Dynamics of microbial populations in the composting process of marine organic waste

It is estimated that marine waste consist of 60% organic material. Therefore, waste must be handled using a composting process. The main determining factor in the composting process is the microorganisms associated with the C/N ratio. Therefore, this research was conducted to observe the dynamics of microbial populations during the decomposition of marine organic waste. The research method for calculating microbial populations (bacteria and fungi) is the dilution method using a colony counter with the Pour Platedilution technique (CFU/g). The results showed that the microbial population, both fungi and bacteria, fluctuated from week 1 to 10 of the composting process. This is related to the temperature during the composting process and the C/N ratio. The highest population of bacteria and fungi was found in the sixth week of composting at 300 x 107 CFU/g where the process entered the thermophilic phase with a C/N of 55.


Introduction
In 2016, the Ministry of Environment and Forestry considered the waste problem a concern.Indonesia is even the second largest producer of plastic waste discharged into the sea after China.The composition of marine waste consist of about 60% organic waste and 14% plastic waste [1].In 2022, the Ministry of Environment and Forestry achieved a 38% reduction and are still on track, with 16 ministries/agencies working together to tackle marine waste.Currently, about 80% of marine waste in Indonesia originates from land, and up to 30% is plastic waste.
The types of waste present in the sea are organic and inorganic.Hazardous non-organic waste is a plastic waste of large, macro, micro, and nano sizes.During this time, huge organic debris on the seabed can decompose and create toxic gases, which then kill a large number of fish.If fish are consumed by humans, there are concerns that the impact will gradually affect human health.Therefore, waste must be treated on land to avoid flowing into rivers and seas.When waste is dumped into the ocean, it is difficult to handle and will be conveyed far, even across oceans [2].
Composting, particularly with the inclusion of extra organic materials like cow dung and rice straw, may be a solution for garbage management, particularly marine organic waste, which is the main source of trash at Kuala Indah Beach in Sei Suka District, Batubara Regency, Indonesia [3].The Indonesian National Standard (SNI 19-7030-2004) for quality characteristics is met by this organic fertilizer, allowing for its production.
Living things, like as microbes, are crucial to the composting process.The role of bacteria in creating compost is that decomposers can break down raw materials so that they become materials that are easily absorbed by plants.When decomposed in the absence of oxygen (anaerobic), organic matter will turn into ammonia, hydrogen sulfide (H2S), methane (CH4), and other simple compounds.Dry leaves and mostly pine branches make up the bulk of the marine organic trash along Kuala Indah Beach in Sei Suka District, Batubara Regency, according to research findings from [3].Microorganisms can utilize organic matter if it is soluble in water.The optimal range humidity for microbial metabolism is 40-60%.Microbial activity will decline below 40% and further decline below 15% humidity.On the other hand, if the humidity level is higher than 60%, nutrients will be washed away, the air volume will be reduced, which will diminish microbial activity, and anaerobic fermentation will take place, which will result in unpleasant aromas [4].
The microbial population is closely related to the ratio of carbon and nitrogen as a source of nutrition.The C/N ratio is crucial to the composting process because it provides the nutrients needed by bacteria to build their body cells.The goal of composting is to have organic matter's CN ratio as close as possible to soil's C/N ratio (>20).The initial tests of compost showed that the C/N ratio changed throughout decomposition, going up.It is because the blend of organic components employed has a varied makeup.The organic matter C/N ratio was 42.46 at the start of the composting process.From the first to the seventh week of the composting process, the C/N value dropped.This is due to the reduced amount of carbon used as the energy source for the microorganisms that decompose organic materials.Therefore, this study aimed to observe the dynamics of microbial populations during the composting process of marine organic waste [4,5].

Site selection
The marine organic waste for compost was taken from Kuala Indah Beach, Sei Suka District, North Sumatra, Indonesia.This location was selected based on an initial field survey.

Collection of waste
Waste collection is carried out by selecting organic marine waste such as dry leaves, pine branches, and other organic materials and separating it from inorganic waste such as plastic.

