 to day-12 was 87.31±0.03 mg/L

to16.93±0.07 mg/L, 87.45±0.02 mg/L to 19.07±0.04 mg/L, and

99.60±0.11 mg/L to 18.42±0.15 mg/L, respectively.

4.3.3.2.1 Grouping of PNSB strains based on COD reduction



the results are given tables 45 to 48. The variations among the strains

with regard to COD reduction from samples collected from various

stations from day-0 and day-12 are given in the tables 45 to 48.

For the post bioremedial reduction of COD from the harvest

discharge collected from the station Vadakkupoygainallur, on day-0

nine different groups were identified with similar means. On day- 12

many of the PNSB strains isolated from the groups (based on day-0)

and hence the number of the groups became eight. With regard to the

post bioremedial analysis of COD in the sample collected from the

stations viz., Pappakovil ,Sethubavachatram and Karankadu, the groups

identified with similar means on day-0 was, six (Pappakovil and

108

Sethubavachatram) and five (Karankadu). On the day-12 for the above

mentioned stations, the number of groups became eleven (Pappakovil

and Sethubavachatram) and seven (Karankadu).

With regard to the COD reduction from Vadakkupoygainallur

sample the strain BRP12 was most effective followed by BRP6 , BRP

5, BRP 3, BRP 9, BRP4, BRP7, BRP2, BRP1, BRP10, BRP8, BRP11,

and control. The percentage of reduction of COD by BRP12 was 76.03

%, while in control it was 31.27 %. With regard to the COD reduction

from Pappakovil sample the strain BRP12 was most effective

followed by BRP6 , BRP 5, BRP 3, BRP 9, BRP4, BRP7, BRP2,

BRP1, BRP10, BRP8, BRP11, and control. The percentage of

reduction of COD by BRP12 was 76.03 %, while in control it was

31.27 %.

With regard to the COD reduction from Sethubavachatram

sample the strain BRP12 was most effective followed by BRP6, BRP5,

BRP3, BRP9, BRP4, BRP7, BRP2, BRP1, BRP8, BRP10, BRP11 and

control. The percentage of reduction of COD by BRP12 was 78.19%,

while in control it was 27.51 %. With regard to the COD reduction

from Karankadu sample the strain BRP12 was most effective followed

by BRP6, BRP5, BRP3, BRP9, BRP4, BRP2, BRP7, BRP1, BRP10,

BRP8, BRP11 and control. The percentage of reduction of COD by

BRP12 was 81.51%, while in control it was 43.41 %.

4.3.3.3 Post bioremedial analysis of TSS

The effect of PNSB strains in reducing the TSS level in the

harvest discharges, across different days by different strains are given

in the tables 49 to 52. There was an overall reduction of TSS starting

from the day-0 today-12. The ability of BRP strains to reduce the TSS

were not constant through day- 0 to day-12, as evident from post Hoc

109

analysis. However among the 12 PNSB strains BRP5 showed

maximum reduction of TSS in the harvest discharges of all stations

under study.

Post bioremedial analysis of TSS from the station

Vadakkupoygainallur , the TSS on day-0 was 119.40±0.07 mg/L, and

on the day-12 the level of TSS reduced to 82.16±0.04 mg/L, by the

strain BRP5, likewise in other stations viz., Pappakovil,

Sethubavachatram, and Karankadu the level of TSS reduction by the

strain BRP5 from day-0 to day-12 was 132.43±0.04 mg/L to

93.31±0.16 mg/L, 94.70±0.00 mg/L to 65.42±1.63 mg/L, and

97.37±0.03 mg/L to 69.34±0.04 mg/L, respectively.

4.3.3.3.1 Grouping of PNSB strains based on TSS reduction




with regard to TSS reduction from samples collected from various


For the post bioremedial reduction of TSS from the harvest

discharge collected from the station Vadakkupoygainallur, on day-0,

three different groups were identified with similar means. However on

day 12 many of the PNSB strains isolated from the groups (based on

day-0) and hence the number of the groups became 10. The percentage

of reduction of TSS (Vadakkupoygainallur) by BRP5 was 31.189%,

while in the control it was 17.27%. With regard to the post bioremedial

analysis of TSS in the sample collected from the stations viz.,

Pappakovil, Sethubavachatram and Karankadu, the groups identified

with similar means on Day-0 was, 1 (Pappakovil), 3

(Sethubavachatram) and 2 (Karankadu). On the day-12 for the above

110

mentioned stations, the number of groups became, 12 (Pappakovil), 9

(Sethubavachatram) and 12 (Karankadu).

With regard to the TSS reduction from Vadakkupoygainallur

sample the strain BRP5 was most effective followed by, BRP3, BRP4,

BRP12, BRP9, BRP8, BRP11, BRP7, BRP1, BRP10, BRP6, control

followed by BRP2. The percentage of reduction of TSS by BRP5 was

31.189 %, while in control it was 17.27 %. With regard to the TSS

reduction from Pappakovil sample the strain BRP5 was most effective

followed by BRP11, BRP3, BRP7, BRP12, BRP6, BRP9, BRP1,

BRP10, BRP4, BRP2, BRP8 and control. The percentage of reduction

of TSS by BRP5 was 29.54 %, while in control it was 11.04 %. With

regard to the TSS reduction from Sethubavachatram sample the strain

BRP5 was most effective followed by BRP11, BRP7, BRP9, BRP12,

BRP6, BRP4, BRP1, BRP3, BRP2, BRP10, BRP8 and control. The

percentage of reduction of TSS by BRP5 was 30.92%, while in control

it was 7.88%.

With regard to the TSS reduction from Karankadu sample the

strain BRP5 was most effective followed by BRP11, BRP6, BRP3,

BRP12, BRP10, BRP2, BRP9, BRP4, BRP8, BRP1, BRP7 and

control. The percentage of reduction of TSS by BRP5 was 28.79%,

while in control it was 9.18%.

4.3.3.4 Post bioremedial analysis of Nitrate

The effect of PNSB strains in reducing the nitrate level in the

harvest discharges, during the period of study are given in the tables 53

to 56. In all the stations with regard to nitrate there was a slight

increase in the nitrate value on day-2 and gradually the levels of nitrate

subsided in the subsequent days leading to total reduction of nitrate.

On the day-8 the sample collected from the station

111

Vadakkupoygainallur, one strain BRP7 showed total reduction of

nitrate, but nitrate still existed in the samples inoculated with other 11

strains of PNSB, and level of nitrate among the strains ranged from

(0.026±0 mg/L to 1.073±0.03 mg/L). Likewise the level of nitrate on

day-8 in control stood at 0.937±0.01 mg/L.

But on the day-10 there was complete reduction of nitrate in

harvest discharge inoculated with rest of 11 strains viz., BRP1, BRP2,

BRP3, BRP4, BRP5, BRP6, BRP8, BRP9, BRP10, BRP11, BRP12.

