DIVERSITY OF DECOMPOSER BACTERIA IN ECO ENZYME FERMENTATION PROCESS OF ORGANIC MATERIALS USING OXFORD NANOPORE TECHNOLOGY (ONT) AND ITS EFFECTIVENESS IN INHIBITING E-COLI IN FISH POND WITH WATER MINERAL SOIL

Objective: This study aims to determine the characteristics of eco enzymes in the form of aroma, color, pH eco enzymes and identify the diversity of bacteria that play a role during the fermentation process. Method/design/approach: The research was carried out from August to December 2022, the first stage of making eco enzymes fermentation 3 months of organic waste three variants of Citrus sinensis Osb peel, Ananas comosus peel, and Citrullus lanatus peel, combined with 2 variants of vegetable residue of fern plants; Stenochlaena palutris and Diplazium esculentum. Eco enzyme yields were organoleptic tested and measured bacterial diversity with the Oxford Nanopore Technology (ONT) technique. Results and conclusion: Eco enzyme harvest organoleptic test results: pH 3.74, ethanol content 2.41%, color 1502 Pt.Co and the characteristic smell of fermentation. Bacterial diversity Test of bacterial diversity that plays a role during the eco enzyme fermentation process was carried out using the Oxford Nanopore Technology (ONT) technique, identified the diversity of lactic acid bacteria (BAL) at the taxon level, Phylum 7, Order 19, Familia 37, Genus 98 and species 269 lactic acid batteries (BAL). Eco enzyme 1 liter in 1000-1 liter of mineral groundwater fish pond can inhibit E-coli 36.1% of the original population. Research implications: This research contributes to the body of knowledge on eco enzymes, bacterial diversity in fermentation processes, and their potential applications in waste management and antibacterial activities. The implications of this study can pave the way for further research, innovations, and practical implementations in the field of eco-friendly technologies and sustainable waste management practices Originality/Value: The originality of this research lies in its focus on eco enzymes and bacterial diversity, while the value lies in its contribution to waste management practices, antibacterial applications, and advancements in environmentally friendly technology.


INTRODUCTION
Reuse of fruit peels and vegetable residues is not commonly found, generally the skin of fruits and vegetable residues is simply thrown away, and becomes a pile of waste that is not utilized, so it can become a problem for the environment and health. Organic waste of fruit peels and vegetable residues from both markets and households can be used as a potential source of alternative raw materials to produce eco enzyme products. Eco Enzyme is a kind of organic compound considered as an all-purpose natural cleansing liquid fermented from the remaining skin of fruits and vegetables, sugar and water. According to Arun et al., (2015) eco enzymes contain protease, lipase and amylase enzymes.
According to Vama and Cherekar (2020) eco enzyme liquids can be utilized as antifungal, anti-bacterial, insecticidal agents and cleaning agents. Eco enzymes can also be used as household cleaning fluids (such as floors, dishes, toilets), vegetable and fruit cleaners, insect repellents and plant feeders. The benefits of eco enzymes as disinfectants are possible because the alcohol and acetic acid (CH3COOH) content contained in eco enzymes can kill germs, viruses and bacteria. study on decomposer bacteria in eco-enzyme fermentation and the importance of medicinal plants in human history are interconnected through the search for alternative solutions to combat drug-resistant bacteria. Exploring the active principles present in plants, such as those used in eco-enzymes, provides a viable option to address the challenge of microbial resistance and promote sustainable and effective approaches to disease prevention and treatment (Bones et al., 2022).
The various benefits that can be obtained from eco enzymes make the study of this product very potential to be explored more deeply, especially related to the identification of the diversity of bacteria that decompose organic matter during the eco enzyme fermentation process. Therefore, it is necessary to identify the diversity of bacteria contained in eco enzymes made from leftover fruit peels and local vegetable residues in Central Kalimantan so that the eco enzymes produced can be used safely.
Measurement of bacterial diversity through 16S rRNA gene sequencing has become a widely used approach in the field of environmental microbiology (Zanatta et al., 2022), especially since the advent of high-throughput sequencing technology. One of the developments of the technology is Oxford Nanopore Technology (ONT) (Bahram et al., 2018). The basic principle of ONT sequencing is to pass one strand of DNA through the nanopore membrane and apply the voltage difference across the membrane. Nucleotides passing through the membrane will affect the electrical resistance of the pore so that measurements of electric current over time can show the sequence of DNA bases passing through the pore (Wick et al., 2019). ONTs provide long read sequencing that includes the full sequence of the 16S rRNA gene (V1-V9 region) through a fast, inexpensive, and high process. Since all informative sites of the 16S rRNA gene are considered, full-length 16S rRNA sequences offer higher levels of taxonomic and phylogenetic resolution for bacterial identification (Bahram et al., 2018).

