Temporal and Spatial Distribution of Sulfate Reducing Bacteria in Shrimp Culture Pond Sediment

Sulfate reduction, a key process in aquatic sediments is carried out by a group of anaerobic microorganism called sulfate reducing bacteria (SRB). High numbers of sulphate reducers in shrimp aquaculture pond sediment deteriorates the soil and water quality, causing physiological stress, thereby reducing the immunity of cultured animal. An attempt was made to evaluate the temporal and spatial distribution of SRB horizontally at different locations viz., water pumping area (WPA), sluice gate (SG) and pond center (PC), and vertically from sediment water interface to 10 cm depth in Pacific white shrimp, Penaeus vannamei culture ponds. Physico-chemical characteristics of water and soil were correlated with the number of SRB. Distribution of SRB was significantly higher (p ≤ 0.05) in the pond sediment nearer to SG (874 MPN/g), followed by PC (272 MPN/g) and WPA (99 MPN/g) and at sediment water interface (751 MPN/g) compared to 10 cm depth (114 MPN/g) of Original Research Article Thulasi et al.; AJEE, 3(4): 1-16, 2017; Article no.AJEE.35287 2 pond profile. Factors like dissolved oxygen and soil redox potential (oxidation to reduction scale), and organic carbon content in soil had a significant negative and positive correlation with SRB numbers. Phylogenetic relationship of SRB targeting SRB groups showed the presence of Desulfonema, Desulfosarcina, Desulfatibacilum, Desulfobotulus, Desulfomicrobium, Desulfococcus, Desulfovibrio and Clostridium genera in shrimp culture pond sediments.


INTRODUCTION
Pond ecosystem is a complex environment in which, microorganism present in water, sediment and gut of the cultured animal interact each other which in turn has impact on growth, survival, nutrient cycling and disease control [1,2]. Walker and Winton, [3] explained that poor soil and water quality along with bacterial and viral pathogens are the main cause for mortalities in shrimp industry. High nutrients load derived from left over feed and fecal materials that accumulate on pond bottom are a major concern, which leads to the formation of toxic compounds like NH 3 , NO 2 and H 2 S. These toxic compounds deteriorate the water and soil quality, causing physiological stress thereby reducing the immunity of cultured animal [4].
In shrimp culture pond sediments, sulfate reduction is a major process which is carried out by a group of anaerobic microorganisms called sulfate reducing bacteria (SRB). This leads to the formation of hydrogen sulfide, a colorless gas with rotten egg smell and buildup of this toxic substance affects the shrimp health, thereby decreasing the production. Hsu and Chen [5]. documented that P.vannamei is more susceptible to Vibrio sp., and considerable reduction in immune parameters was found under sulfide stress. Generally in organically enriched sediments, the rate of sulfate reduction depends on factors like availability of organic compounds [6], depth related gradient [7] and the quality and quantity of accumulated organic matter [8]. Oxygen acts as a barrier for the movement of reduced toxic compounds into the water column [9.10]. In aquatic sediments, as oxygen is consumed in 1-5 mm layer, sulfate reduction process becomes dominant in sediment water interface (SWI) and deeper layers [11]. All the factors mentioned above are not uniformly distributed and varies at different places in a pond and also from the neighboring ponds, the cause for variation in sulfate reduction and SRB population.
Several studies have focused on studying the diversity and composition of SRB in environments such as hyper saline lakes [12], freshwater [13], marine sediments [14], estuarine sediments [15] and aquaculture fish ponds [16,17]. Few reports pertaining to the population of SRB in shrimp culture ponds are available with MPN (Most probable number) quantification [18,19]. In the previous studies, it was found that higher SRB in the range of 0.8-4.4 × 10 9 cells g -1 dry sediment was found in fish farm sediment and it was positively correlated with organic enrichment in the pond sediments [20]. Similarly, SRB in the range of 9,300-42,000 cm −3 was detected in the sediment of experimental ponds stocked with penaeid shrimp and fish [21]. Other than this MPN quantification, studies on distribution of SRB in different places of the pond and their phylogenetic relationship in shrimp culture sediments are not available. The fact that only few studies are available with regard to SRB and its distribution in shrimp culture sediments, emphasizes the need for more studies on SRB and the role various pond environmental factors in influencing the process of sulfate reduction. Hence, we made an attempt to study the phylogenetic relationship and distribution of SRB in shrimp culture ponds. In this study, temporal and spatial distribution of SRB horizontally at different places and vertically along the depth in brackishwater shrimp culture ponds. In addition to this, various pond environmental factors that influence the sulfate reducing bacteria was studied.