On the contrary in the control nitrate could be observed (0.071±0.01

mg/L) on day-12. With regard to the sample from the station

Pappakovil on the day-8, strains BRP7, BRP9, BRP10 and BRP12,

showed complete reduction of nitrate, but nitrate still existed in the

samples inoculated with other 8 strains of PNSB, and level of nitrate

among the strains ranged from (0.042±0.01 mg/L to 0.462±0.01mg/L).

Likewise the level of nitrate on day-8 in control stood at 1.369±0.01

mg/L. On the day-10 the nitrate was completely absent in the rest of

the strains viz., BRP1, BRP2, BRP3, BRP4, BRP5, BRP6, BRP8 and

BRP11. But in the control on the day-10 the level of nitrate stood at

0.083±0.01 mg/L, and on the day-12, in the control, nitrate was totally

absent. With regard to the sample collected from the station

Sethubavachatram on day-8 strains BRP1, BRP2, and BRP9, showed

complete reduction of nitrate, but nitrate still persisted in samples

inoculated with other strains of PNSB, where the level of nitrate on

day-8 among the strains ranged from (0.033±0 mg/L to 1.144±0.1

mg/L). Likewise the level of nitrate on day-8 in control stood at 3.178

±0.01 mg/L. On the day-10 rest of the strains viz.,BRP3, BRP4,

BPR5,BRP6, BRP7, BRP8, BRP10, BRP11, BRP12, showed absence

of nitrate excluding control where the level of nitrate in the control

stood at 0.472 ±0.00 mg/L, but on the day-12 nitrate was completely

absent in the control also.

112

As with regard to sample collected from the station Karankadu

on the day-8 strains BRP3, BRP5, BRP6, BRP7,BRP9, BRP10, BRP11

and BRP12 showed complete reduction of nitrate, but in other strains

nitrate still existed and the levels of nitrate among the strains ranged

between (0.062±0.00 mg/L to 1.119±0.05 mg/L). Likewise the level of

nitrate on day-8 in control stood at 3.207±0.1 mg/L. On the day-10

nitrate was completely reduced in the rest of the strains viz.,BRP1,

BRP2, BRP4 and BRP8, except the control, where the level of nitrate

stood at 1.792±0.11 mg/L, but on the 12th

day the nitrate was absent in

the control also.

4.3.3.4.1 Grouping of PNSB strains based on nitrate reduction




with regard to nitrate reduction from samples collected from various


For the post bioremedial reduction of nitrate from the harvest

discharges collected from the station Vadakkupoygainallur, on day- 0,


day- 8, many of the PNSB strains were isolated from the groups (based

on day-0) and hence the number of the groups became ten. Likewise in

the samples from the stations Pappakovil and Sethubavachatram on

day-0, two groups were identified with similar means, in the sample

from the station Karankadu, three groups with similar means were

obtained on day-0. However on day-8 the groups became five for

sample from the stations Pappakovil and Karankadu and four for

samples from Sethubavachatram. With regard to the nitrate reduction

on day-8 from Vadakkupoygainallur sample the strain BRP7 was most

effective followed by BRP6, BRP5, BRP10, BRP2, BRP9, BRP8,

113

BRP12, BRP4, BRP3, BRP1, control and BRP11. The percentage of

reduction of nitrate by was BRP7 100%, while in control it was 80.11

%. With regard to the nitrate reduction on day-8 from Pappakovil

sample the strains BRP7, BRP9, BRP10 and BRP12 were most

effective followed by BRP3, BRP5, BRP6, BRP11, BRP8, BRP4,

BRP2, BRP1 and control. The percentage of reduction of nitrate on

day-8 by the strains, BRP7, BRP9, BRP10 and BRP12 was100 %,

while in control it was 70.96%. With regard to the nitrate reduction on

day-8 from Sethubavachatram sample the strains BRP1, BRP2 , BRP9

were most effective followed by BRP7, BRP12, BRP6, BRP5, BRP8,

BRP4, BRP10, BRP3, BRP11 and control. The percentage of reduction

of nitrate on day-8 by the strains BRP1, BRP2, BRP9 was 100%, while

in control it was 41.37%. With regard to the nitrate reduction on day-8

from Karankadu sample the strains BRP3, BRP5, BRP6, BRP7, BRP9,

BRP10, BRP11 and BRP12 were most effective followed by BRP8,

BRP1, BRP4, BRP2 and control. The percentage of reduction of nitrate

on day-8, by the strains BRP3, BRP5, BRP6, BRP7, BRP9, BRP10,

BRP11 and BRP12 was 100 %, while in control it was 39.27 %.

4.3.3.5 Post bioremedial analysis of Nitrite

The effect of PNSB strains in reducing the nitrite level in the

harvest discharges, during the period of study are given in the tables 57

to 60. There was an overall reduction of nitrite starting from the day-0,

and complete reduction of nitrite after day-2, including the control. The

ability of BRP strains to reduce the nitrite were not constant through

day- 0 to day-2, as evident from post hoc analysis.

With regard to reduction of nitrite in the sample collected from

the station Vadakkupoygainallur, on day-0, level of nitrite ranged from

0.518±0.018 mg/L to 0.537±0.007 mg/L , on day-2 the strain BRP9

114

showed maximum reduction of nitrite than other strains of PNSB

where the level of nitrite was reduced to 0.013±0.029 mg/l.


the station Pappakovil, on day-0, level of nitrite ranged from

0.612±0.021 mg/L to 0.644±0.015 mg/L, on day-2 the strain BRP7

showed maximum reduction of nitrite than other strains of PNSB

where the level of nitrite was reduced to 0.021±0.006 mg/l. With

regard to reduction of nitrite in the sample collected from the station

Sethubavachatram, on day-0, level of nitrite ranged from 0.344±0.016

mg/L to 0.377±0.008 mg/L, on day-2 the control showed maximum

reduction of nitrite than PNSB strains of PNSB where the level of

nitrite was reduced to 0.031±0.01 mg/l.


the station Karankadu, on day-0, level of nitrite ranged from

0.411±0.004 mg/L to 0.447±0.02 mg/L, on day-2 the strain BRP1

showed maximum reduction of nitrite than PNSB strains of PNSB

where the level of nitrite was reduced to 0.022±0.007 mg/l.

4.3.3.5.1 Grouping of PNSB strains based on nitrite reduction




with regard to nitrite reduction from samples collected from various

stations from day-0 and day-12 are given in the tables 57 to 60. For the

post bioremedial reduction of nitrite from the harvest discharge

collected from the station Vadakkupoygainallur, on day-0, two

different groups were identified with similar means. However on day-2

many of the PNSB strains isolated from the groups (based on day-0)

and hence the number of the groups became five.

