Vertical distribution of bacteria populations at the finfish marine culture in North Bali Coast, Indonesia

. One of the indicators of aquatic biology is the bacterial population. (cid:3) This study was conducted to determine the vertical distribution of bacterial populations in coastal waters at the marine fish farming in North Bali. Three locations of water samples in depths of 5, 10, 20, and 30 m samples were determined. They are shoreline near the hatcheries at Penyabangan and Gerokgak and water near the net cages at Pegametan Sumberkima. Three replications of the sample collection were done in the surface, middle, and bottom at each sampling point. Samples were collected monthly during the two seasonal transitions from April to June and August to October. The distribution of bacteria was determined by the culture-dependent method (CDM) by measuring the number of bacteria based on bacterial cultivability on agar media. The results showed a lower bacterial population (10 1 -10 2 cfu/mL) was observed in August. An even distribution of total bacteria (10 3 cfu/mL) was seen at Pegametan Bay. Higher Vibrio spp. was found at a depth of 10 m than 20 and 30 m. While Pegametan Bay station had the highest Vibrio spp. number. Halotolerant bacteria tend to increase to 5 x 10 2 cfu/mL from April to June at a depth of 5.


Introduction
Global marine aquaculture is experiencing rapid development, including Indonesian aquaculture.The marine fish farming center is located in Nort Bali at Gerokgak District, Buleleng Regency.The growth of the aquaculture business in Gerokgak has contributed to the district's local revenue, job opportunities, and increased community income [1].However, the growth of mariculture businesses has also documented adverse effects on the water conditions [2].Using processed feed with high protein content in intensive cultured systems produces waste products in the form of dissolved compounds containing phosphorus or nitrogen and solid phase phases containing suspended solids [3,4].Solid phases in wastewater usually contain total nitrogen (TN) up to 7%-32% and total phosphorus 30%-84% [5].The environment receives high organic carbon and nutrients from feeding activity Corresponding author: kmahardika@yahoo.comthrough remaining feed, fecal production, or fish excretion and respiration [6].Several studies informed that C, N, and P, from feed about 23%, 23 and 53% are accumulated at the bottom of the sediment and have a severe impact in its neighboring area up to 1 -1.5 km from the farm.At the same time, C and N compounds will support the production of bacteria in the sea [7].
Pathogenic bacteria are one of the bioindicators of water contamination [8].Our earlier research discovered that the marine aquaculture center in Gerokgak District had significant bacteria and Vibrio spp.populations from March to May and from October to November.Seasonal shifts, such as going from the rainy to the dry season or vice versa, are directly associated with fluctuations in the bacterial population [9].The Gerokgak District's coastal waters exhibit bacterial variations characterized by rising ammonia levels and falling water temperatures [10].According to [11], the vertical distribution of TN, TP, and ammonia concentrations in coastal waters in the Gerokgak District is largely homogenous.This condition occurs up to a depth of 30 m with a distance of 900 m from the shoreline, which is also an outlet for fish farming activities.It was also reported that the vertical distribution of TN, TP, and ammonia concentrations in marine fish farming areas tends to have the same trend as the seawater temperature in that area.This condition leads to a question on the vertical population of planktonic bacteria in the water collum.Considering that the hatcheries in Gerokgak Village draw water from the bottom of the coastal waters with depths ranging from 4.1-9.6 (average 6.25 m), the determination of vertical distribution of bacterial population in the coast near the center of fish farming in North Bali need to be studied.

Sampling stations and periods
This research was conducted in 2019 at the center of fish marine farming in Gerokgak District, Buleleng Regency, Bali Province.Water sampling was carried out with water quality measurements from our previous study [11].The study was divided into two sampling periods following the sea surface temperature distribution pattern previously reported by [10].The first period was in April, May, and June, when the pattern of changes in sea surface temperature gradually changed from high to low-temperature conditions.The second period was August, September, and October, when the seawater temperature moved from low to high.Sampling was done once a month.The three selected areas are coastal waters in Gerokgak Village (Station A) and Penyabangan Village (Station B) which are hatchery areas for grouper (Epinephelus spp.), white snapper (Lates calcarifer), and milkfish (Chanos chanos), and Pegametan Bay (Station C), Sumberkima Village which is a grow-out area for grouper and white snapper in floating net cages.

Sampling point distribution
The sampling coordinate point was determined following the determination of the coordinates reported by [11] using an echosounder, and the position coordinates of each depth point were detected using a global positioning system (GPS).At the Gerokgak and Penyabangan locations, the sampling points were determined perpendicular to the shoreline at 5, 10, 20, and 30 meters (Figure 1A & B).
In Pegametan Bay, three sampling points were determined around an active floating net cage with a distance of 100 m between sampling points (Fig. 1C), according to Septory et al. [11].Water sampling was carried out on the surface, middle, and bottom of the water using a Nansen water sampler.The middle layer for sampling was calculated as half of the in situ sampling depth at each depth.

