Bacterial abundance of cacao agroforestry, cacao monoculture and secondary forest land uses in north Luwu, South Sulawesi

Expanding the agricultural sector in North Luwu influences land use modification to enhance plantation output. Continuous land use and clearance can jeopardize the survival of native land vegetation, altering C and N cycles, community composition, population, and soil microbial activity. Microorganisms are essential for maintaining the health and quality of the soil. The abundance of soil microorganisms significantly impacts ecosystem function, so it can help boost agricultural output. This demonstrates that data on the abundance and diversity of soil microorganisms can be used to determine soil fertility. However, more information is needed about the diversity of soil microbes in North Luwu. This study seeks to determine the abundance of soil microorganisms, particularly for various land uses. Random soil samples were collected from cacao monoculture, cacao agroforestry, and secondary forest. The C: N ratio and the total number of microorganisms were then measured in the collected soil samples. Cacao agroforestry with the highest C: N ratio had 1.8 x 108 CFU/g of total soil bacterial, the highest bacterial abundance among the three land covers, followed by secondary forest and cacao monoculture with the lowest C: N ratio and lowest bacterial abundance 2 x 107 CFU/g. These results indicated that the soil C: N ratio influences the bacterial abundance of various land uses. The highest abundance of nitrate-fixing bacteria was found in secondary forests and the lowest in cacao agroforestry, even though the %N indicated by both was the same.


Introduction
North Luwu Regency is an area that makes the agricultural sector a strategic regional development order.The agricultural sector is the most significant contributor to non-oil and gas foreign exchange, accounting for 47.02 % of the regional gross domestic product.As a result, plantations producing the development objectives of cacao, oil palm, pepper, coffee, cloves, sago, and coconut produce more [1].
The increase in production is in line with the land conversion that threatens the development of the agricultural sector in the North Luwu Regency [2].As a consequence of ongoing land use and clearing, native vegetation is reduced in size and eliminated, which affects soil microbe population, activity, and community composition [3,4] as well as C, N, and P cycling [5].Understanding which factors play a significant role in the development of the soil microbial community will enhance our knowledge of the effects of soil management on soil carbon and nutrient cycling.The soil C: N ratio was the most 1230 (2023) 012081 IOP Publishing doi:10.1088/1755-1315/1230/1/012081 2 influential factor in determining the soil microbial community structure [6].The carbon-to-nitrogen ratio of organic matter refers to the proportion of carbon to nitrogen present [7].
Soil microbes are crucial participants in biogeochemical cycles, and soils are the source of energy [8].Soil microorganisms are essential to the Earth's ecosystem and influence global climate by regulating CO2, CH4, N2O, and other greenhouse gases.Numerous studies have documented the connection between soil bacteria and climate change.It has been verified that soil microorganisms' regulation of carbon, nitrogen, phosphorus and other element cycles has played a significant role in climate change feedback [9].In addition, soil bacteria can promote plant growth [10,11].Nitrogen-fixing microorganisms in the soil can boost plant absorption of nitrogen (N), phosphorus (P), and potassium (K), among other nutrients.Because N, P, K, and other elements are the primary nutrients that affect plant growth and reproduction, the presence of nitrogen-fixing microbes can influence the enhancement of plant community growth [12].
Nitrogen-fixing bacteria can be found in soil and plant roots [13,14].Nitrogen-fixing bacteria consist of non-symbiotic bacteria that fix free nitrogen in the air without associating with other organisms and symbiotic bacteria that fix nitrogen by associating with other organisms.Symbiotic nitrogen-fixing bacteria only provide nitrogen to their host, while non-symbiotic bacteria provide ready-to-use nitrogen in the soil.Some non-symbiotic nitrogen-fixing bacteria are the genus Azotobacter [15] and Bacillus [16].Non-symbiotic nitrogen-fixing bacteria such as Azotobacter and Bacillus can fix nitrogen mediated by nitrogenase activity [17][18][19].
The abundance of soil bacteria, especially nitrogen-fixing bacteria, significantly influences ecosystem function and can help increase productivity in the agricultural sector.However, the community structure of soil bacteria is very vulnerable to changes in soil conditions.Directly and indirectly, changes to soil conditions, mainly by human activities can disturb the physical and chemical properties of the soil, thus affecting the presence of soil bacteria and changing their ecological functions [20].The abundance and diversity of soil microbes vary depending on the land cover type [21].
Soil management is crucial for sustaining soil quality in agroforestry systems for cacao production.As a sustainable land management practice, agroforestry has demonstrated its function in enhancing soil microbial dynamics, which would positively affect soil health [22].The composition of bacterial communities differed significantly between organic and conventional management, as well as agroforestry and monoculture.Taxa associated with conventionally managed systems were more taxonomically diverse than those associated with organic management [23].Silva-Parra [24], showed that secondary forest and cacao agroforestry had more total bacteria than rice monoculture and pineapple monoculture.
Soil bacterial abundance data can be one of the references in determining soil fertility on a land.However, there needs to be more data on the microbial abundance in North Luwu, especially on different land covers.Therefore, research on soil microbial abundance analysis on different land covers in North Luwu needs to be done to get an overview of bacterial abundance on each land cover that can potentially increase agricultural sector production.