115

With regard to the post bioremedial analysis of nitrite in the

sample collected from the stations viz., Pappakovil ,Sethubavachatram

and Karankadu, the groups identified with similar means on day-0 was

three for Pappakovil and Sethubavachatram and two for Karankadu.

However on the day-2 for the above mentioned stations, the number of

groups became four for Pappakovil, two for Sethubavachatram and

five for Karankadu. With regard to the nitrite reduction from

Vadakkupoygainallur sample the strain BRP9 was most effective


BRP4, BRP11, control, BRP8 and BRP6. The percentage of reduction

of nitrite by BRP12 was 97.54%, while in control it was 82.93%.

With regard to the nitrite reduction from Pappakovil sample the


BRP8, BRP10, control, BRP9, BRP6, BRP5, BRP2, BRP4 and

BRP12. The percentage of reduction of nitrite by BRP7 was 96.65%

while in control it was 91.58%. With regard to the nitrite reduction

from Sethubavachatram sample the control was most effective


BRP7, BRP9, BRP11, BRP4, BRP2. The percentage of reduction of

nitrite by control was 91.36%, while in BRP5 it was 90.80 %.

With regard to the nitrite reduction from Karankadu sample the


BRP8, BRP3, BRP12, BRP6, BRP10, BRP7, control, BRP5, BRP9.

The percentage of reduction of nitrite by BRP1 was 94.72 %, while in

control it was 87.70 %.

4.3.3.6 Post bioremedial analysis of TAN

The effect of PNSB strains in reducing the total ammonia

nitrogen (TAN) level in the harvest discharges, during the period of

116

study are given in the tables 61 to 64. There was an overall reduction

of TAN starting from the day-0, and complete reduction of TAN after

day- 2, including the control. With regard to reduction of TAN in the

sample collected from the station Vadakkupoygainallur, on day-0,

level of TAN ranged from 0.328±0.006 mg/L to 0.381±0.012mg/L ,

on day-2 the strain BRP10 showed maximum reduction of TAN than

other strains of PNSB where the level of TAN was reduced to

0.042±0.007 mg/L. With regard to the reduction of TAN in the sample

collected from the station Pappakovil, on day-0, level of TAN ranged

from 0.151±0.002mg/L to 0.171±0.009 mg/L., on day-2 the strain

BRP6 showed complete reduction of TAN. With regard to the

reduction of TAN in the sample collected from the station

Sethubavachatram, on day-0, level of TAN ranged from 0.266±0.013

mg/L to 0.284±0.003 mg/L., on day 2 the strain BRP12 reduced the

level of TAN considerably to 0.059±0.009 mg/L. With regard to the

reduction of TAN in the sample collected from the station Karankadu,

on day0, level of TAN ranged from 0.162±0.003mg/L to 0.176±0.002

mg/L, on day 2 the strain BRP5 reduced the level of TAN to

0.041±0.002 mg/L.

4.3.3.6 .1 Grouping of PNSB strains based on TAN reduction




with regard to nitrate reduction from samples collected from various

stations from day-0 and day-12 are given in the tables 61 to 64. For the

post bioremedial reduction of TAN from the harvest discharges

collected from the station Vadakkupoygainallur, on day- 0, two

different groups were identified with similar means. However on day-

2 many of the PNSB strains were isolated from the groups (based on

day-0) and hence the number of the groups became four. The strain

117



percentage of reduction of TAN by the strain BRP10 was 88.33%,

while in control it was 79.94%.

For the post bioremedial reduction of TAN from the harvest

discharges collected from the station Pappakovil, on day-0, five

different groups were identified with similar means. However on day-

2 many of the PNSB strains were isolated from the groups (based on

day-0) and hence the number of the groups became two. The strain

BRP6was most effective followed by BRP7, BRP11, BRP5, BRP12,


percentage of reduction of TAN by the strain BRP6 was 100%, while

in control it was 50 %.

For the post bioremedial reduction of TAN from the harvest

discharges collected from the station Sethubavachatram, on day-0,

two different groups were identified with similar means. However on

day-2 many of the PNSB strains were isolated from the groups (based

on day-0) and hence the number of the groups became five. The strain



percentage of reduction of TAN by the strain BRP12 was 78.70%,

while in control it was 67.74 %. For the post bioremedial reduction of

TAN from the harvest discharges collected from the station

Karankadu, on day-0, five different groups were identified with similar

means. However on day-2 many of the PNSB strains were isolated

from the groups (based on day-0) and hence the number of the groups

became three. The strain BRP5 was most effective followed by BRP2,

BRP7, BRP3, BRP6, BRP8, BRP11, BRP1, BRP4, BRP12, control,

118

BRP9 and BRP10. The percentage of reduction of TAN by the strain

BRP5 was 75.44 %, while in control, it was 60.79 %.

4.3.3.7 Post bioremedial analysis of phosphates

The effect of PNSB strains in reducing the phosphate level in

the harvest discharges, during the period of study are given in the

tables 65 to 68. In all the stations with regard to phosphate there was a

gradual decrease starting from the day-0 to day-12, including the

control. In the stations excepting Vadakkupoygainallur, the level of

phosphates, completely got reduced on day10.

In the station Vadakupoygainallur, on day-0 the level of

phosphates ranged from 0.721± 0.002 mg/L to 0.730±0.002 mg/L, but

on the day-8, the strains viz., BRP5, BRP11 and BRP12, showed

complete reduction of phosphates. The complete reduction of

phosphates in the samples collected from the station

Vadakupoygainallur in all the inoculated PNSB strains occurred on the

day-12, excepting the control where the level of phosphates stood at

0.089±0.004mg/L. In the station Pappakovil, on day-0 the level of

phosphates ranged from 0.951±0.017mg/L to 0.980±0.008 mg/L, but

on the day-8, the strains viz., BRP5, BRP7 and BRP12 showed

complete reduction of phosphates.

The complete reduction of phosphates in the sample collected

from the station Pappakovil, in all the inoculated PNSB strains

occurred on the day-10, excepting the control where on day-12, in the

control, level of phosphates stood at 0.076±0.004mg/L. In the station

Sethubavachatram, on day-0 the level of phosphates ranged from

0.472±0.006 mg/L to 0.482±0.000 mg/L, but on the day-6, the strain

BRP3 showed complete reduction of phosphates. The complete

reduction of phosphates in the sample collected from the station

119

Sethubavachatram, in all the inoculated PNSB strains occurred on the

day-10, excepting the control, where on day-12, in the control, level of

phosphates stood at 0.018±0.004. In the station Karankadu, on day-0

the level of phosphates ranged from 0.622±0.006 mg/L to 0.631±0.005

mg/L, but on the day-8, the strains BRP1, BRP2, BRP5, BRP7, BRP8,

BRP9, BRP10 and BRP12 showed complete reduction of phosphates,

excepting the strains BRP3, BRP4, BRP6 and BRP11, but on the

day10 all the rest of the strains showed complete reduction of

phosphates, excepting the control, where on day-12, in the control,

level of phosphates stood at 0.025±0.012 mg/L.