Collection of water sample
Water samples were collected at one point in each coordinate (depth 5, 10, 20, and 30 m).Water from the top, middle, and bottom were taken at 10 m, 20 m, and 30 m depth coordinates.Water at 5 m depth coordinate only taken from surface and bottom.The water sample from the Nansen water sampler was put into a 15 mL screw cap tube in a sterile condition.Sample from each point the coordinates were stored on separate tubes.During transportation, the sample was carried using a cold box for about 2 hours at a temperature of 4°C.Sample from each point the coordinates were stored on separate devices.Bacterial enumeration was processed at the Pathology Laboratory at the Institute for Mariculture and Fisheries Extension, Gondol, Bali.

Culture-dependent method (CDM)
The culture-dependent method was performed by serially diluting 1 mL of the sample into 9 mL of sterile seawater.The diluted sample was homogenized by vortexing for about 10 seconds.The solution from the end dilution was taken for bacterial enumeration.The medium used in this research were tryptic soy agar (TSA) with 2% NaCl for total bacteria, tryptic soy agar without NaCl for halotolerant bacteria, and thiosulfate citrate bile salts-sucrose agar (TCBSA) for Vibrio spp., and MacConkey agar for rod shape gram-negative bacteria.Each medium was inoculated with 100 uL of solution, then incubated at 30°C for 24 hours.The grown colonies were counted after incubation using a colony counter.
Total bacteria and Vibrio spp.were isolated from seawater samples at depths (coordinates) of 10, 20, and 30 m with three samples per coordinate covering surface, middle, and bottom water.Meanwhile, water samples for halotolerant and rod-shaped gram-negative bacteria were isolated from seawater at 5 and 10 m depths with a total sample of 2 per depth, including surface and bottom water.

Data analysis
Determination of the bacterial number was counted using the formula: Number of colonies (cfu/mL) = colonies growth in the medium x P x 10 Noted: P = dilution 10 = volume of inoculated samples cultured in the medium (100 µL converted to mL = x10) The number of colonies (cfu/mL) was transformed into log10 and presented in graphical form.