THEORETICAL FRAMEWORK
Agenda 2030 for Sustainable Development (SDGs) is a development agreement approved by 193 countries, including Indonesia, to promote sustainable development based on human rights and equality. Indonesia implements four pillars of development, including environment, social, economic, and governance. One of the challenges faced is waste management that exceeds landfill capacity and has an impact on the environment. Data shows that Indonesia's national waste production reaches 175,000 tons per day, with about 60-70% of organic waste not being transported to landfills. Waste management issues include negative impacts on health, the environment, and socio-economic aspects. Disposing of waste into water bodies can cause floods and reduce environmental quality. Waste management policies include institutional aspects, financing, legal regulations, community roles, and operational technical aspects (Vilas,2023).
Waste management poses a significant challenge for urban governments (Mayboroda, V., & Spirin, 2023). The main problems include large waste volumes, limited infrastructure, and inadequate management. The community's paradigm about waste needs to change to separate organic and inorganic waste to support recycling and reduce waste volume. Waste management in Indonesia's three major cities highlights the importance of community participation in waste management.
One way to process and utilize organic waste is by transforming it into eco-enzymes. Eco-enzyme is a solution of complex organic substances produced through the fermentation of organic materials, sugar, and water. The development of eco-enzymes aims to replace the use of synthetic chemicals and maintain environmental sustainability. The raw materials for eco-enzymes generally come from fruit peels, vegetables, and fruit skins, following the principles of green chemistry and renewable resources.
One tip for utilizing and processing organic waste in the Eco Enzyme Production module (2020), shared by the Eco Enzyme Nusantara community, is to convert it into ecoenzymes. Eco-enzyme is a solution of complex organic substances produced through the fermentation of organic materials, sugar, and water. Organic waste refers to any waste material that is derived from plants or animals and is biodegradable. It includes a wide range of materials such as food scraps, yard waste, agricultural waste, paper products, and sewage sludge. Organic waste contains high levels of carbon and nutrients, making it valuable for recycling and composting (Severo et al., 2017;Barboza et al., 2021, de Paula Araújo et al., 2022. It is developed in an effort to reduce the use of synthetic chemicals and protect the environment sustainably. It is commonly applied in sustainable and environmentally friendly chemistry, following the principles of green chemistry, using renewable resources such as fruit peels and vegetable waste. In some cases of fermentation, white-colored fungi (pitera) or brown jelly-like layers (mam-enzyme) occur, which are the sources of enzymes. The enzymes in eco-enzymes include protease, amylase, and lipase enzymes. (Mugitsah, 2020).
Fermentation is a chemical process in food substances caused by enzymes produced by microorganisms or inherent in the food. Microbes commonly involved in the fermentation process of food products include Lactobacillus bulgaricus, which contributes to aroma formation, and Streptococcus thermophilus, which contributes to flavor formation. Lactobacillus bulgaris forms a mutualistic symbiosis with Streptococcus thermophilus, converting lactose into lactic acid, and its growth is stimulated by the presence of amino acids, particularly valine, lysine, and histidine. Lactobacillus bulgaris grows rapidly due to the stimulation of formic acid and CO2 produced by Streptococcus thermophilus. (Maulidya, 2007). Arini (2017) states that during the fermentation process, acidity increases with the increasing number and activity of microorganisms that convert lactose into lactic acid. The difference in acidity levels is followed by an increase in starter concentration and the breakdown of lactose into acetic acid. Acetic acid (CH3COOH) produced through fermentation can kill germs, viruses, and bacteria. In addition, nitrates (NO3-) and carbon dioxide (CO3) are produced, which are needed by the soil as nutrients. The enzyme content itself consists of lipase, trypsin, and amylase. The economic benefit of eco-enzyme production is reducing the cost of purchasing cleaning and pest control materials. (Tim Litbang, 2021).
The fermentation process of eco-enzymes, as described by Mugitsah (2020), is anaerobic respiration carried out by bacteria to obtain energy from carbohydrates/proteins/lipids in the absence of oxygen, and during this process, vinegar or acetic acid is produced.
The anaerobic fermentation process occurs in two phases: a. In the first phase, facultative anaerobic bacteria initiate the hydrolysis and disintegration of organic matter. Complex organic compounds are broken down into organic acids, CO2, H2S, and alcohol. Dissolved oxygen is consumed by bacteria, and nitrate and sulfate are reduced, resulting in a decrease in pH value. b. In the second phase, methane bacteria digest the products of metabolism from the first phase, and the end products are methane gas, CO2, and mineral salts.
If the fermentation of materials is successful, it will produce a brownish eco-enzyme liquid with a pH of 3.5-4 and a distinct fermented smell. The recommended combination ratio for eco-enzyme production, as stated in the module, is 10:3:1 (water: fruit and vegetable waste: brown sugar/molasses). Eco-enzyme liquid can be used as a versatile cleaner, natural hormone, and plant nutrition. It contains amylase, protease, and lipase enzymes that are useful for processing waste containing carbohydrates, proteins, and fats. Research by Madhumitha and Kalaiyarasi (2020) suggests that to reduce pollution from household and industrial waste in the Yamuna River in India, eco-enzyme liquid can be used productively and economically to address environmental issues cost-effectively. Other research shows that adding eco-enzyme liquid to pet food and drinks can enhance immunity and improve meat quality.