Sample Collection
Sediment samples were collected from three brackishwater shrimp culture farms located in Mamallapuram, Tamil Nadu, India. From each farm, four ponds were selected for this study. Pacific white shrimp Penaeus vannamei was the cultured species with a stocking density of 40 to 45 nos /m 2 in each pond and the culture period was 95 to 100 days. All the ponds followed zero water exchange culture with standard management practices. To study the horizontal and temporal distribution of SRB in the culture pond, sediment samples were collected from three locations viz., water pumping area (WPA), sluice gate (SG) and pond center (PC) at different days of culture (DOC) throughout the culture period (22, 37, 51, 65, 80 and the day of harvest). In addition, to study the temporal and vertical distribution of SRB, sediment samples were collected from the above mentioned locations at three time intervals (22 & 51 DOC and harvest time, by inserting PVC core sampler from SWI and cores of 0-0.5 cm, 0.5-1.5 cm, 1.5-2.5 cm, 2.5-5.0 cm, 5.0-7.5 cm and 7.5-10 cm were separated. In order to have a uniform representative sample, soil samples of each core of same depth from all the sampling points in a pond were pooled together. All the samples were transported to laboratory in ice cold condition.

Sediment and Water Quality Analysis
Measurement of pH, dissolved oxygen (DO) salinity and turbidity in pond water, and redox potential (E h ) in pond sediment was done onsite using multi-parameter water quality analyzer (Hach, USA) at all the sampling places. Water samples were analyzed for total sulfide-S, nitrite-N, nitrate-N, total ammonia nitrogen (TAN) [

Cultivation and Enumeration of SRB
Cultivation of SRB was done using Postgate medium B with lactate as electron acceptor [25] Resazurin (0.001 g/L) and thioglycollic acid (0.1 ml/L) were added as oxygen indicator and reducing agent, respectively. pH of the medium was adjusted to 7.5 and sterilized by autoclaving at 121°C for 20 min. A 5% ferrous ammonium sulfate was filter sterilized and added separately at 0.1 ml for every 5.0 ml of the autoclaved medium. Enumeration of viable SRB was done by MPN method using three tube MPN method following FDA-BAM protocol [26]. Headspace oxygen was removed completely by passing oxygen free nitrogen and incubated for the period of two weeks. Formation of black colour precipitate was taken as positive growth and quantification of SRB was done using FDA-BAM table. To study the phylogenetic relationship of SRB, 5 ml of culture from all the highest positive MPN dilutions were transferred to the enrichment media and cultured as mentioned above.

Cloning and Sequencing
After PCR amplification, unpurified products were purified and cloned with TA (pcrII) cloning vector. The ligation products were introduced into E.coli Top10 chemically competent cells, and these cells were plated onto LB medium containing ampicillin and incubated overnight at 37°C. White colonies were randomly selected and screening of positive clones was performed. Clones were analyzed for insert and the purified clones were sequenced using ABI 3730 sequencer.

Sequence Analysis
Sequences containing chimeras were checked with Decipher [28]. Sequences were aligned using MUSCLE program [29] and aligned sequences were grouped into Operational Taxonomic Units (OTU) with 97% as the distance cut-off value [30] using RDP (Ribosomal database project) pipeline tool [31]. From each OTU only one representative sequence was taken to construct phylogenetic analysis. Neighbor joining method was used to construct the phylogenetic tree using, MEGA 5.2.2 [32] Bootstrap analysis was performed with 1000 replicates to verify the reliability of the constructed phylogenetic tree.

Nucleotide Accession Number
Representative sequence from each OTU was published in DDBJ database under the accession numbers AB983657-AB983706.

Statistical Data Analysis
One-way repeated measures ANOVA was performed to find out the significance in variation of SRB between different places of the pond and days of culture using SPSS 16.0. Pearson correlation analysis was done to find out the relation between physico-chemical parameters of water and soil and SRB population.

