The effect of bioactivator formulation on nutritional quality and functional microbial content in goat feces biofertilizer

Changes in lifestyle and the negative impact of chemicals make people aware of the dangers of consuming food products containing chemical residues so many people switch consuming organic food. Black rice is one of the kinds of rice that is widely consumed as an organic functional food because of its antioxidant and dietary fiber content. Black rice has been rarely cultivated because it longer harvests age and lower productivity than white rice. The way to increase the production and quality of black rice is by applying manure or biofertilizer, which contains the nutrients needed by plants. This study aimed to find bioactivator formulation that has the highest nutrients and functional microbes’ content in biofertilizer. This study used a completely randomized design with five bio activator treatments: Bacillus, Trichoderma, Saccharomyces, Bacillus + Trichoderma, Bacillus + Saccharomyces, which have replicated three times. The results showed that the bio activator formulation had a significant effect on the total bacteria, very significant effect on the organic C content, total N-fixing bacteria, P solubilizing microbes, N and P content.


Introduction
Lifestyle changes and the negative impact of chemicals make people aware of the dangers of consuming food products that contain chemical residues [1][2].This opens the opportunity to develop organic farming that produces organic plant and food in Indonesia.The way to increase the production and quality of the organic plant can be achieved by applying organic manure or biofertilizers containing the nutrients plants need.Previous studies have shown that application of goat manure can increase chlorophyll content, growth rate, and yield of black rice compared to cow and chicken manure [3].This is because goat manure contains 2.325% total N, 4.045% P2O5, and 2.977% K2O [4].This content is almost two times higher than the nutritional content in chicken and cow manure.The presence of these elements will tend to increase the chlorophyll content of plants.The high content of chlorophyll found in goat manure treatment that positively correlated with the grain weight of black rice plants [3].
Optimizing goat manure can be achieved by adding bioactivators as organic matter decomposing organisms.Biofertilizers contain microbial inoculants or carriers containing living cells of efficient strains for nitrogen fixation, phosphate solubilization, and cellulose decomposition, which are helpful for increasing soil fertility and plant growth by increasing the number and biological activity of beneficial microorganisms present in the soil [5].Bioactivator are genetically natural and not engineered, which can ferment organic matter, improve the quality of organic matter as fertilizer, 1230 (2023) 012069 IOP Publishing doi:10.1088/1755-1315/1230/1/012069 2 improve soil quality, and produce energy [8].This research is an advanced stage of research carried out in the previous year, exploring the basic material of livestock manure, with a focus on adding bioactivators.The production of goat fertilizer previously used Trichoderma and Bacillus as bioactivators.However, it has not yet identified the best type of bioactivator from a single microorganism or consortium which can result a good biofertilizer [3].This research aimed to find an appropriate bioactivator formulation from the microbe consortium that can be combined with humic acid as an additive in goat manure fermentation.

Materials
The raw material used in this study was organic goat feces that had been mature and mashed.Goat feces naturally contain microorganisms that can decompose organic matter.These microorganisms are Bacillus sp, Pseudomonas, and Staphylococcus, which have the potential to compost organic matter [6] so that fermentation can occur even without adding bioactivators, but it generally takes a longer fermentation time.The following content of the goat feces based on analysis used in this research can see in Table 1 below.The bioactivators used for this study were single and consortium in the form of Bacillus, Trichoderma, and Saccharomyces.Humic acids are applied in feces to maintain the stability of the acidity of the fertilizer substrate so that natural microorganisms contained in goat feces can survive [3].Decomposition by soil microbes is either aerobic (aerobic Bacteria and fungi) or anaerobic bacteria.Aerobic degradation of the plant microbial residues contains humic acid in the soil.The abundance of humic acid and fulvic acid promotes the bonding properties of the humus [7].The content of the humic acid based on analysis used in this research can see in Table 2.