Research by Bhavani et al. (2020) demonstrates that applying eco-enzymes to water bodies (rivers) leads to changes in the substance values of dissolved oxygen (DO), biochemical oxygen demand (BOD), and chemical oxygen demand (COD) in the water. The water bodies become cleaner, odor disappears, and the COD level reaches the permitted level after treatment. Nazaitulshiha et al. (2019), who studied the advantages of eco-enzyme liquid fermentation from orange and tomato waste, showed a higher percentage reduction in sludge water from industrial waste, including total dissolved solids, total phosphorus, ammonia nitrogen, and COD, using orange waste eco-enzyme compared to tomato waste eco-enzyme after 10 days of treatment. This is because the citric acid from the fermentation contains biocatalytic enzymes such as protease, amylase, and lipase.
Research by Tang and Tong (2011) shows that eco-enzyme, or what they call "enzyme waste," can reduce pollutants and wastewater pollution from household/residential areas in a shorter time before being discharged into water bodies. They also mention that in wastewater treatment, enzymes with biological additives tend to lower the temperature to enhance bacterial activity. Biological additives such as laccase enzymes have been widely used and explored in the preliminary treatment of wastewater, especially wastewater with high lipid and fat content.
In line with the above research, the results of Zero Waste Indonesia (ZWID) research (2020), the first online community in Indonesia that encourages Indonesians to adopt a zero waste lifestyle, claim that eco-enzymes can release ozone gas (O3) and reduce carbon dioxide (CO2) that trap heat in the atmosphere, thus reducing greenhouse effects and global warming. According to Vama et al. (2020), eco-enzyme liquid is easily biodegradable and not harmful to humans and the environment. Furthermore, eco-enzyme liquid has disinfectant properties due to the presence of alcohol or acetic acid produced from the bacterial metabolism process found in fruit and vegetable waste.
The research conducted by Geetha et al. (2017) shows that eco-enzyme liquid derived from lemon peel extract at a concentration of 15% can inhibit the growth of pathogenic bacteria such as E. coli, S. aureus, S. pyogenes, S. typhi, and P. aeruginosa, as well as fungi like Aspergillus niger, Fusarium sp, and Cladosporium. Maula et al. (2020) found that eco-enzyme liquid at a concentration of 100%, sprayed on tomatoes and strawberries, effectively maintained the freshness of tomatoes for six days and strawberries for five days when stored at room temperature without packaging. Rocyani et al. (2020) studied the conversion value of ecoenzymes from pineapple (Ananas comosus) fermentation, which had a pH of 3.15, and from papaya (Carica papaya L.), which had a pH of 3.29.
Considering the enormous potential for fish production and consumption in fish ponds in Palangka Raya City, it is essential to ensure effective feeding and waste management in fish farming, as well as water quality maintenance to prevent feed pollution. Hendry et al. (2018) emphasize that efforts to increase aquaculture production must consider the environmental carrying capacity, particularly water quality and pollution caused by excessive feeding. One way to reduce the pollution load of waste from Dumbo catfish (Clarias gariepinus) in Palangka Raya City, as recommended by Bambang and Restu (2018), is by converting the waste into bloodworm biomass (Chironomidae larvae) as natural feed for freshwater fish, which provides suitable nutrition for the fish.
Assessing the quality status of fish ponds can be done by measuring and verifying the field variables that make up water quality, as recommended by Ratnaningsih et al. (2020). The 6 components of water quality assessment studied in this research include physical, biological, and chemical qualities measured by: a. pH: to determine the acidity level of the water. b. Total Dissolved Solids (TDS): to measure the concentration of dissolved solid particles in the water that are not visible to the naked eye. c. Dissolved Oxygen (DO): to measure the amount of oxygen dissolved in the water.
Higher DO values indicate better water quality. The maximum DO at a temperature of 20°C is 9 ppm/l. d. Biological Oxygen Demand (BOD): to measure the amount of dissolved oxygen required by microorganisms to decompose organic matter in the water. Higher BOD values indicate poorer water quality and lower DO levels. Good BOD levels range from 1-2 mg/l (green level) and 3-5 mg/l (yellow level). e. Chemical Oxygen Demand (COD): to measure the amount of oxygen required by chemical compounds to decompose organic matter. The COD value in unpolluted surface water is around 20 mg/l. f. Total Ammonia (NH3-N), consisting of free ammonia (NH3) and ionized ammonia (NH4+): generated from the breakdown of organic matter (feed remnants, feces, and dead aquatic organisms) by decomposing bacteria. Free ammonia is toxic to aquatic organisms, and its toxicity increases with decreasing dissolved oxygen levels. g. Nitrite (NO2-): a nitrogen waste product resulting from the oxidation/reduction process of ammonia with the help of oxygen and bacteria. The nitrite level in ponds is typically between 0.05-0.06 mg/l. Concentrations above this range can be toxic to some aquatic organisms. h. Nitrate (NO3-): a form of combined nitrogen found in natural waters, resulting from oxidation under low oxygen conditions. The nitrate content standard is 0.1 mg/l. Escherichia coli is a gram-negative bacterium that is short and rod-shaped, with a length of approximately 2 µm, diameter of 0.7 µm, and width of 0.4-0.7 µm. It is facultative anaerobic. Escherichia coli forms round, convex, and smooth colonies with distinct edges. Escherichia coli is classified based on its virulence characteristics, and each group causes infections in the intestines and other parts of humans and animals.