Measurement of moisture content and organic matter C and N in soil samples
The soil samples obtained were dried in an oven at 105 0 C to measure soil moisture content and dry weight.Measurements were taken every 24 hours until stable measurement data were obtained.The obtained measurement results were inputted into the following equation [26]: Each sample was also air-dried at room temperature before being analyzed for organic matter C and N in the Soil Chemistry and Fertility laboratory, Soil Science Department, Faculty of Agriculture, Hasanuddin University.C content analysis using the Walkey Black method and N content using the Kjeidahl method.

Isolation and enumeration of soil microbes
Soil samples were isolated using stratified dilution.A 10-gram soil sample was placed in a 90 ml physiological NaCl solution for a 1:10 dilution.Transfer the sample as much as 1 ml to the subsequent dilution, and do so until the dilution series 10 -6 .Furthermore, 1 ml of sample was taken aseptically from each dilution and inoculated on Nutrient Agar (NA) medium (Oxoid) for total microbes and Asbhy's mannitol agar (HIMEDIA) for total Nitrogen-fixing bacteria.Enumeration was done by indirectly counting the number of bacteria by counting the number of colonies that grew in each dilution series with the help of a colony counter.Petri dishes that were counted were those with 30-300 colonies.The colony count results were then converted through the Total Plate Count (TPC) method to obtain the total number of microbes per gram of soil dry weight [26] Total Population (CFU)g -1 soil dry= (colony count)x (Df) dw of soil Note : Df : The dilution factor of the petri dish whose colonies were counted Dw : Dry weight of soil sample (g) = wet weight x (1 -moisture content)

Analysis of Organic C and N in Three different land use in North Luwu
The results of soil organic matter carbon and nitrogen levels analysis at three locations in North Luwu are shown in Table 1.Based on the observation, CA showed the highest C% and C: N ratio.In most cacao-growing regions of the globe, soil organic C and total N stocks are typically high in cacao agroforestry systems [27][28][29].The analysis of soil N% revealed a range between 0.11-0.16%, with the greatest percentage corresponding to cacao monoculture land cover.Most nitrogen is in the topsoil, and less than 10% of the total N stock is in cacao and shade trees [27].dilution The results of isolating the three soil samples on NA media (Figure 1) revealed that CA had the highest total bacterial abundance at 1.8 x 10 8 CFU/g dry soil, followed by SF and CM (Table 2).In monoculture cacao land, the bacterial population density was 10 7 CFU/g, while in secondary forest and agroforestry cacao land cover, it was approximately 10 8 CFU/g.Fertile soil has at least a minimum bacterial population abundance of 10 7 CFU/g [30]; this indicates that the three land uses are still fertile states based on the number of abundant bacterial populations.The abundance of microorganisms in soil is highly variable, and environmental factors significantly influence their distribution.Soil nutrients are one factor influencing the abundance of microorganisms in a field.According to prior research [31], dissolved organic carbon and total nitrogen were positively correlated with microbial abundance, whereas pH and soil moisture content were negatively correlated with microbial abundance.The higher the C: N ratio, the greater the total microbial count.Sample CA exhibited the highest microbial count and the highest C: N ratio of 23.C: N ratios between 20 and 30 create an equilibrium between mineralization and immobilization, with the optimal ratio being 24 [7].Cacao agroforestry systems can contain large quantities of organic C, total N, and organic P in the soil profile and large quantities of microbial biomass C and N and mineralizable N in the topsoil [32].In their study, Gawol et al. [33] found that variations in the C: N ratio influence bacterial diversity in the IDFBC ecozone; however, changes in the C: N ratio have a negligible effect on soil microbial communities.