Composting and compost production
In order to hasten the composting process and track the microbial variety of the necessary parameters, EM4 (Effective Microorganisms-4) is a microbial activator and molasses are added to marine organic waste.10 ml of EM4 mixed with 1 liter of water is poured onto marine organic waste every week.Turning is carried out at the required frequency and tests are carried out specifically on piles (after turning) over 10 weeks.Microbial populations (bacteria and fungi) were observed weekly using the standard MPN dilution method.

Isolation and identification of microbial cultures from compost
Using a spatula, combine 10 g of composted marine waste with 90 mL of sterile water, then shake to combine (10 -1 dilution).The compost sample (10 g) was diluted in 90 mL of sterile water and shaken until homogeneous (10 -1 dilution), then 10 mL of 1 dilution solution was taken using a micropipette of 1 mL into a test tube containing 9 mL of sterile water (10 -1 dilution) this was repeated until we got dilutions of 10 -6 , 10 -7 and 10 -8 . 1 ml of each of the three dilutions was pipetted to isolate on non-selective media for fungi (Potato Dextrose Agar) and bacteria (Nutrient Agar), then incubated for several days.Fungi can generally reproduce well in media containing high carbohydrates with a pH range of between 5-6, while media containing protein with a pH of around 7 is a good medium for bacterial growth.Utilizing a colony counter and the Pour Platedilution method, the microbial population of each isolate was determined after inoculation and incubation for three to five days by the following formula (CFU/g): CFU/g = (number of colonies × dilution factor)/mass of culture plate