Research Site
The research was carried out from August to December 2022, eco enzyme fermentation was carried out on Laboratory at Palangka Raya of University, Central Kalimantan. The identification of diversity of bacterial was carried out at the Microbiology Laboratory, Brawijaya University-Malang.

Materials
Rainwater, brown sugar, organic waste from fruit peels consists of three variants; Citrus sinensis Osb, Ananas comosus, and Citrullus lanatus. Vegetable residues of fern plants; Stenochlaena palutris and Diplazium esculentum. Tools: plastic containers, analytical scales, measuring cups and cutter.

Method
The method used is an experimental method for making eco enzyme liquids and testing the diversity abundance of decomposing bacteria during the fermentation process.

Eco enzyme liquid manufacturing
The remaining fresh skins of local fruits and vegetables are collected, cut into small pieces and mixed with other eco enzyme-making ingredients in a ratio of 10 water: 1 brown sugar and 3 rinds of fruits and vegetables. The fruit peel tried in this study consisted of three variants: Citrus sinensis Osb, Ananas comosus and Citrullus lanatus. Vegetable residues fern plants Stenochlaena palutris and Diplazium esculentum with a composition of 90% fruit peel and 10% vegetable residue, fermented in a closed plastic containers at room temperature. Harvesting eco enzymes is carried out 90 days after fermentation.