Isolation and enumeration of soil microbes
Furthermore, the total population of non-symbiotic nitrogen-fixing soil bacteria was calculated using Asbhy's mannitol agar media.Asbhy's mannitol agar is an N-free medium to cultivate non-symbiotic Nfixing Azotobacter bacteria from soil [34].Accumulation of soil nitrogen also increases microbial abundance because increased utilization of nitrogen resources reduces the carbon limitation of microorganisms [35].3 displays the results of isolating three samples with Asbhy's mannitol agar media.The soil sample from the secondary forest (SF) had the highest total microbial population with 4.3 x 10 7 CFU/g dry soil, while CA had the lowest with 4.2 x 10 6 CFU/g dry soil.The availability of organic carbon energy sources in the rhizosphere environment influences the high and low total population of nonsymbiotic N 2 -fixing microbes.Non-symbiotic N 2 -fixing bacteria use organic matter as a carbon source to obtain energy; research by Indriani et al. [36] showed in an experiment using organic matter that the population of non-symbiotic N2-fixing bacteria had the highest population at a higher dose of organic matter, namely 4.5% compared to the population of 1.5% organic matter dose.
Nitrogen in the cacao agroforestry and secondary forest had similar values, but cacao agroforestry showed the lowest bacterial abundances.CM with the highest N% did not show the highest microbial abundance.The effect of C and N gradients on soil can cause significant effects on soil microbial community structure.Liu et al. [37] in their study showed that the correlation between total nitrogen and fungal biomass was not significant.The interactions between soil microbes and the C and N fractions are complex and require in-depth understanding.

Conclusion
Based on the results of the research, it can be concluded that the CA sample with the highest C: N ratio had the maximum bacterial abundance of 1.8 x 10 8 CFU/g, whereas the CM sample with the lowest C: N ratio had the lowest bacterial abundance of 2 x 10 7 CFU/g.Based on observations of the abundance of nitrate-fixing bacteria, nitrate-fixing microorganisms in the three land cover types varied, although the N% in each land cover type was identical.

Figure 1 .
Figure 1.Microbial growth on NA media (a) CM 10 -5 dilution (b) SF 10 -6 dilution and (c) CA 10 -6dilution The results of isolating the three soil samples on NA media (Figure1) revealed that CA had the highest total bacterial abundance at 1.8 x 10 8 CFU/g dry soil, followed by SF and CM (Table2).In monoculture cacao land, the bacterial population density was 10 7 CFU/g, while in secondary forest and agroforestry cacao land cover, it was approximately 10 8 CFU/g.Fertile soil has at least a minimum bacterial population abundance of 10 7 CFU/g[30]; this indicates that the three land uses are still fertile states based on the number of abundant bacterial populations.

Table 1 .
Analysis of C and N levels in cacao agroforestry, cacao monoculture, and secondary forest land covers in North Luwu.

Table 2 .
Total microbial population of samples CM, SF and CA.

Table 3 .
Total population of N-fixing bacteria in cacao agroforestry, cacao monoculture, and secondary forest land covers in North Luwu.