Changes in Physico-chemical Parameters of Water and Soil
The average water and soil parameters at different days of culture (DOC) in the culture pond are given in Table 1 and the trend of significant parameters at different sampling places in a pond is shown in Fig. 1 through 4. pH, DO (ppm), TAN (ppm), total sulfide-S (ppm) and sulfate-S (ppm) content in water during the culture period ranged from 6.78-8.31, 5.43-8.69, 0.055-0.174, and 0.004-0.09 and 1367-1626, respectively. There was no significant difference between days of culture with respect to pond water pH, salinity, turbidity, TAN, nitrite-N, nitrate-N and sulfate-S (p ≥ 0.05), whereas DO and total sulfide content in pond water and, redox potential and organic carbon content in pond soil differed significantly (p ≤ 0.05) with the progress of culture period (Table 1).
Redox potential in pond soil ranged from 150 to -167 mV during the culture period. Soil redox potential (E h ) which is the indicator of soil health, was in the positive side 150 & 61.6 mV at the start of the culture i.e. at DOC 22 and 37 respectively, and then it gradually decreased to negative values towards the end of the culture (-123 ± 53 mV). This shows that bottom sediment was in oxidized state during the early phase of culture, which facilitates the aerobic decomposition of organic matter rather than anaerobic decomposition. During the early phase of culture, accumulation of organic carbon and availability of substrates will be less, the reason for the E h values on oxidation scale, whereas E h values on reduction scale during the later phase of the culture period, is due to the excess availability of substrates obtained from accumulation of unused feed and fecal material. It is evident by the increase in organic carbon content in pond soil from 0.2% at DOC 22 to 0.41 % at harvest time. Wiyito et al. [4] studied the effect of redox potential in which it was found that negative redox potential generates reduced compounds specifically when the redox value is above -200 mV. It reduces the dissolved oxygen concentration in SWI and increases the sulfide concentration in water column.

Changes in Physico-chemical parameters of water and soil at different places of the pond
When a different place within a pond is taken into account, E h was highly variable between the three places. Lowest average E h values of -19.83, -9.3, -167 and -123 mV were observed nearer to SG at DOC 51, 65, 80 and harvest time, respectively ( Fig. 1). Similar trend of variation was observed in E h values at the other two sampling points at different days of culture. Similarly, total sulfide content was highly variable at three places with high value at SG (0.116 ppm) followed by PC (0.094 ppm) at harvest time ( Fig. 2). DO content varied between sampling points within the range of 4.37 to 8.34 mg/L. On the whole, low DO values were found nearer to SG at DOC 65 (4.4 mg/L) and DOC 80 (4.37 mg/L) compared to PC at DOC 65 (5.37 mg/L) and DOC 80 (8.21 mg/L) and WPA at DOC 65 (6.42 mg/l) and DOC 80 (8.31 mg/l) (Fig. 3). Organic carbon content in soil ranged from 0.13-0.65% throughout the culture period. Fluctuations were observed between sampling points with lowest organic carbon content at WPA (0.13%) during early days of culture and high nearer to SG (0.65%) at DOC 80 (Fig. 2). Overall, there existed a significant difference (p< 0.05) in organic carbon, redox potential, dissolved oxygen and sulfide levels between the three places of the pond. Significantly high organic carbon content (0.49%) and sulfide (0.05 ppm) and redox potential on reduction scale (-102 mV) and low DO (5.63 ppm) were observed at sluice gate compared to other two sampling places ( Table 2).

Changes in Physico-chemical parameters along the depth of pond
In case of vertical distribution, OC was fluctuating along the depth and there was no particular trend observed throughout the culture period. Comparing all the cores, OC content (%) was found to be high in SWI (0.5 cm) at DOC 22 (0.26 ± 0.03) and at harvest time (0.55 ± 0.03), whereas at DOC 51 high OC was found in 1.5 cm cores (0.61 ± 0.014) compared to 0.26 ± 0.04 in SWI (Fig. 4). Likewise sulfate content in soil was also highly variable along the depth, throughout the culture period. Maximum sulfate concentration (mg/L) was observed in 5.0 cm core at DOC 22 (1218± 29.69), 7.5 cm layer at DOC 51 (1733 ± 32.10) & 1.5 cm layer at harvest time (1354± 16.37) (Fig. 4).

Horizontal distribution of SRB at different places of the pond
Distribution of SRB varied between different places of the pond, high numbers registered at SG followed by CP and WPA (Fig. 5