Eco enzyme liquid bacteria diversity test
Bacterial cells are obtained through the screening of previously made eco enzyme crops, carried out in stages using filter paper, and nitrocellulose membranes of pores 0.45 and 0.20 µm. The filtering process is carried out aseptically using a vacuum pump. Bacterial cells tethered to a 0.20 µm pore nitrocellulose membrane subsequently extracted their DNA using the FastDNA Spin Kit (MPBIO) (Tenriawaru EP et al., 2022). The deoxyribonucleic acid (DNA) obtained was further measured in concentration and purity using NanoDrop spectrophotometer and Qubit fluorometer. DNA library preparation was performed using an ONT Kit and 27F and 1492R primers (primers for the V1-V9 region at 16S rDNA). DNA base sequences were amplified using MyTaq HS Red Mix 2X (Bioline, BIO-25048). Amplicon (PCR results) as many as 2 µL analyzed using 1% agarose gel and compared to a 1 kb marker of 2.5 µL. Nanopore sorting is operated by MinKNOW software version 22.05.7. Base calling was performed using Guppy version 6.1.5 with a high accuracy model (Wick et al., 2019). The quality of FASTQ files is visualized using NanoPlot (de Coster et al., 2018). DNA sequences are classified using the Centrifuge classifier (Kim et al., 2016). The Bacterial and Archaea Index was constructed using the NCBI 16S RefSeq (https://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/) database. Analysis and visualization were performed using Pavian (https://github.com/fbreitwieser/pavian), Krona Tools (https://github.com/marbl/Krona), and RStudio using R version 4.2.0 (https:///www.Rproject.org/).
The results of the eco enzyme harvest in the first study, heapplied as much as 1 liter in 1000-1 liters of bio floc pond water catfish enlargement (Pangasius djambal) taken from the fish pond with mineral soil water at the Anugerah fish pond, Sei Gohong Village, Bukit Batu District, Palangka Raya City, Central Kalimantan. Observation of E-coli test parameters was carried out before eco enzyme application and 20 days after eco enzyme application without pool water change. The test of E-coli bacteria content was carried out at the Unilab Jakarta Laboratory, the test results were compared with water quality standards for fisheries businesses in accordance with PP

Eco Enzyme Organoleptic Test Results
Harvesting eco enzymes is carried out 90 days after fermentation. The organoleptic tests of eco enzyme harvests from the combinations and variants of organic matter above are: pH 3.74, ethanol content 2.41%, color 1502 Pt.Co and the characteristic smell of fermentation. The eco enzyme harvest is then filtered to obtain bacterial cells through testing with the Oxford nanopore technique.

Phylum-level bacterial composition
The abundance of bacteria is presented in bar charts, krona visualizations, and Sankey diagrams. The abundance of bacteria is presented at each taxon level. Certain taxons that have the highest abundance are presented in the form of krona visualizations.          Information: KM0 = Mineral groundwater fish enlargement pond before eco enzyme application KM1 = Fish enlargement pond mineral groundwater 20 HSI 1-liter eco enzyme 1000-1 pond water hsi = Day after eco enzyme implementation