Results
The biofertilizer treatment significantly affected the N content of the biofertilizer (P<0.01).The highest N content in goat feces biofertilizer was found in the treatment with the bioactivator combination of the Bacillus + Trichoderma + Saccharomyches (B6) consortium.In contrast, the lowest nitrogen content was found in the B3 (Saccharomyces) treatment.Bioactivators with single treatment (B1 and B2) showed non-significantly different results, except for B3, which was significantly different with all treatments.Bioactivators with 2 combinations of microbes (B4 and B5) showed lower nitrogen yields when compared to B6.The effect of bioactivator on nitrogen content is shown in Table 3.

Observations:
N, P, K, C organic, total bacteria, total N fixing, total phosphate solvent Bioactivator application and advanced fermentation: 1).Every 100 ml of the bioactivator formula is mixed into 100 kg of goat manure, stir until homogeneous 2).Aerobic fermentation for 7 days.
The treatment of the bioactivator also had a very significant effect on the P content of the biofertilizer (P<0.01).The biofertilizer with the highest phosphorus content was found in the Bacillus + Trichoderma + Saccharomyces (B6) consortium bioactivator treatment.The effect of bioactivator on phosphorus content is shown in Table 4. Description: The number followed by the same letter is not significantly different according to DMRT 5%.
Based on the data, the potassium content in all bioactivator treatments showed no significant difference (P<0.05).The treatment of a single bioactivator and microbial consortium showed results that were not statistically different.The effect of bioactivator on potassium content is shown in Table 5.The organic C content of goat feces biofertilizer showed very significant results (P<0.01).The highest biofertilizer organic C content was found in the Bacillus + Trichoderma (B4) and Bacillus + Trichoderma + Saccharomyces (B6) consortium bioactivator treatments, while the lowest organic C content was in the Bacillus + Saccharomyces (B5) treatment.The three single bioactivator treatments showed non significantly differences in organic C content (B1, B2, and B3).The effect of bioactivator on organic C content is shown in Table 6.The number followed by the same letter is not significantly different according to DMRT 5%.
Based on the results of the data analysis, the microbial content of N fixing has a very significant result (P<0.01).The combination of the microbial consortium Bacillus + Trichoderma + Saccharomyces (B6) showed the highest total N fixation results compared to the treatment of a single bioactivator or a combination of 2 activators.The effect of bioactivator on N fixing total content is shown in Table 7. Description: The number followed by the same letter is not significantly different according to DMRT 5%.
The P solubilizing content has a very significant result (P <0.01).The treatments with the highest total P solvent were Bacillus (B1), Bacillus + Trichoderma (B4), and Saccharomyces (B3) bioactivators.In contrast the treatment of bioactivator B2 and B5 showed the lowest total P solvent compared to other treatments.In general, Bacillus and its combinations showed good total P solvent yields, except for combination with Saccharomyces (B5).The effect of bioactivator on P solubilizing content is shown in Table 8.Description: The number followed by the same letter is not significantly different according to DMRT 5%.
The total bacterial content has a significant result (0.01<P>0.05).Bacteria were found in all biofertilizer treatments, including those containing fungi and yeast, Trichoderma (B2), and Saccharomyces (B3).However, the abundance of bacteria in these two treatments was the lowest compared to the other treatments.The single bioactivator treatment gave a higher total bacterial yield on Bacillus (B1) and Bacillus + Trichoderma+ Saccharomyces (B6) treatments.Meanwhile, treatments B4 and B5 showed the lowest total bacterial abundance.The effect of bioactivator on total bacteria abundance is shown in Table 9 and Figure 2.   Microorganisms in biofertilizers that act as bioactivators convert complex organic materials into simpler compounds [5].Goat manure (GM) is an excellent raw material for anaerobic digestion because of its high total nitrogen content and fermentation stability [8].The total N element content in the goat feces biofertilizer increased when compared to the total N content of the unfermented feces base material, except for treatments containing Saccharomyces (B3 and B5).The highest N content in goat feces biofertilizer was found in the treatment with the Bacillus, Trichoderma, and Saccharomyches (B6) bioactivator consortium.The N content was higher in the combination of the 3 microbial consortiums than alone, indicating a better synergism in the process of providing N.This is thought to be due to the role of Bacillus, which is able to fix nitrogen and provide nitrogen from the decomposition process of organic matter [7].In addition, Bacillus can function to suppress the growth of diseases caused by fungi.Bacteria Bacillus sp.[9,10] can also increase the growth of rubber plants through the growth regulators they produce so that more latex is produced [9].The use of Trichoderma plays a role in the process of breaking down the residues of plants by means of humic acid synthesis which is important in the development of soil structure.In addition, Trichoderma also acts as an antagonist against fungal pathogens so that plants can avoid harmful fungal attacks [11].Saccharomyces is classified as yeast which is suspected of producing enzymes like urease [12], which may play an important role in the mineralization or ammunition of nitrogen [13].But until now, the role of fungi and yeast in nitrogen cycling in Antarctic soils, is still poorly understood to date [7].