4.3.3.7.1 Grouping of PNSB strains based on phosphate reduction




with regard to phosphate reduction from samples collected from

various stations from day-0 and day-12 are given in the tables 65 to 68.

For the post bioremedial reduction of phosphates from the

harvest discharges collected from the station Vadakkupoygainallur, on

day-0, four different groups were identified with similar means.

However on day-8 many of the PNSB strains were isolated from the

groups (based on day-0) and hence the number of the groups became 7.

The strains BRP5, BRP11 and BRP12 were most effective followed by

BRP10, BRP9, BRP8, BRP7, BRP6, BRP1, BRP3, BRP4, control and

BRP2. The percentage of reduction of phosphates on day-8 by the

strains BRP5, BRP11 and BRP12 was 100 %, while in control it was

85.28 %. For the post bioremedial reduction of phosphates from the

harvest discharges collected from the station Pappakovil, on day-0,



120

on day-0) and hence the number of the groups became 5. The strains

BRP5, BRP7 and BRP12 were most effective followed by BRP11,

BRP4, BRP1, BRP9, BRP6, BRP10, BRP3, BRP2, BRP8 and control.

The percentage of reduction of phosphates on day-8 by the strains

BRP5, BRP7 and BRP12 was 100 %, while in control it was 93.57%.


harvest discharges collected from the station Sethubavachatram, on

day-0, two different groups were identified with similar means.

However on day-6 many of the PNSB strains were isolated from the

groups (based on day-0) and hence the number of the groups became

10. The strain BRP3 was most effective followed by BRP11, BRP8,

BRP12, BRP4, BRP5, BRP9, BRP6, BRP1, BRP7, BRP10, control

and BRP2. The percentage of reduction of phosphates on the day-6 by

the strain BRP3 was 100%, while in control it was 82.78 %.


harvest discharges collected from the station Karankadu, on day-0, a

single group alone was identified with similar means. However on


on day-0) and hence the number of the groups became 5. The strains

BRP1, BRP2, BRP5, BRP7, BRP8, BRP9, BRP10 and BRP12 were

most effective followed by BRP4, BRP6, BRP3, BRP11 and control.

The percentage of reduction of phosphates on the day-8 by the strains

BRP1, BRP2, BRP5, BRP7, BRP8, BRP9, BRP10 and BRP12 was

100%, while in control it was 88.53 %.

4.3.3.8 Enumeration of PNSB strains post bioremediation

With regard to post bioremedial enumeration of PNSB strains

across various stations viz., Vadakkupoygainallur, Pappakovil,

Sethubavachatram and Karankadu yielded brown/brownish red

121

colonies on modified Biebl and Pfennig‟s (1981) agar medium. The

number of PNSB colonies increased in the samples subjected to

bioremediation using 12 PNSB strains including the control, as days

progressed. The enumeration of PNSB from the samples subjected to

bioremediation across various days has been summarized in the table

69 to 72.

122

4.4. DISCUSSION

Intensive shrimp aquaculture systems rely on high protein feed

pellets to produce high rates of growth, but a large proportion of the

pellets are not assimilated by the shrimps (Primavera, 1994).

Approximately 10% of the feed is dissolved and 15% remains uneaten.

The remaining 75% is ingested, but 50% is excreted as metabolic

waste, producing large amounts of gaseous, dissolved and particulate

waste (Lin et al., 1993). Subsequently, the effluent contains elevated

concentrations of dissolved nutrients (primarily ammonia), plankton

and other suspended solids (Ziemann et al., 1992).

The types of shrimp farming like extensive, semi intensive and

intensive culture systems, with varied stocking densities, and non

utilization of formulated high protein feed pellets along with fecal and

detritus of shrimps , increases the potential for environmental impacts

in the marine environment (Phillips et al., 1993). The dissolved

nutrients and organic material in shrimp ponds stimulate rapid growth

of bacteria, phytoplankton, and zooplankton (Lin et al., 1993). Raj et

al. (1997) observed that the concentrations of TSS, BOD, COD and

nutrients in the farm effluents might increase towards the end of each

crop. Major sources of TSS in ponds are suspended soil particles and

particulate organic matter resulting from phytoplankton and detritus

(Boyd and Tucker, 1998).

Concerns about the possible adverse impacts of aquaculture

discharge have become a risk factor for the industry (Braaten, 1991).

Biochemical oxygen demand, chemical oxygen demand, total

suspended solids and nutrients of discharge water is important to the

ecology of receiving waters because large quantities of high BOD

water may result in critically low oxygen conditions (Teichert-

Coddington et al., 1999). This along with high levels of suspended

123

solids could result in suffocation of fish. Besides increase in nutrient

levels may stimulate algal blooms (Nyanti et al., 2011), eutrophication,

organic enrichment and turbidity in the receiving waters (Eng et al.,

1989; O' Connor et al., 1989; Prakash, 1989). The physico-chemical

analysis of harvest discharge in the present study reveal that TSS was

highest followed by COD, BOD, nitrate, phosphate nitrite and Total

ammonia nitrogen (Table 40).

Discharging such nutrient rich aquaculture effluents into inland

water bodies and oceans is a matter of serious concern due to the

adverse effect that it brings in the form of eutrophication and

subsequent damages to those waters (Pulefou et al., 2008). Recent

reviews of intensive shrimp aquaculture have emphasised the need for

more effective controls on the quality of effluent water discharged into

the environment (Phillips et al., 1993; Primavera, 1994). Effective use

of effluent treatment system will reduce the concentration of the TSS,

BOD, COD and nutrients in the farm effluents (Raj et al., 1997).

Various types of treatment of shrimp farm harvest discharges and

effluent from shrimp farms have been tried by various researchers and

their details are as follows, physical treatments like using ultra-low

pressure asymmetric polyethersulfone (PES) membrane by Ali et al.

(2005), was found to remove the total ammonium and total phosphorus

efficiently. Sedimentation of shrimp farm effluent using sedimentation

ponds, can reduce the load of nutrients in shrimp farm effluents (Jones

et al., 2001; Nyanti et al., 2010; 2011). Constructed wetlands can

perform satisfactorily as recirculation filters in large-scale shrimp

aquaculture operations, reducing the impact of effluent on local water

bodies, conserving large quantities of water and providing valuable

ecological habitat (Tilley et al., 2002). Biological treatment methods

using hydrophytes like watermeal, Wolffia arrhiza by Suppadit et al.