E-coli Content
The bacterial diversity test that played a role during the eco enzyme fermentation process was carried out using the Oxford Nanopore Technology (ONT) technique, identified the diversity of lactic acid bacteria (BAL) at the taxon level, Phylum 7, Order 19, Familia 37, Genus 98 and species 269. Lactic acid batteries (BAL) are produced from carbohydrates and cellulose from ingredients during the eco enzyme fermentation process, occurring under aerobic and anaerobic conditions. Lactic acid bacteria (BAL) mainly need enough carbohydrates to meet the needs of their growth and production of organic acids. Diarlin et al., (2013). Fermentation by lactic acid bacteria, especially from the top 5 genera and species above, causes an increase in the concentration of amino acids, namely aspartic acid, glutamic acid, proline, and valine which contribute to the smell, color and pH of the eco enzyme recipe.
The addition of carbohydrate sources from brown sugar and from organic matter of fruit peels and vegetable residues in the manufacture of eco enzymes supports lactic acid bacteria to grow and produces lactic acid which is sufficient in inhibiting the growth of putrefactive microbes. Fermentation by BAL resulting in lactic acid production as well as low pH will inhibit the growth of other microbes. The fermentation process lowers the pH of organic matter so that it inhibits other groups of bacteria, or causes disturbances to putrefactive bacteria and pathogens, Nandini, et al (2021), besides that BAL will convert glucose or simple carbohydrates into alcohol, acetic acid, carbonic acid, and lactic acid, because glucose or carbohydrates contained in organic matter will produce a lower pH of 3.74 as in harvest the above eco enzymes.
At the genus level there are 5 top genera having the highest abundance, namely the genus Laktobacillaceae reaching 99.6% of the 98 identified genera, the genus Basillaceae 0.09%, the genus Enterocococeae 0.05%, the genus Strecptococceae 0.05% and the genus Carnobacteriaceae 0.03%. The bacterial genus Lactobacillus, and the bacteria Strecptococceae are a group of beneficial lactic acid bacteria and as probiotic bacteria for the digestion of humans and animals (Laily, 2008). Enterococceae bacteria are anaerobic bacteria that are able to survive in extreme conditions and also produce enterosin chemical compounds that are effective against pathogens. Streptococceae bacteria are useful in relieving the symptoms of inability to digest sugar (lactose) in dairy products, lowering stomach acid and overcoming digestive problems. Streptococcus thermophiles produces ATP (adenosine trifaosfat) from respiration. While the genus Cornobakteriaceae is a bacterium naturally ubiquitous gramnegative which is detrimental, although it is rare that this type of bacteria can contaminate cooked and uncooked food and drinks, and is able to survive in very dry conditions, (Tentiawaru EP., et al., 2022 andLaily. 2008) At the species level there are top 10 species of bacteria with high abundance, namely Lentilactobacillus parabuchneri species 46.89% or 23.2k, Liquorilactobacillus vini species 21.66% or 10.7k, Limosilaktobacillus fermentum species 12.96% or 6.42k, Lacticaseibacillus paracaseri species 6.48% or 3.21k and Liquorilactobacillus ghanensis 4.21% or 2.08k, Lentilactobacillus otakiensis species 0.90% or 448, Lentilactobacillus neat species 0.56% or 275, Lentilactobacillus kefiri species 0.46% or 230 species of Acetobacter indonesiensis 0.46% or 299, and species of Liquorilactobacillus nagelii 0.43% or 211.
These species are fermented bacteria that produce lactic acid and acetic acid during the fermentation process, besides that they can act as bacterial inoculants to improve the aerobic stability of preserved forage-based feed. The function of lactobacillus parabuchneri lactic acid bacteria for the health of the human body is that it can relieve irritation in the gastrointestinal tract, cure heart and respiratory diseases and also maintain skin health. Lactobacillus parabuchneri which has a probiotic function also contributes to the health of the human body such as lowering cholesterol levels, antioxidant activity and high anti-microbials so that the lactic acid bacteria can be said to have a good probiotic function to be developed further. In addition, the application of phylogenetic or kinship between other types of bacteria in the food industry can be used to detect and track the presence of food contamination so that it can be predicted what types of microorganisms can become destructive bacteria. . Lactobacillus vinni species, is a gram-positive BAL bacterium that is beneficial and serves as an E-coli inhibitor that lives in the digestive tract of both humans and animals. Lactobacillus fermentun bacteria are probiotic bacteria species that have the ability to degrade adenosine compounds which are derivatives of purines that cause uric acid. Laily, (2008).
The dominant bacteria identified in the eco enzyme harvest come from lactic acid bacteria. Rashid et al., (2013), Lactic Acid Bacteria (BAL) can inhibit and kill other bacteria by producing proteins called bacteriocin, such as nisin produced by Lactobacillus lactis ssp. lactis. Nisin can inhibit the growth of several bacteria, namely Bacillus, Clostridium, Staphylococcus, and Listeria. In addition to bacteriocins, antimicrobial compounds (inhibitors of other bacteria) that can be produced by BAL, such as hydrogen peroxide, organic acids, diacetyl, acetoin, reuterin, and bacteriocin. Antimicrobial compounds such as bacteriocins can be an alternative treatment in the prevention of bacterial diseases in fish. Bacteriocins are ribosomal synthesized protein substances produced by bacteria that exhibit antimicrobial activity against bacteria closely (Trivedi et al., 2013 andYolanda et al,.2017 ). The intestines of fish proved to be the natural habitat of probiotic microorganisms, as they are typically found in the digestive tract of many species of fish from different environments (Buntin et al., 2008).
