Water Quality Improvement by Wild Bacilli Isolated from Marine Environments

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Rungnapa Keawkong
Onanong Siriboon
Sunipa Chankaew
Yutthapong Sangnoi
Arnon Uppabullung


Huge feed volume and shrimp density loading in the intensive shrimp culture decreased water quality. Ammonia and nitrite, which are toxic substances, are occurred from feed and shrimp excretion. Bacillus and related genera (class Bacilli) are bacteria capable of transforming ammonia to nitrite and then nitrite to nitrate by nitrification. In this study, wild marine Bacilli were isolated from samples collected from seagrass beds Andaman Sea, Thailand. Bacilli strains were obtained by using the heat-shock method. Three bacilli bacteria were consisting of Bacillus sp. BC02 (DNA-based), Bacillus sp. BC05 (morphology-based) and Virgibacillus sp. BC06 (DNA-based) was obtained. This study aims to investigate these isolates in shrimp wastewater improvement compared with the commercial seed of Bacillus spp. product (PM-1). Seven wastewater treatments were separately tested by adding different formulas of bacteria. Each treatment was added for 1% (w/v) of 107 CFU/mL density and incubated for 7 days. Treatment of BC02+BC05 showed a significant TSS decrease (66.56%) and produced the highest nitrate concentration. The highest increase of OTP (84.97%) was found in the treatment of BC02+BC05+BC06. PM-1 product has presented the best BOD lessening (54.35%) and showed a non-significant reduction of ammonia (98.60%) with the highest nitrite (0.685 ± 0.009 mg/L) at the end of the experiment. Virgibacillus (BC06) has resulted in the highest significant reduction of COD (60.39%). Thus, it might be summarized that three marine isolates of Bacillus, Virgibacillus, and commercial PM-1 product have excellent potential to improve wastewater quality with no significant diferent. Marine Bacilli can be substitution used for commercial PM-1 products.

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Hlordzi, V.; Kuebutornye, F. K. A.; Afriyie, G.; Abarike, E. D.; Lu, Y.; Chi, S.; Anokyewaa, M. A. The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquac. Rep. 2020, 18, 100503.

Iber, B. T.; Kasan, N. A. Recent advances in shrimp aquaculture wastewater management. Heliyon. 2021, 7, e08283.

Sangnoi, Y.; Chankaew, S.; O-Thong, S. Indigenous Halomonas spp., the potential nitrifying bacteria for saline ammonium waste water treatment. Pak. J. Biol. Sci. 2017, 20, 52–58.

Bull, E. G.; Cunha, C. L. N.; Scudelari, A. C. Water quality impact from shrimp farming effluents in a tropical estuary. Water Sci. Technol. 2021, 83.1, 123–136.

Sanchez-Zazueta, E.; Martínez-Cordero, F. J.; Chávez-Sánchez, M. C.; Montoya-Rodríguez, L. Quantitative risk assessment of WSSV transmission through partial harvesting and transport practices for shrimp aquaculture in Mexico. Prev. Vet. Med. 2017, 146, 27–33.

John, E. M.; Krishnapriya, K.; Sankar, T. V. Treatment of ammonia and nitrite in aquaculture wastewater by an assembled bacterial consortium. Aquaculture. 2020, 56, 735390.

Yang, X.-P.; Wang, S.-M.; Zhang, D.-W.; Zhou, L.-X. Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying–denitrifying bacterium, Bacillus subtilis A1. Bioresource Technol. 2011, 102, 854–862.

Zhang, Q. L.; Liu, Y.; Ai, G. M.; Miao, L. L.; Zheng, H. Y.; Liu, Z. P. The characteristics of a novel heterotrophic nitrification–aerobic denitrification bacterium, Bacillus methylotrophicus strain L7. Bioresource Technol. 2012, 108, 35–44.

Chankaew, S.; O-Thong, S; Sangnoi, Y. Nitrogen removal efficiency of salt-tolerant heterotrophic nitrifying bacteria. Chiang Mai J. Sci. 2018, 45, 11–20.