(2008), was found to reduce the nutrients especially nitrogenous

124

substances to an considerable extant. Jones et al. (2001) utilized,

macro algae belonging to the members of Gracilaria edulis to treat

shrimp farm effluent and found that these members can absorb and

utilize dissolved nutrients in the effluent. Likewise the utilization of

marine micro algal species such as Skeletonima costatum and

Chaetoceros coarctatus, has significantly reduced the levels of

nutrients in the shrimp farm effluent (Venkatesan et al., 2006).

Constructed microbial mats have been has been found to be efficient in

treating /bioremediating, the effluents from shrimp ponds as well as

from other aquaculture ponds and these microbial mats have mixed

populations of autotrophic (cyanobacteria and purple photosynthetic

bacteria), nitrifying bacteria and mixed heterotrophic bacterial

communities which can be employed to cleanse the effluents free from

nutrients as well other polluting factors (Paniagua-Michel and Garcia,

2003; Bender and Phillips, 2004; Lezama-Cervantes and Paniagua-

Michel, 2010). The presedimented shrimp farm effluent was subjected

to oyster filteration by Jones et al. (2001) and found that the oysters

could reduce the levels of TSS in the effluent.

The conventional technologies applied to remove those

pollutant nutrients are impractical for sensitive areas, generally costly

to operate for developing countries, often lead to secondary pollution

and to incomplete utilization of natural resources (Phang, 1990; Dela-

Noue et al., 1992). The current approach to improving water quality in

aquaculture is the application of microbes/enzymes to the ponds,

known as bioremediation. When macro and microorganisms and /or

their products are used as additive to improve water quality, they are

referred to as bioremediators or bioremediating agents. The practice of

bioremediation or bioaugmentation, is applied in many areas, but

success varies greatly, depending on the nature of the products used

and the technical information available to the end user. The bacteria

125

that are added must be selected for specific functions that are amenable

to bioremediation (Moriarty, 1998).

The present study focuses on bioremediating potential of PNSB

that are native to the shrimp ponds, as they are capable of decomposing

organic matter, H2S, NO2 and other harmful wastes of pond

(Worawattanamateekul and Ray, 2011). The harvest discharge from

the shrimp ponds in the present study supported high level of

heterotrophic bacteria and also PNSB. This is evident from the total

heterotrophic bacterial counts as well as enrichment studies for PNSB.

The higher heterotrophic bacterial counts in the harvest discharges may

be due to the abundance and availability of nutrients derived from

excess feed, shrimp excreta and other dead and decaying organic

matter (Abraham et al., 2004). Enumeration of PNSB strains in the

present study (Table 5), reveal that harvest discharges contained

significantly higher level of PNSB populations.

In the present endeavor to bioremediate shrimp farm harvest

discharge using indigenous purple non sulfur bacteria (PNSB), samples

were collected across 4 stations viz., Vadakkupoygainallur,

Pappakovil, Sethubavachatram and Karankadu, located along the south

eastern coastal districts of Tamil Nadu, India. The physicochemical

parameters of the harvest discharge were analyzed and summarized in

the table 40. When compared with the effluent standards prescribed by

the Aquaculture authority of India (2001) for the discharge of effluents

from shrimp ponds (brackish and direct sea water) the pH was well

within the permissible limits, but with regard to BOD, COD and TSS

from the brackish water ponds from the stations viz.,

Vadakkupoygainallur and Pappakovil, their levels were higher than the

permissible limits. But on the contrary, in the direct sea water ponds

126

(Sethubavachatram and Karankadu) the levels of BOD, COD and TSS

fell within the range set by the Aquaculture authority of India (2001).

But with regard to dissolved phosphates in all the shrimp pond

harvest discharges, their levels were higher than the limits prescribed

by the Aquaculture authority of India (2001). BOD reflects the organic

matter in the pond effluent and the main sources could be uneaten feed

and waste excreted by the shrimps. Likewise the COD values were

much higher than BOD values possibly due to continuous oxidation

of organic matter in the pond by microorganisms over the culture cycle

and those that were more resistant to biological oxidation were

oxidized by chemical means because more compounds can be

chemically oxidized than biologically oxidized (Metcalf and Eddy,

1991). As Moriarty (1998) reported bacteria that are amenable to

bioremediation must be added at a high enough population density and

under right environmental conditions to achieve the desired outcome

viz., reducing the level of parameters responsible for pollution.

The harvest discharge served as culture medium in the

bioreactor and the harvest discharge was optimized for raw carbon and

inoculum concentration for obtaining high population of PNSB.

Microbial degradation of any waste depends on the amount of carbon,

nitrogen, and phosphorus available for their activity. If there is too

little nitrogen present, the bacteria will be unable to produce necessary

enzymes to utilize the carbon. If there is too much nitrogen,

particularly in the form of ammonia, it can inhibit the growth of the

bacteria (Fontenot et al., 2007). The addition of raw molasses in the

shrimp ponds is a common practice in shrimp ponds all along

Tamilnadu. The addition of molasses to shrimp ponds bring about

reduction of TAN in the shrimp pond water, and there by maintains

optimal C: N ratio, for the efficient degradation of nutrients by

127

heterotrophic bacteria (Samocha et al., 2007; Fontenot et al., 2007;

Avnimelech, 1999).

In the present study clarified molasses syrup at different

concentrations viz., 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5 % was

added separately into different screw capped bottles containing the

harvest discharge. As the harvest discharge was not sterilized, a

relatively higher proportion of inoculum was necessary to repress the

growth of other microorganisms. To maintain the predominance of

phototrophic bacteria throughout the culture, heavy inoculation was

required to achieve the cultivation within short period (Sasaki et al.,

1991).

So in the present study inoculum concentration of 10%, 15%

and 20% was used for optimization studies. Based on the values

obtained, inoculum size of 10% and the raw carbon volume of 0.2%

yielded good growth starting from the 6th

day onwards for all the

representative PNSB strains and the growth was on par with higher

sizes of inoculum as well as higher concentration of raw carbon source.

Thus it was determined that a 10% inoculum and 0.2% of raw carbon

source was found to be optimal, in order to carry out bioremediation of

harvest discharges. Sawada and Rogers (1977) and Getha et al.,

(1998b) reported that most of the PNSB members grew well when

supplied with 0.2% of carbon source.

In the present study of bioremediation using PNSB strains the

number of PNSB colonies increased as days progressed on course of

bioremediation (Table 69 - 72). This shows that the shrimp farm

harvest discharges have acted as a suitable medium for the growth and

development of PNSB strains when the conditions were optimized and

in turn this could have an impact in the reduction of pollutants in these

waste waters. With regard to the bioremediation of harvest discharges

128

using PNSB strains, an overall trend of reduction in the values of

physicochemical parameters viz., pH, Temperature, Biological oxygen

demand (BOD), Chemical oxygen demand (COD), Total suspended

solids (TSS), Nitrite, Nitrate, Total ammonia nitrogen (TAN), and

dissolved phosphates could be observed. However the strains varied in

their ability to bring about reduction.