Results and discussion
The total bacteria in Gerokgak, Penyabangan, and Pegametan, stations have a highly similar distribution at a depth of 10, 20, and 30 m.Total bacteria from the Gerokgak Station, Penyabangan Station, and Pegametan Station have almost the same distribution among the depths (10, 20, and 30 m).The total bacteria from surface, middle, and bottom water also had the same distribution (Figure 2).The low bacterial population (10 1 -10 2 cfu/mL) was seen only in August and varied between (10 2 -10 4 cfu/mL) in other months.Total bacteria in Pegametan Bay had almost the same value in the 10 3 cfu/mL range within three sampling points.The increase in total bacterial values occurred in May and some in September.Fig. 2. Vertical distribution of total bacteria during April to June and August to October 2019 at the three sampling stations: Gerokgak (A), Penyabangan (B), and Pegametan Bay (C) PB 1; sampling point 1, PB 2; sampling point 2, PB 3; sampling point 3.
The population of bacteria from Gerokgak station is almost equal to that of bacteria from Penyabangan station, with an average value is 10 2 cfu/mL.The population of these bacteria is lower than the population of bacteria in Pegametan Bay (average of 10 3 cfu/mL).
Total Vibrio spp.fluctuates between surface, middle, and bottom water (Figure 3).Likewise, higher total Vibrio spp. was seen at a depth of 10 m than at 20 and 30m.The highest number of Vibrio spp.occurred in June at Gerokgak Village and Penyabangan Village, especially in the middle and bottom water.While the highest number of Vibrio spp. in Pegametan Bay occurs in May in all water columns.The high number of Vibrio spp. in May-June is influenced by decreasing seawater temperatures [10].Generally, water from Pegametan Bay had the highest total Vibrio spp.This is probably due to the sampling points near the floating net cages.The pattern of total bacteria showed similarity to the previous study in 2018.The similarity was also demonstrated by Vibrio spp.This pattern was reported to follow the trends of temperature and ammonia [9].It is further reported that the total population of bacteria and Vibrio spp. in the Gerokgak station, Penyabangan station, and Pegametan Bay are still within normal limits with a range from 10 3 -10 6 cfu/mL and Vibrio spp.ranged from 10 2 -10 3 cfu/mL.In our previous study, V. Harvey, V. alginolyticus, and V. parahaemolyticus were the main Vibrio spp species observed in coastal waters in the marine fish culture centers in Gerokgak District [9].The three Vibrio spp species have been reported to be pathogenic in farmed fish [12].V. harveyi is a severe pathogen of fish and invertebrates [13].Vibriosis infection can occur in hatcheries and grow-out fish in floating net cages, but consumptionsized fish are more susceptible to this disease.This is influenced by various factors, especially fish sources and the condition of the environment, such as water quality, aquaculture practice, and virulence factors of Vibrio spp.[14].Vibrio spp.found in fish at hatcheries and floating net cages in Gerokgak District were reported to cause wounds or ulcers [15].Fish infected with Vibrio spp.show similar clinical signs reported by [16], such as loss of appetite, lethargy, and ulcers on the skin.The other bacteria found in coastal waters in Gerokgak District in 2018-2019 were Bacillus cereus, and Staphylococcus sciuri [9].Therefore, in this study, observations of Vibrio spp.were focused on the fluorescent Vibrio (V.harveyi).The results showed that V. harveyi was found in coastal waters at three sampling locations (Figure 4).V. harveyi was found in April, May, and October in coastal waters in Gerokgak Village, and in April, May, June, August, and October in coastal waters in Penyabangan Village.However, V. harveyi was found in Pegametan Bay throughout the sampling month with almost equal distribution in all parts of the water column.This indicates that V. harveyi has been present in the coastal waters of North Bali with a population of 1,0 x 10 1 to 1,1 x 10 2 cfu/mL).
Halotolerant bacteria isolated from 5 and 10 m depths in Gerokgak station and Penyabangan station and three sampling points in Pegametan station (surface and bottom water) showed an abundance of halotolerant bacteria in these waters up to 5 x 10 2 cfu/mL (Figure 5).Halotolerant bacteria were found at all sampling locations from April to June and October.These bacteria were detected in coastal waters from the rainy to the transitional seasons.Halotolerant bacteria can grow without or without salt, even in high concentrations.They keep a low concentration of ions to synthesize solutes to stabilize the osmotic level in the cytoplasm and the outside medium.This mechanism helps them adapt to changing saline environments [17].The existence of halotolerant bacteria indicates the movement of organic matter from land to marine waters, and the population of these bacteria is generally lower than that of heterotrophic bacteria.The organic matter accumulation in coastal water at North Bali was shown by particulate organic matter (POM) values between 75.20 ± 2.57 and 92.97 ± 0.59% [10].The lower density of halotolerant bacteria indicates that activities on land do not affect marine waters, and organic elements from the sea are very high [18].Halotolerant bacteria such as Halomonas spp., Marinobacter spp., Pseudomonas spp., Vibrio spp., Enterobacteriaceae, Clostridium spp.and Corynebacterium spp.[16,19,20].
Gram-negative rod-shaped bacteria populations were found in Gerokgak Station, Penyabangan Station, and Pegametan Bay (Figure 6).The population of rod-shaped gramnegative bacteria was found to be more in August and September, which is the dry season.The distribution of gram-negative rod-shaped bacteria from Penyabangan station was seen to a depth of 5 m, while in Gerokgak station, it was found to a depth of 10 m.Water layer stratification might contribute to the vertical exchange of heat, carbon, and oxygen, potentially changing microbial life [21].The bacteria in the highest population (7 x 10 1 cfu/mL) were found in the three coastal waters, possibly influenced by household waste and marine aquaculture.
Gram-negative rod-shaped bacteria growing on MacConkey agar are generally classified as coliform.Coliform bacteria can be found in the aquatic environment, in soil, and on vegetation and are often considered an indicator of fecal contamination.This pollution occurs due to the disposal of waste, livestock manure, and food scraps from agriculture and households into river and coastal waters [22].Coliform bacteria include Enterobacter, Citrobacter, Klebsiella, Hapnia, and Escherichia.

Conclusion
It is concluded that the total bacteria and Vibrio spp. in the marine finfish area of Gerokgak District were higher, mainly in April-June and September-October.The total Vibrio spp. was higher on the surface water than in the middle and bottom layers.However, the bacterial population is still within safe limits.Halotolerant bacteria were found to be more abundant in the rainy and transitional seasons (April-June and October).In contrast, Rod-shaped gramnegative bacteria were more frequently found in summer (August and September).

Fig 1 .
Fig 1. Selected sampling stations for Bacterial population measurement on the North Bali coast Gerokgak Village (A), Penyabangan Village (B), and Pegametan Bay, Sumberkima Village(C)

Fig. 3 .
Fig. 3. Vertical distribution of Vibrio spp.during April to June and August to October 2019 at the three sampling stations: Gerokgak (A), Penyabangan (B), and Pegametan Bay (C).

Fig. 4 .
Fig. 4. Vertical distribution of Vibrio harveyi in the population of Vibrio spp.during April to June and August to October 2019 at the three sampling stations: Gerokgak (A), Penyabangan (B), and Pegametan Bay (C).

Fig. 5 .
Fig. 5. Vertical distribution of halotolerant bacteria during April to June and August to October 2019 at the three sampling stations: Gerokgak Village (A), Penyabangan Village (B), and Pegametan Bay (C).

Fig. 6 .
Fig. 6.Vertical distribution of rod-shaped gram-negative bacteria during April to June and August to October 2019 at the three sampling stations: Gerokgak (A), Penyabangan (B), and Pegametan Bay (C).