Effect of bioactivators on phosphorus content in goat feces biofertilizer.
Bacillus acts as a Psolubilizing bacterium so, it dissolves the P contained in the fertilizer better.Phosphate solubilizing bacteria solubilize inorganic forms of phosphate because of the release of organic acids into the medium, changing the pH and increasing the availability of phosphorus for the host plant [14].The main mechanism of phosphate solubilization is the production of organic acids.Gluconic, formic, 2-keto gluconic, citric, oxalic, lactic, isovaleric, succinic, glycolic, and acetic acids are among some produced by phosphate-solubilizing microorganisms [15].The lower pH of the medium suggests the release of organic acids by the P-solubilizing microorganisms via the direct oxidation pathway that occurs on the outer face of the cytoplasmic membrane [15] Thus, treatment with a combination of bacillus will produce a biofertilizer with a higher P content than other biofertilizers.In general, the total phosphorus content was lower when compared to the basic fecal P content and humic acid.This is thought to be caused by the high-water content of the biofertilizer produced (data not shown), which varied between 56-64% [16] due to the high humidity outside the experimental area, which affected the P solubilization process to become immobilized by microorganisms that play a role in the process.Intensive microbial immobilization of 33P after rewetting (up to 41 %) demonstrated a conversion of dissolved P to Pmic potentially available for plant nutrition [17].Nevertheless, the combination of the three microbial consortia showed the highest total P yield, which indicated a synergistic effect of the three microorganisms in P dissolution.The combination of Bacillus and Trichoderma was primarily able to increase the availability of P in biofertilizers.Bacillus acts as a P-solubilizing bacterium so that it can better dissolve the P contained in the fertilizer [18].This is also an explanation for the abundance of phosphate-solubilizing microorganisms in the combined Bacillus and Trichoderma (B4) biofertilizer.