These bacteriocins were active against common Gram-positive pathogens tested, such as B. cereus and S. aureus K. common properties with other nisin-like bacteriocins stable to heat and sensitive to proteases. exhibits antagonistic activity against gram-negative pathogens, such as S. thyphimurium. These results suggest that nisin-like bacteriocins can have a broad spectrum of antagonisms indicating inhibition against gram-negative pathogens. These strains and the bacteriocins they produce have great potential for use in applications such as catfish feed, or other feed formulations in aquaculture. In addition to having properties similar to nisin, being active against gram-negative bacteria can expand its antimicrobial properties that are useful in food preservation and safety (Azahar., et al 2017). Widiyanti (2019), microbiologically the presence of E-coli can be a reference to determine the feasibility of water before it is used for various purposes Water can be a medium of disease transmission to humans and animals, E-coli bacteria found in maintenance water ponds can be considered an index of pollution by feed and feces because it survives in water relatively longer. Eco enzyme can be used as additives in wastewater treatment, to remove ammonia nitrogen and phosphorus, thus the utilization of waste enzymes is currently developing as part of a viable strategy to treat contaminated water, (Tang et al., 2011., Rajmi et al.,2017 The highest abundance of three eco enzyme crop species, namely Lentilactobacillus parabuchneri species 46.89%, Liquorilactobacillus vini species 21.66%, and Limosilactobacillus fermentum species 12.96% as species that are mostly responsible for the production and accumulation of histamine, a toxic biogenic amine, L parabucheneri It is also known to form biofilms on the surfaces of industrial equipment, is resistant to cleaning and disinfection, and can act as a reservoir of histamine-producing contaminants in milk and cheese, Sarqius, et al., (2023). According to Passoh et al., (2007) L. vini is a bacterium producing yeast and ethanol, unconventional yeast is able to defeat Saccharomyces cerevisiae used traditionally. at the ethanol plant. The formation of floc can reduce the surface exposed to ethanol and help bacteria to survive in the pressure conditions of the ethanol process and can reduce the absorption of sugars, nutrients by cells and at the same time protect (Tiukova et al., 2014). Even L.vini bacteria have been isolated from several ethanol production plants in Sweden and Brazil, ethanol plants produce low pH (3.6) and low sugar and ethanol concentrations up to 100 g / L, (Lucena et al., 2010;Passoh et al., 2007). Lactobacillus fermentum, is one species of probiotic bacteria that is given as a food supplement or drink and provides health effects for humans and animals. As probiotic bacteria, these bacteria are able to produce lactic acid, which decreases pH (Vernazza et al., 2006., Rajmi et al.,2017. This decrease in pH occurs due to the activity of probiotic bacteria in converting protein compounds and sugars into lactic acid, this acid condition that is formed is able to inhibit the growth of other bacteria, especially pathogenic bacteria that grow in the normal pH (Longhout, 2000). The advantages of Lactobacillus sp as a probiotic bacterium help improve the intestinal defense system, both by forming colonies on the intestinal mucosa and helping the absorption of nutrients (Laily 2008), this is fish rearing in fish ponds. The results of (Siska et al., 2013), study showed Lactobacillus fermentum and Lactobacillus salivarius can also reduce the number of E. coli pathogenic bacteria by 68.13% from the initial population. Further more, the research of (Yolanda et al., 2017) bacterial isolates lactic acid from packaged and homemade kimchi capable of inhibiting the growth of both test bacteria, S. aureus and E. coli.
Thus, eco enzyme produced from fermentation of 3 variants of fruit skin waste and 2 vegetable variants, an abundance of 269 species of bacteria, effectively inhibited the population of E-coli bacteria by 36.1% of the initial population of 23,000 in catfish bio floc breeding ponds, mineral soil pond water media. The presence of E-coli bacteria is still quite a lot in the magnifying pond because of the high content of E-coli carried from the previous fishing pond water media and because there is no change in the water of the ama cell enlargement pond 20 days of research. The inhibitory power of eco enzyme against E-coli bacteria in mineral groundwater-type fish rearing ponds can be recommended for peat soil pond water bio floc ponds that are widely developed by farmers in the Central Kalimantan region.

CONCLUSION
Three months fermentation harvest of eco enzyme from organic matter from the composition of waste 3 variants of fruit peels and 2 variants of vegetables, identified lactic acid bacteria (BAL) at the taxon level, Phylum 7, Order 19, Family 37, Genus 98 and species 269. At the species level taxon there are top 3 species of bacteria with high abundance, namely Lentilactobacillus parabuchneri species 46.89%, Liquorilactobacillus vini species 21.66%, Limosilactobacillus fermentum species 12.96%. These species are bacteria that are able to inhibit pathogenic bacteria. Experiments with the application of eco enzyme liquid 1 liter 1000-1 L fish enlargement pond water with mineral soil pond water media, able to inhibit the population of E-coli bacteria 36.1% from the initial population. The reduction in the population is considered as an inhibitory power of eco enzyme against the population of pathogenic bacteria E-coli.

ACKNOLEDGEMENTS
This study was assisted by funding by Palangka Raya University, the authors would like to thank Prof. Dr. Salampak, MS as UPR Rector for the technical assistance.