Vertical distribution of SRB along the depth of pond
The average number of SRB at DOC 22, 51 and harvest time was 30, 431, 684 MPN/g, respectively (Fig. 7). On DOC 22 from SWI to 10 cm the number of SRB was 53 MPN/g at SWI and decreased up to 0.5 cm (30 MPN/g) and then remained constant throughout 10 cm cores. At DOC 51, number of SRB was higher in 0.5 and 1.5 cm cores (˃1100 MPN/g), decreased from 2.5 to 5.0 cm (150 MPN/g) and further decreased to 30 MPN/g from 7.5 to 10 cm cores.
In contrast, at DOC 95, number of SRB was found to be constant (˃1100 MPN/g) in 0.5-2.5 cm cores and then decreased to 290 MPN/g in the remaining cores. A significant difference was found between the numbers of SRB (p ≤ 0.05) along the depth of the pond except in 0.5 cm and 1.5 cm cores (Fig. 8).
On the whole, number of SRB was found to be high nearer to SG followed by PC and WPA and their distribution along the depth is found to be decreasing with high numbers at SWI. Similar kind of result, with maximum number of SRB in top 3 cm layer (6.6 ± 1.0×10 8 cells cm -3 ) and then decreasing numbers below 3cm layer (1.7 ± 0.8×10 6 cells cm -3 ) was observed in Wadden Sea sediment [33]. Urakawa et al. [7] studied the variation of microbial communities along the depth in marine sediments. They observed large number of SRB in surface layers compared to the deeper depth because of organic matter availability. Generally SRB is expected to be high in deeper sediments due to anaerobic condition in lakes and marine waters. During shrimp culture, unused feeds and waste materials will be deposited on surface sediments. In the presence of sulfate, these complex macromolecules will be utilized by SRB whereas, in absence of sulfate, SRB ferment organic acids through fermentative bacteria by which fermentation compounds like Bars represent means ± SD lactate, acetate and propionate will be produced. Holmer and Storkholm [34] observed high sulfate reduction rate in surface layers of fish farmed sediments, where accumulation of organic matter was high. Thus, SWI provides ideal condition for the anaerobic dissimilatory sulfate reduction and fermentative growth of SRB. Presence of sulfide levels of 0.004, 0.028 and 0.09 ppm during DOC 22, 51 and harvest time, respectively (Fig. 2) indicates the effective sulfide production in the pond sediment.

Correlation between SRB and Physico-chemical Parameters of Pond Water and Soil
Significant negative correlation was found between SRB numbers and dissolved oxygen (r = -0.633) and redox potential values from oxidation to reduction scale (r = -0.672). A significant positive correlation was found between SRB numbers and organic carbon content (r = 0.812) and sulfide in water (r = 0.525). In case of vertical distribution, significant positive correlation was found between organic carbon in soil and SRB (r= 0.651) and a weak correlation (r= 0.109) was observed with sulfate. Many studies have reported that organic carbon as a main limiting factor in shaping the abundance and distribution of SRB. Kondo et al [35] studied SRB from Japanese fish farm sediments with different level of organic enrichment in which fish pond with high organic pollution found to be more diverse in SRB. Kawahara et al [17] observed that organic enrichment influences the bacterial communities and clones related to SRB were found to be in higher frequency from organically enriched surface sediments (0-4 cm) of fish farm in Wakasa Bay of Japan. Santander et al. [36]  d studied the bacterial communities in milk fish cages from Philippines in which they observed many sulfate reducing bacteria and other spore forming genera. They further explained that organic load derived from higher fish feed and its left over constituents, created reduced environment through which anaerobic bacteria flourish. It is evident from this study that organic matter accumulated in the form of unused feed and faecal materials play a major role in sulfate reduction.

PCR Amplification
DNA was isolated from highest positive (˃1100/g) MPN dilutions and amplification of partial fragments of 16S-rDNA genes using group specific primer yielded expected base pair in all the enrichment cultures for group 5 & 6. No bands were found in groups 1, 2, 3 and 4. More than 100 clones in group 5 & 6 were screened for an insert. Twenty seven clones in group 5 and 43 clones in group 6 were positive. All the 70 clones were sequenced in which eight sequences were found to be chimeric and removed from further analysis. The remaining clones were grouped into 50 OTU's (16 in group 5 & 34 in group 6) using 0.03% as distance cutoff value.