Effect of bioactivators on potassium content in goat feces biofertilizer.
Potassium content in all bioactivator treatments did not show a significant difference (P<0.05).This is presumably because the livestock ration than the mineralization more influences the potassium.Because the raw material of goat feces used were the same, the total potassium yield showed no significant difference (ranging from 3.05-3.22%).In accordance with the statement of Wienhold and Giiley that the availability of K from manure is almost 100%, manure can be used like potassium fertilizer [19].Goat manure is a high source of potassium when compared to other manures, such as poultry [20].
3.2.4.Effect of bioactivators on organic C content of goat feces biofertilizer.Organic carbon is the carbon atoms in the structure of organic compounds.[21].The total organic carbon in the biofertilizer has increased compared to basic feces, where the highest increase in carbon was in the Bacillus + Trichoderma (B4) and Bacillus + Trichoderma + Saccharomyces (B6) treatments.Carbon availability in biofertilizer is influenced by the type of raw materials and microorganisms contained therein.Microorganisms Bacillus subtilis, Bacillus licheniformis, Trichoderma viride, Aspergillus niger, and yeast with the proportion (1:1:1:1:2) showed the highest increase in total carbon (12.21%) [22].Consequently, the preservation of carbon might result not only from the enrichment of the humic acid carbon and the decomposition of hemicellulose and lignin, but also the increased amount of OTU and abundance of Lactobacillus.This result provides a ratio of microbial agents to preserve the carbon during goat manure aerobic composting.Microorganisms require adequate amounts of carbon and some nitrogen from the media (fertilizer/soil) to maintain an ideal C/N ratio for their cells [23].
3.2.5.Effect of bioactivators on total N-fixing bacteria in goat feces biofertilizer.The combination of Bacillus + Trichoderma + Saccharomyces (B6) showed the highest total N fixing results compared to other treatments.The Trichoderma sp consortium has a positive correlates with Bacillus sp. and Sacharomyces sp [24] Trichoderma likes materials with high cellulose content and can coexist with Lactobacillus sp and yeast [25].Adding the bioactivator Saccharomyces cerevisiae as much as 5% can properly decompose goat urine and litter [25].Biofertilizer research consisting of a microbial consortium (Lactobacillus, Saccharomyces and Cellulomonas) was able to increase root length, number of nodules, root weight, and leaf area in soybean plants [26].Biofertilizers have been shown to be able to increase soil fertility and productivity of rice plants [27,28].Of the three types of the microbial consortium, Bacillus subtilis acts as a nitrogen-fixing bacteria and inorganic nitrogen solubilizing [29], so treatments containing Bacillus tend to have a higher total N fixing.In addition, although Saccharomyces and Trichoderma are not N-functional microorganisms, the presence of these microorganisms or their combinations is thought to support the positivity of non-symbiotic N-fixing microorganisms other than Bacillus such as Azospirillum sp, Azotobacter sp.
3.2.6.Effect of bioactivators on total P solubilizing bacteria in goat feces biofertilizer.Besides functioning as an N fixing, Bacillus also acts as a P solubilizing bacterium [18], so the total abundance of phosphate solubilizing was abundant in the Bacillus (B1) and Bacillus + Trichoderma (B4) treatments.Bacillus can solubilize phosphorus from goat feces so that it becomes available for plants [30].These microorganisms also dissolve slightly soluble phosphates by lowering the pH of the surrounding environment [30].The primary mechanism for the solubilization of phosphate minerals is the production of organic acids, and acid phosphatase plays a major role in the mineralization of organic phosphorus in soil [31].In addition, the Trichoderma (B2) and Saccharomyces (B3) treatments also could solubilize phosphate [32], but the abundance and solubilization ability of phosphorus showed lower results when compared to Bacillus.
3.2.7.Effect of bioactivators on total goat feces biofertilizer bacteria.Bacteria were found in all biofertilizer treatments, including the treatment of fungi and yeast Bacillus + Trichoderma (B4) and Bacillus + Saccharomyces (B5).However, the abundance of bacteria in the two treatments was the lowest when compared to the other treatments.This is because the goat feces material used is thought to contain natural microorganisms, including types of bacteria.Microorganisms contained in the feces are Bacillus sp, Pseudomonas, Staphylococcus which have the potential to compost organic matter [11].
The single bioactivator treatment gave a higher total bacterial yield on Bacillus (B1) and Bacillus + Trichoderma + Saccharomyces (B6) treatment.The decrease in total bacteria in B4 and B5 indicates an antagonism with the combined microorganisms, so it is necessary to consider combining bacillus with other microorganisms when considering the total abundance of bacteria.

Conclusion
Based on all results of parameters collected from the nutritional content of goat manure and the abundance of microorganisms from each biofertilizer, it was found that the Bacillus + Trichoderma + Saccharomyces (B6) treatment gave the best results.The consortium of three microorganisms that were bacteria, fungal, and yeast gave the best results on total nitrogen, phosphorus, organic C, N fixing, and total bacteria compared to other treatments.This research proved that these microorganisms can synergize well and produce good goat feces biofertilizer when combined.

Figure 1 .
Figure 1.Stages of the biofertilizer production process.

Table 1 .
Basic content of goat feces.

Table 2 .
Content and characteristics of humic acid.This research was conducted in two place.The bioactivator formulation was carried out in Trunojoyo University Laboratory, while the fertilizer fermentation process was carried out in Banyuwangi State Polytechnic.The overall research activity stage can see in the following diagram: 3

Table 6 .
Organic content of goat feces biofertilizer

Table 7 .
N fixing total of goat feces biofertilizer