With regard to BOD and COD reduction, the strain

Rhodobacter capsulatus (BRP12) was most efficient. The reduction of

biological oxygen demand and chemical oxygen demand by the

members of Rhodobacter capsulatus has been studied by Sawada et al.

(1977) in the wool scour liquor, antibiotic factory effluent and abattoir

waste water, where they efficiently reduced the level of BOD and

COD.

Rhodovulum sulphidophilum (BRP5) showed maximum

reduction of TSS in the harvest discharges collected from all the 4

stations compared to other strains. In the present study using purple

non sulfur bacteria, TSS reduction could be observed in all the

treatments even in control (without PNSB) to a level set by the Coastal

Aquaculture Authority viz.,100mg/L, at the end of 10th

or 12th

day,

except at Pappakovil where only few strains could bring about a

reduction in TSS to permissible limit.

Among them the PNSB strain, Rhodovulum sulphidophilum

(BRP5) had an edge over other strains in reducing TSS. However

further studies are needed to improve the efficiency of PNSB with

regard to reduction of TSS. With regard to nitrate reduction all the

PNSB strains reduced nitrate completely on day-10. However the

strains did not show uniform pattern of nitrate reduction in the harvest

discharge samples from all the 4 stations. The strain Rhodobium

129

orientis (BRP7) reduced the level of nitrate completely on the day-8 in

the harvest discharge collected from Vadakkupoygainallur. Likewise in

the station Pappakovil on the day-8 strains Rhodobium orientis

(BRP7), Rhodobacter sphaeroides (BRP9), Rhodobacter maris

(BRP10) and Rhodobacter capsulatus (BRP12) showed complete

reduction of nitrate. With regard to sample from Sethubavachatram on

day-8 strains Phaeovibrio sulfidiphilus gen.nov., sp. nov., (BRP1 and

BRP2) and Rhodobacter sphaeroides (BRP9) could bring about total

elimination of nitrate and similar result was reported at the station

Karankadu by the strains, Rhodobacter sphaeroides (BRP3),

Rhodovulum sulphidophilum (BRP5), Rhodobacter capsulatus (BRP6),

Rhodobium orientis (BRP7), Rhodobacter sphaeroides (BRP9),

Rhodobacter maris (BRP10), Rhodovulum strictum BRP11 and

Rhodobacter capsulatus (BRP12). Total elimination of nitrate was

found not only in PNSB treated samples but also in control where no

PNSB was added.

This suggests that PNSB may not have contributed significantly

to nitrate reduction. With regard to nitrite and TAN total reduction

could be observed even in control suggests that bioagumentation with

PNSB may not have any significant effect. However the strains do

exhibit variation in their performance. With regard to the reduction of

nitrogenous parameters like nitrite and TAN their complete absence in

the samples after day-2 may be attributed to the addition of the

molasses, which might have aided in its reduction in the samples, as

observed by Avnimelech (1999), where the addition of carbonaceous

substrate might have enhanced the reduction of nitrogen in shrimp

ponds. At high carbon to nitrogen ratios (C:N) heterotrophic

microorganisms would dominate over autotrophic microorganisms and

would assimilate total ammonia nitrogen, nitrite and nitrate

130

(Avnimelech et al., 1982; 1994; Avnimelech, 1999; Browdy et al.,

2001; Burford and Lorenzen, 2004).

With regard to phosphate reduction most of the PNSB strains

reduced the level of phosphates on day-10. However the strains did not

show uniform pattern of phosphate reduction in the effluent samples

from all the 4 stations. In the stations excepting Vadakkupoygainallur,

there was a complete reduction in the level of phosphates by the PNSB

strains, on day10. The strains Rhodovulum sulphidophilum (BRP5),

Rhodovulum strictum (BRP11), Rhodobacter capsulatus (BRP12)

showed complete reduction of phosphates in the sample collected from

the station Vadakkupoygainallur on day-8. Likewise the strains

Rhodovulum sulphidophilum (BRP5), Rhodobium orientis (BRP7) and

Rhodobacter capsulatus (BRP12) showed complete reduction of

phosphates in the sample collected from the station Pappakovil and

strains Phaeovibrio sulfidiphilus gen., sp.nov., (BRP1), Phaeovibrio

sulfidiphilus gen., sp.nov., (BRP2), Rhodovulum sulphidophilum

(BRP5), Rhodobium orientis (BRP7), Rhodobium marinum (BRP8),

Rhodobacter sphaeroides (BRP9), Rhodobacter maris (BRP10) and

Rhodobacter capsulatus (BRP12) in the samples from the Karankadu

on day-8 respectively. The strain Rhodobacter sphaeroides (BRP3)

completely reduced the level of phosphates on day-6, in the sample

collected from Sethubavachatram.

Takeno et al. (1999) reported on the removal of phosphates in

oyster farm mud sediments by the purple non sulfur bacteria

Rhodobacter sphaeroides. Kim et al. (2004) reported on the removal of

phosphates by Rhodopseudomonas palustris in swine waste water. The

simultaneous removal of phosphates nitrates and COD by purple non

sulfur bacterial members like Rhodopseudomonas palustris and

Rhodobacter sphaeroides has been reported by Nagadomi et al.

131

(2000), and also suggested that photosynthetic bacteria seemed to

contribute to the simultaneous removals of nitrate and phosphate.

From the above reports, it is clear that the addition of Purple

non sulfur bacteria has had an positive influence with regard to the

reduction of polluting parameters in shrimp pond harvest discharge,

where the strain Rhodobacter capsulatus (BRP12) has aided in the

reduction of BOD and COD and it is evident that in the uninoculated

sample the reduction of polluting parameter was rather slow.

From the present study it is evident that the polluting parameters

like BOD, COD, TSS, and phosphates, can be efficiently lowered to a

considerable limit by PNSB. But with regard to TSS, even though

PNSB strains have lowered the level of TSS, the reduction cannot be

considered as significant since reduction of TSS to permissible limits

was achieved even in control. But with phosphates there was a

considerable reduction when compared to the control. However

extensive works in future in the actual pond environment, are needed

to study the effectiveness of introduced PNSB in reducing the level of

pollutants/nutrients, to use them as potential bioremediators in shrimp

culture environment.

132

5. SUMMARY AND CONCLUSION

Shrimps ponds are suitable for the growth and development of

purple non sulfur bacteria (PNSB) as there is excess accumulation of

nutrients in the shrimp pond bottom due to feed, detritus and the

excreta from the shrimps and this leads to the establishment of PNSB

members in shrimp ponds.