Results and discussion
The graph of the dynamics of microbial activity during composting (bacterial and fungal populations) and the number of microbial populations that have been calculated using the colony counter method with the Pour Platedilution technique (CFU/g) is presented in Figure 1.A graph of microbial population evolution (bacteria and fungus) is created based on the composting of marine organic waste.Composting goes through numerous stages, including the thermophilic stage, in which the temperature starts to rise.The role of thermophiles is similar to that of psychrophiles; they are most active between 21 and 32 °C.The primary component will be played by thermophilic bacteria that continue to breakdown during the composting process when the compost pile's temperature rises over 45 °C.The initial thermophilic phase lasts for a few days on average, after which the temperature of the compost mixture rises over 40 °C and the thermophilic phase starts [6,7].
The thermophilic phase of composting proceeds rapidly and maximum temperatures are reached after two weeks.The thermophilic phase lasts about 3 weeks.Temperatures above 50 °C for more than 3 weeks are the maximum temperatures for composting.Aeration, C/N ratio, and humidity all affect when the thermophilic phase begins.The compost mixture must be added and moistened to lengthen this period, which might last 10 to 30 days.At this point, the decomposition rate quickens when the compost pile heats up to 62 °C (>50 °C) [7,8].
Once the maximum temperature is reached, the compost temperature begins to decrease and a slow cooling phase occurs until it reaches room temperature.In the final stages, around week 10, the compost begins to look black, resemble dirt, and no longer smells.The degree or completion of the composting process is referred to as maturity.The greatest technique to judge maturity is to measure two or more key criteria because maturity is not based on a single attribute.The relative stability of the substance has some bearing on the compost's maturity, but other chemical aspects of the compost's composition also have an effect on plant development.Certain organic acids, free ammonia, and other water-soluble substances that might inhibit seed germination and root growth can be found in some immature composts.The mature product of compost must be devoid of any elements that could be cytotoxic for all intended purposes [9].
Compost is deemed "mature" after it reaches a certain amount of stability and maturity, therefore measuring these two factors is crucial.To assess the consistency and maturity of compost samples, several physical, chemical, and biological tests have been suggested.The stability of the compost generated is evaluated using variables like C/N temperature and dissolved organic carbon (DOC), while seed germination and solvency emissions are put to the test.For maturity analysis, compost calculations are available [10].The entire composting process is monitored for microbial activity.In the early stage, bacteria and fungus (30 10 7 CFU/g and 26 10 7 CFU/g, respectively) predominated.The microbial population grows dramatically during the mesophilic phase (first to third week) (bacteria 238 10 7 CFU/g and fungus 190 10 7 CFU/g).The majority of the bacterial population was present at high temperatures in the thermophilic stage (fourth to seventh week), but the fungal population was significantly less (295 10 7 CFU/g).Additionally, the quantity of bacteria and fungus rose when the composting temperature was discovered to be higher than 50 o C (fifth to sixth week).This is because microbes begin to reproduce very well by utilizing and processing organic materials into energy sources, at this time the peak of the decomposition process occurs, where the C/N ratio is very high.
Microbial respiration is the process by which bacteria utilise the energy included in chemical and organic materials (such as sugar, leaves, paper, fruit, and plant waste) in order to build cells and transport resources throughout the body.Microbial communities' metabolic activity has a direct impact on respiration.When there is a lot of bioavailable organic stuff, microorganisms breathe more frequently.As a result, the respiration pattern is tracked based on CO2 generation, O2 absorption, and heat release during the composting process.The respiratory index is basically a test or method used to evaluate CO2 production, O2 absorption, or heat release [11].
In the cooling phase (eighth week to tenth week) stabilization occurs because the temperature decreases slowly until it is constant, there is a bacterial population of 39 × 10 7 CFU/g and fungus 33 × 10 7 CFU/g which begins to decline.Finally, during the maturation phase of organic matter into mature compost, the population of bacteria and fungi decreased (30 × 10 7 CFU/g).This is due to the reduction of nutrients for microbes, especially carbon sources needed by microbes to grow and multiply, while during the cooling phase the organic material is almost completely mature.
When the compost reaches room temperature and has a C/N ratio of less than 20, which indicates that it is mature and ready for application to plants, the temperature is starting to stabilize.The C/N graph during composting is shown in Figure 2. The compost's maturity and quality may be determined by looking at its C/N ratio.Organic carbon (C) for energy and growth, and nitrogen (N) for proteins as the building blocks for metabolic cells are needed for the breakdown process that takes place during composting.Between 30 and 40 is the range for the effective C/N ratio in composting.In addition to using N to synthesize proteins, microorganisms employ C molecules as an energy source.Bacteria receive sufficient C for energy and N for protein synthesis at C/N ratios of 30 to 40.Bacteria will not have enough N for protein synthesis if the C/N value is too high, which will slow down breakdown [12].
Low C/N ratio organic fertilizers will have a high concentration of ammonia (NH3) generated by ammonia bacteria.This substance can undergo further oxidation to produce nitrites and nitrates, which are readily assimilated by plants.Nitrogen will readily escape into the atmosphere if the C/N ratio is too low as it will also produce ammonia gas.The organic carbon content of the raw material has an impact on the shift in the C/N ratio, and it tends to go down.The C/N ratio falls near the end of incubation because the change in nitrogen content is comparatively steady.Observations of the initial C/N ratio interaction showed a decrease until the fourth week and remained relatively stable, approaching the standard compost C/N value of approximately 18-25 over the past week.In comparison to values of 40 and 30, a greater carbon content at a C/N ratio of 50 led to a bigger shift in C/N values.Organic matter decreased the C/N ratio and organic C concentration.The substrate is oxidized to CO2 and utilized as an energy source by microorganisms.The availability of carbon during decomposition is necessary as an energy supplier for optimal microbial functioning [13,14].

Conclusions
Throughout the composting process, the microbial population's dynamics change.From week 1 through week 10 of the composting process, the microbial population, including fungus and bacteria, varies.This is connected to both the C/N ratio and the temperature experienced during the composting process.At 300 x 10 7 CFU/g during the sixth week of composting, when the process had reached the thermophilic phase with a C/N of 55, the largest population of bacteria and fungus was discovered.

Figure 1 .
Figure 1.Graph of microbial population (bacteria and fungi) during the decomposition of marine organic waste

Figure 2 .
Figure 2. Graph of the C/N ratio during the composting process of marine organic waste