Phatthongkleang, T., Sangnoi, Y.; O-Thong, S.; Uppabullung, A.; Keawtawee, T. The efficiency of Bacillus spp. to remove ammonia in shrimp aquaculture. Wichcha J. NSTRU. 2019, 38, 1–15.

Kawman, K.; Uppabullung, A.; Kaewtawee, T.; Sangnoi, Y. Effect of probiotic used on phytoplankton community structure in white shrimp (Litopenaeus vannamei) pond. ASEAN J. Sci. Tech. Report. 2022, 25, 39–49.

Olmos, J.; Acosta, M.; Mendoza, G.; Pitones, V. Bacillus subtilis, an ideal probiotic bacterium to shrimp and fish aquaculture that increase feed digestibility, prevent microbial diseases, and avoid water pollution. Arch. Microbiol. 2020, 202, 427–435.

Purivirojkul, W.; Maketon, M.; Areechon, N. Probiotic properties of Bacillus sphaericus and Bacillus subtilis in black tiger shrimp (Penaeus monodon Fabricius) culture. Kasetsart J. (Nat. Sci.) 2005, 39, 262–273.

Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027.

Strickland, J. D. H.; Parsons, T. R. A Practical Handbook of Seawater Analysis, 2nd ed.; The Alger Press Ltd.: Fishery Research Board, Canada, 1972; pp. 49–131.

APHA. Standard Methods for the Examination of Water and Wastewater, 19th ed, American Public Health Association Inc.: New York, 1995.

APHA. Standard Methods for the Examination of Water and Wastewater, Part 3, Determination of Metals, 17th ed, American Public Health Association Inc.: Washington DC, 1989.

Boyd, C. E.; Tucker, C. S. Water Quality and Pond Soil Analysis for Aquaculture. Agricultural Experiment Station, Alabama, 1992.

Kumar, P.; Kamle, M.; Borah, R.; Mahato, D. K.; Sharma, B. Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture. Egypt. J. Biol. Pest Control. 2021, 31, 95.

Maeda, M.; Mizuki, E.; Nakamura, Y.; Hatano, T.; Ohba, M. Recovery of Bacillus thuringiensis from marine sediments of Japan. Curr. Microbiol. 2000, 40, 418–422.

Maeda, M.; Mizuki, E., Hara, M.; Tanaka, R.; Akao, T.; Yamashita, S.; Ohba, M. Isolation of Bacillus thuringiensis from intertidal brackish sediments in mangroves. Microbiol. Res. 2001, 156, 195–198.

Dash, H. R.; Mangwani, N.; Das, S. Characterization and potential application in mercury bioremediation of highly mercury-resistant marine bacterium Bacillus thuringiensis PW-05. Environ. Sci. Pollut. Res. 2014, 21, 2642–2653.

Anyanwu, N. G.; Ariole, C. N. Probiotic potential of an indigenous marine Bacillus thuringiensis on shrimp (Penaeus monodon) culture infected with Vibrio mimicus. J. Applied Sci. 2019, 19, 173–179.

Pang, S. T.; Ransangan, J.; Hatai, K. Isolation, identification and preliminary characterization of candidate probiotic bacteria from the intestine of domesticated goldfish (Carassius auratus). J. Fish. Environ. 2020, 44, 39–52.

Mendoza-Estrada, L. J.; Hernández-Velázquez, V. M.; Arenas-Sosa, I.; Flores-Pérez, F. I.; Morales-Montor, J.; Peña-Chora, G. Anthelmintic effect of Bacillus thuringiensis strains against the gill fish trematode Centrocestus formosanus. Biomed Res. Int. 2016, ID 8272407.

Dawson, D.; Salice, C. J.; Subbiah, S. The efficacy of the Bacillus thuringiensis israelensis larvicide against Culex tarsalis in municipal wastewater and water from natural wetlands. J. Am. Mosq. Control Assoc. 2019, 35, 97–106.