Desulfovibrio
spp., which belongs to deltaproteobacter, is dominant in organic rich marine habitats. In the presence of sulfate, fermentation products like lactate, malate and fumarate can be used as electron donors and even in the absence of sulfate, it can utilize pyruvate and formate [40]. Saas et al [41] studied the vertical distribution of SRB in the oligotrophic lake sediments, in which many strains related to Desulfovibrio sp., and Desulfomicrobium sp., were found in oxic-anoxic layers and these bacteria, being catalase positive organism, shows higher oxygen tolerance capacity and they are well adapted to live near sediment water interface. Desulfovibrio has also been isolated commonly from corals affected by Black Band disease [42,43]. Around eleven clones related to Desulfovibrio desulfuricans (with 97% similarity) were isolated from diseased corals in Northern Red sea [44]. Interestingly two clones DSV17 & DSV19 isolated in this study were similar to Desulfovibrio sp., isolated from coral montipora in Japan.
Cluster 3 had six clones (DSV32, 36, 34, 23, 20 and 33) in which DSV32 and DSV23 formed monophyletic cluster with Clostridium sp., (AB470961) and Clostridium aestuarii (DQ126679) isolated from tidal flat sediment which is closely related to type species Clostridium butyricum. DSV36 & DSV34 formed independent lineage within family Clostridiales, a group of spore forming bacteria under phylum Firmicutes which have the potential to switch to an energy conserving metabolism with low sulfate or even with no sulfate [15]. Tamminen et al, [45] also observed clones similar to Clostridia from Rainbow trout fish farms with black muddy sediment near Baltic Sea. It is further explained that clones related to Clostridia, more likely to have originated from fish gut micro biota. Moreover, this is possible because many of the soil probiotics used in aquaculture contains Clostridium butyricum as one of the essential component. It is interesting to note that Clones related to group Desulfotomaculum (Group-1), Desulfobulbus (Group-2), Desulfobacterium (Group-3), Desulfobacter (Group-4) which are reported to have isolated from both marine and fresh water sediments are completely absent in this study. This is possibly because of substrate availability and organic enrichment in the shrimp culture sediments. Kawahara et al, [17] suggested that composition and activity of SRB is influenced by amount of organic enrichment in aquaculture. Castine et al, [46] studied the effect of organic load in tropical finfish cage farm in which, they found genus related to Desulfovibrio, Desulfobacter, Desulfococcus, Desulfonema in higher abundance. They further suggested that anaerobic zones created by organic matter load plays a key role in selection of bacteria involved in sulfate reduction. Understanding the microbial interaction in pond bottom is necessary in shrimp aquaculture because more than any other aquatic species, shrimp dwell on the bottom and ingest pond-bottom soil [47]. Avnimelech [48] estimated that 50% of the pond bottom in shrimp culture is covered by reduced sediment with a typical black color and smell of hydrogen sulfide which affects the shrimp growth, activity and health.
Abraham et al, [19] studied the population of SRB in shrimp culture systems of India, in which significant difference was found in population of SRB in semi intensive and traditional ponds because of varying degree of substrate availability and anaerobic processes. Suplee and Cotner [18] studied sulfide fluxes in shrimp ponds over a period of 17 months during which they found organic matter availability and its reactivity was the predominant factor in determining the sulfide fluxes in shrimp pond. They also compared sulfate reduction in old and newly constructed shrimp ponds, in which significant difference was observed between two ponds because of higher organic matter deposition in old ponds over the years.
Farm management practices which includes stocking, feeding, and inputs application during the culture period plays a key role in shaping up the microbial community in pond bottom. Generally in shrimp culture sediments, as the days of culture increases, pond bottom becomes more and more reduced due to application of feed rich in protein, accumulation of nutrients, unused feed and fecal materials. This provides suitable environment for anaerobic microorganism to flourish and in addition to this, brackishwater shrimp ponds contain high level of sulfate in sea water which provides enormous scope for sulfate reduction to take place. Moreover, in organic matter rich sediments, methanogenic archaea compete with SRB for common substrates like acetate, H 2 and other organic compounds. Since, SRB has more affinity towards these common substrates, they generally out compete methanogens which further favours the process of sulfate reduction [49].
Despite high saline sediments having huge scope for sulfate reduction; there are studies which demonstrated sulfide production in low saline aquatic sediments [50,51]. Liu et al, [52] quantified the sulfate reducing prokaryotes in paddy sediments. They observed sulfate reduction in rice paddy sediments equal to marine sediments and it is further mentioned that in low sulfate environments, sulfate reduction happens via fermentative growth of SRB. This indicates that sulfate reduction will be an equally important process even in low saline shrimp culture sediments as the organic matter accumulation and anaerobic condition are same vertically from SWI.

CONCLUSION
In this study, we evaluated the distribution of SRB horizontally and vertically and its phylogenetic diversity brackishwater shrimp culture ponds. In conclusion, SRB was higher nearer to SG followed by PC and WPA and at sediment water interface compared to deeper depths. Factors like dissolved oxygen in water, redox potential and organic carbon content in soil had a key role in sulfate reduction and were correlated with SRB numbers. Genera like Desulfonema, Desulfosarcina, Desulfatibacilum, Desulfobotulus, Desulfomicrobium, Clostridium, Desulfococcus and Desulfovibrio were found to be present in brackishwater shrimp culture sediments. Future investigations with regard to the role of various organic matter sources on sulfate reducing bacteria communities will certainly help in understanding the process of sulfate reduction and its mitigation in shrimp culture sediments.
to Director, CIBA for providing the necessary facilities to carry out the research.