Besides these PNSB other heterotrophic bacteria also are

present in shrimp ponds. Among these heterotrophic bacteria the

members of Vibrio sp., cause extensive damage to the shrimps leading

to diseases in the growing shrimps. Likewise the discharges that are let

out from the shrimp ponds are rich in nutrients and pollute the nearby

marine environment, thereby causing extensive environmental damage,

which has become a matter of concern worldwide.

Hence the present study focuses its attention on the diversity of

PNSB from shrimp ponds (brackish and direct sea water) from the east

coast of TamilNadu spanning three districts viz.,

Nagapattinam,Thanjavur and Ramanathapuram and their utility as

biocontrol agents against pathogenic Vibrio spp., as well as potential

bioremidiators of harvest discharges.

The detailed study of this research include: enumeration of

PNSB from shrimp ponds (water and soil sediments), enrichment of

water and soil sediments from shrimp ponds, isolation, purification and

characterization of PNSB strains from shrimp ponds, reporting of

novel taxa if any by polyphasic taxonomic approach, microbiological

screening of shrimps and harvest discharges with reference to

heterotrophic bacteria, screening of Vibrio spp., from harvest

133

discharges, healthy and diseased shrimps, characterization and

identification of Vibrio spp., antagonistic activity of purple non sulfur

bacterial strains against Vibrio spp, extraction of crude intracellular

extracts from positive PNSB strains and bioassay against pathogenic

Vibrio spp., physico-chemical analysis of harvest discharges,

optimization studies and bioremediation of harvest discharges using

the native strains of PNSB.

The details of the research pertains to three different aspects of

PNSB, and are presented with separate introduction cum review of

literature, materials and methods, results and discussion, besides a

general introduction and summary. The various methodologies adopted

include.

Enumeration of purple non sulfur bacteria (PNSB) from shrimp

ponds:

- Paraffin wax overlay of pour plate

Enrichment of water and sediment soil samples using Biebl and

pfennigs, media

Purification of PNSB by argon argon flushing

Maintenance of PNSB stock cultures

Characterization and identification of Purple non sulfur bacterial

strains includes the following:

- Gram staining

- Flagella staining

- TEM (negative staining for flagella)

- TEM (sectioning) for ICM structures

- Whole cell absorption spectrum

- Separation of pigments by column chromatography

- Carotenoid composition

- Cellular fatty acid composition

134

- Determination of Growth

- Photolithoautotrophy

- Photoorganoheterotrophy

- Chemolithoautotrophy

- Chemoorganoheterotrophy

- Fermentative mode

- Utilization of organic/inorganic compounds as electron

donor and/or carbon source

- Utilization of various sulfur sources

- Utilization of various nitrogen sources

- Vitamin requirement

- Saline requirement and tolerance

- Growth at different temperatures

- Growth at different pH

- Gelatin liquefaction

- Indole production from L-tryptophan

- DNA mol % determination

- DNA extraction and purification

- HPLC

- Amplification of 16S rRNA gene

- Agarose gel electrophoresis

- PCR amplicon purification

- 16S rRNA gene sequencing and assembling of the 4 partial

sequences

- BLAST search

- Collection of 16S rRNA gene sequences of the type strains

- 16S rRNA gene sequence alignment

- DNA-DNA Hybridization

135

Microbiological screening of shrimps and harvest discharges with

reference to heterotrophic bacteria includes the following:

- Selection of diseased shrimps

- Selection of healthy shrimps

- Isolation and enumeration of heterotrophic bacteria from

infected and healthy tissues of Penaeus monodon.

- Isolation and enumeration of heterotrophic bacteria from

shrimp pond harvest discharge

Screening for Vibrio spp.,

- Colour of the colony on TCBS agar

Characterization and identification of Vibrio spp., includes the

following :

- Growth at various NaCl concentrations

- Decarboxylases and arginine dihydrolase test (ODC, LDC,

ADH)

- Gelatin Liquefaction

- Voges-Proskauer Test

- Indole test

- Utilization of organic substrate as carbon sources

- ONPG(o-nitrophenyl-β-galactopyranoside) test

Biocontrol of Vibrio spp., using PNSB strains includes the

following

- Preparation of crude aqueous extracts from PNSB strains

- Agar well diffusion method

- Extraction of crude intracellular extracts from positive

PNSB strains

- Bioassay of crude extracts by disc diffusion

- Bioassay of crude extracts by broth tube dilution

136

Bioremediation studies

Physicochemical analysis (pre and post bioremediation)

- pH

- Temperature,

- Biological oxygen demand (BOD),

- Chemical oxygen demand (COD),

- Total suspended solids (TSS),

- Nitrite

- Nitrate

- Total ammonia nitrogen (TAN)

- Dissolved phosphates

Optimization studies for bioreactor

- Treatment of cane molasses

- Effect of inoculum size and volume of natural carbon

source on the growth of Purple non sulfur bacterial strains in

shrimp farm harvest discharge

Enumeration of PNSB (Post bioremediation)

-Paraffin wax overlay of pour plate

Significant findings in the present study are enumerated below.

During the enumeration of PNSB from various shrimp pond

locations like inlet, culture pond, draining channel, all the soil

sediment samples showed a sizeable population of PNSB

members, but in water samples from the inlet of shrimp ponds

showed negative presence of PNSB colonies in many

instances.

137

The inlet water samples collected from brackish ponds

harbored more PNSB colonies that its direct sea water

counterpart.

The enumeration of PNSB from shrimp pond samples, yielded

brown to red pigmented colonies. The PNSB cell numbers in

the soil sediment as well as water samples ranged from 0.9 ×

10 2

to 1.0 × 104 cells/gram and 1.2 × 10

2 to 1.88 × 10

4

cells/mL respectively.

All the soil sediment samples kept for enrichment gave 100%

positive enrichment, but water samples gave only 58.2 %

positive enrichment. The percentage negative enrichments

observed in the water samples were 28.57%, 10.71% and

7.14% respectively in various locations like inlet, culture pond

and draining channel.

In the draining channel with stagnant pool of water positive

enrichments could be observed because of its anoxic nature

with sufficient quality of light. But in the case of water samples

collected from the draining channels where the culture waste

water discharge was free flowing the conditions will be oxic

(rather than anoxic) which might not have favored PNSB.

A total number of 210 pure cultures of purple nonsulfur

bacteria were obtained. Based on morphological and cultural

characterization the purified isolates were grouped under 12

PNSB strains. Among them, the PNSB strains BRP3, BRP4,

BRP6, BRP9, BRP10 and BRP12 were identified as belonging

to the Genus, Rhodobacter spp., the strains BRP5 and BRP11

as Rhodovulum spp., and BRP7 and BRP8 as Rhodobium spp.

138

The percentage distribution of PNSB are Rhodobacter spp.,

(34.28%), Phaeovibrio spp., nov. (30%), Rhodovulum spp.,

(26.19%) and Rhodobium spp., (9.52%).