Heyndrickx, M.; Lebbe, L.; Kersters, K.; De Vos, P.; Forsyth, G.; Logan, N. A. Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int. J. Syst. Bacteriol. 1998, 48, 99–106.

Seck, E.; Rathored, J.; Khelaifia1, S.; Croce, O.; Robert, C.; Couderc, C.; Di Pinto, F.; Sokhna, C.; Raoult, D.; Lagier, J.-C. Virgibacillus senegalensis sp. nov., a new moderately halophilic bacterium isolated from human gut. New Microbes New Infect. 2015, 8, 116–126.

Yin, X.; Yang, Y.; Wang, S.; Zhang, G. Virgibacillus oceani sp. nov. isolated from ocean sediment. Int. J. Syst. Evol. Microbiol. 2015, 65, 159–164.

Daroonpunt, R.; Tanasupawat, S.; Kudo, T.; Ohkuma, M.; Itoh, T. Virgibacillus kapii sp. nov., isolated from Thai shrimp paste (Ka-pi). Int. J. Syst. Evol. Microbiol. 2016, 66, 1832–1837.

Zhang, D.-C.; Schumann, P.; Wu, J.; Franca, L.; Neuner, K.; Margesin, R. Virgibacillus flavescens sp. nov., isolated from marine sediment. Int. J. Syst. Evol. Microbiol. 2016, 66, 1138–1143.

Agunbiade, M.; Oladipo, B.; Ademakinwa, A. N.; Awolusi, O.; Adesiyan, I. M.; Oyekola, O.; Ololade, O.; Ojo, A. Bioflocculant produced by Bacillus velezensis and its potential application in brewery wastewater treatment. Sci. Rep. 2022, 12, 10945.

Reddy, K. V.; Reddy, V. K.; Babu, B. S.; Lakshmi, T. V. Applications of Bacillus sp. in aquaculture waste water treatment. Int J. S. Res. Sci. Tech. 2018, 4, 1806–1812.

Lalloo, R.; Ramchuran, S.; Ramduth, D.; Go¨rgens, J.; Gardiner, N. Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. J. Appl. Microbiol. 2007, 103, 1471–1479.

Thurlow, C. M.; Williams, M. A.; Carrias, A; Ran, C.; Newman, M.; Tweedie, J.; Allison, E.; Jescovitch, L. N.; Wilson, A. E.; Terhune, J. S.; Liles, M. R. Bacillus velezensis AP193 exerts probiotic effects in channel catfish (Ictalurus punctatus) and reduces aquaculture pond eutrophication. Aquaculture. 2019, 503, 347–356.

Li, X.; Wang, T.; Fu, B.; Mu, X. Improvement of aquaculture water quality by mixed Bacillus and its effects on microbial community structure. Environ. Sci. Pollut. Res. 2022, 29, 69731–69742.

Karichappan, T.; Venkatachalam, S.; Jeganathan, P. M. Analysis of efficiency of Bacillus subtilis to treat bagasse based paper and pulp industry wastewater – a novel approach. J. Korean Chem. Soc. 2014, 58, 198–204.

Yan, L.; He, Y.; Kong, H.; Tanaka, S.; Lin, Y. Isolation of a new heterotrophic nitrifying Bacillus sp. strain. J. Environ. Biol. 2006, 27, 323–326.

Ren, W.; Wu, H.; Guo, C.; Xue, B.; Long, H.; Zhang, X.; Cai, X.; Huang, A.; Xie, Z. Multi-strain tropical Bacillus spp. as a potential probiotic biocontrol agent for large-scale enhancement of mariculture water quality. Front. Microbiol. 2021, 12, 699378.

Zokaeifar, H.; Babaei, N.; Saad, C. R.; Kamarudin, M. S.; Sijam, K.; Balcazar, J. L. Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2014, 36, 68–74.