The strains BRP3, BRP4 and BRP9 identified as belonging to

the genera Rhodobacter spp., was further identified to their

closest species as Rhodobacter sphaeroides. However they

were showing minor variations in cell size, carbon utilization

and Nacl tolerance.

The strains BRP6 and BRP12 showed variation in carbon

utilization which differed from the ideal biotypes of

Rhodobacter spp., and hence their species identity could not be

established using Bergey‟s manual, so the PNSB strains BRP6

and BRP12 were subjected to 16srRNA sequencing. Based on

sequence similarity, these two strains were identified as

Rhodobacter capsulatus. These strains showed saline tolerance

upto 4%.

The strain BRP10 which was characterized as belonging to

Rhodobacter spp., upto genera level, could not be characterized

at species level based on Bergey‟s manual of systematic

bacteriology (2005), as this strain could not utilize majority of

carbon sources as utilized by other members of Rhodobacter

spp. Hence this strain (BRP10) was subjected to 16srRNA

sequencing, and the sequence results, showed 100% sequence

similarity with Rhodobacter maris JA276T.

The strains BRP5 and BRP11 which showed phenotypic

similarity with Rhodovulum spp., at the genera level, was

139

tentatively identified up to their closest species as Rhodovulum

sulphidophilum (BRP5) and Rhodovulum strictum (BRP11),

based on Bergey‟s manual of systematic bacteriology (2005).

The strains BRP7 and BRP8 were characterized upto species

level as Rhodobium orientis (BRP7) and Rhodobium marinum

(BRP8) based on based on Bergey‟s manual of systematic

bacteriology (2005).

The strains BRP1 and BRP2 showed some ambiguousness with

regard to identification, which necessitated polyphasic

characterization. The characterization based on polyphasic

approach of both BRP1 and BRP2 yielded novel genera

belonging to the family Rhodospirillaceae, proposed as

PhaeoVibrio spp., gen. nov., and a novel type species of this

genera, Phaeovibrio sulphidiphilus gen nov. sp. nov.

Thus from the foregoing findings it is evident that shrimp

ponds harbours not only various species of PNSB but also

confirms the existence of diversity among them.

During the enumeration of total heterotrophic bacteria from the

harvest discharge and shrimps (healthy and diseased), samples

yielded bacterial cells ranging from 8.9 × 105 to 1.05 × 10

6

cell/mg (healthy shrimps), 1.04 × 106 to 9.30 × 10

6 cells /mg

(diseased shrimps), likewise the total heterotrophic bacteria

from harvest discharges from the four stations ranged from

1.00 × 106 to 2.13 × 10

6 cells /ml of sample.

In the present study, the shrimps showing symptoms of various

types of shrimp diseases could be observed. For example, Shell

140

disease was observed in shrimps collected from the stations

Vadakkupoygainallur and Pappakovil, Red disease could be

observed in shrimps in the station Vadakkupoygainallur ,

Pappakovil and Karankadu, necrosis in the abdomen could be

observed in the stations Vadakkupoygainallur and Pappakovil,

loose shell in Sethuvbavachatram and Karankadu, White spot

disease could be found in only one station viz.,

Sethubavachatram and erratic swimming in diseased shrimps

could be observed in two stations viz., Sethubavachatram and

Karankadu.

A total number of 66 pure culture of Vibrio spp., were

characterized and identified. The species identified include,

Vibrio fischeri, V. mediterranei, V.harveyi, V.orientalis,

V.splendidus, V. logei, and V. alginolyticus.

The percentage of prevalence of various strains of Vibrio spp.,

in all the 4 stations are as follows, Vibrio fischeri (7.69%),

Vibrio mediterrranei (28.84%),Vibrio harveyi (34.61%), Vibrio

orientalis (3.84%), Vibrio splendidus (7.69%),Vibrio logei

(13.46%) and Vibrio alginolyticus (3.84%).

Among the pathogenic Vibrio spp., isolated from diseased

shrimps Vibrio harveyi was dominant.

Among the 12 PNSB strains screened for antagonistic activity

by agar well diffusion method, against the strains of pathogenic

Vibrio spp., viz., V. harveyi, V. fischeri, and V. alginolyticus,

only 5 PNSB strains showed antagonistic activity. The positive

PNSB strains are as follows, Rhodobacter spheroids (BRP9),

Rhodobacter capsulatus (BRP12), Rhodobium marinum

141

(BRP8), Rhodovulum sulphidophilum (BRP5) and Rhodobium

orientis (BRP7). However the last two species were negative in

their antagonistic response to V.alginolyticus.

Rhodobacter sphaeroides (BRP9) was found to be most

effective among the positive PNSB strains with regard to

antagonistic activity against the pathogenic Vibrio spp., by disk

diffusion method, using the E1 solvent

(chloroform:methanol:water) extract.

From these findings, it is clear that intra cellular extracts of

Rhodobacter sphaeroides strains can be considered as potential

vibriostatic agent in controlling vibriosis in penaeid shrimps.

The harvest discharges when subjected to physico-chemical

analysis showed that TSS was highest followed by COD, BOD,

nitrate, phosphate, nitrite and Total ammonia nitrogen.

In the present study which focuses on the bioremediation of

harvest discharges using PNSB strains, upon optimization, it

was found out that a 10% inoculum and 0.2% of raw carbon

source was optimal, to carry out bioremediation of harvest

discharges.

The PNSB strain Rhodobacter capsulatus (BRP12) was most

effective in bringing down the level of BOD and COD in the

harvest discharges.

Among the PNSB strains Rhodovulum sulphidophilum (BRP5)

exhibited maximum reduction of TSS in the harvest discharges.

142

PNSB strains were not effective in bringing down the level of

nitrate, nitrite and TAN. However the strains do exhibit

variation in their performance.

With regard to the reduction of nitrogenous parameters like

nitrite and TAN their complete absence in the samples after

day-2 may be attributed to the addition of the molasses.

With regard to phosphate reduction most of the PNSB strains

reduced the level of phosphates on day-10. However the strains

did not show uniform pattern of phosphate reduction in the

effluent samples.

CONCLUSION

The shrimp ponds harbor PNSB members and there exists

diversity among the PNSB populations. PNSB members which are

termed as strictly fresh water species have also been isolated from

shrimp ponds, indicates that purple nonsulfur bacterial members adapt

and thrive in diverse marine environments. In the current process of

studying the diversity of PNSB in shrimp ponds, a novel PNSB

member has been identified by polyphasic taxonomic approach, which

signals that much more intensive explorations has to be undertaken to

bring out various unreported PNSB species that may lay hidden in

these manmade environments. The studies on the potential application

of PNSB strains gives us an insight into their vibriostatic as well as

bioremediating potentialities.

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