A Study of Bioconversion Method for Milk Waste Treatment Using Black Soldier Fly Larvae

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Introduction
Milk waste is one of the environmental problems that has good characteristics as a place to growth and breeding of microorganisms such as bacteria and fungi that can cause disease in humans if further management is not conducted immediately.Milk waste has a characteristic that is very easy to rot and if not managed quickly it will result in the occurrence of a very disturbing and unpleasant odor [1].Significantly, one of the ways that can be done in the management of unused milk waste is to carry out composting methods so that unused milk waste does not pollute the environment.Black soldier fly larvae or commonly known as maggot, has been used by many as a method of composting organic waste through a bioconversion process.In the stages of the composting or bioconversion process carried out by maggot larvae, there is one important stage, namely the formation of biomass which is rich in protein and fat sources which will certainly have a high potential value as animal feed such as poultry and fisheries [2].
Waste has become one of the most common problems found among every stakeholder and all communities in Indonesia [3].According to Law Number 18 of 2008 which regulates Waste Management, the reuse of organic waste into compost is one of the effective ways to reduce the amount of waste generated from the source [4].Based on the types of organic waste produced from various sources, The most commonly practiced organic waste management process is composting.But to carry out the composting p-ISSN : 2528-3561 e-ISSN :  process in decomposing organic waste takes a relatively long time.The larvae of the black soldier fly or commonly known as maggots are one of the most effective methods in managing organic waste, where the management process is carried out by Maggots have an accelerated seepage rate of 62.68-73.98%[5].Proses organic waste treatment using black soldier fly larvae through a bioconversion process, bioconversion is one of the organic waste treatment methods in which maggots absorb nutrients from the waste and convert them into larval biomass [6] [7].
Maggot are organisms derived from eggs phase of BSF or scientifically known as Hermetia illucens, which is the second phase after the eggs and before the pupae stage in the larval form.Depending on the ambient temperature, the larvae of the black soldier fly can actively break down food in 21-24 days [8].The low ambient temperature prolongs the process of environmental degradation.Another study found that BSF larvae were able to decompose 8122.1 grams of household waste, 1859.7 grams of melon waste, 1320.3 grams of chicory waste and 1683.3 grams of tofu waste [5].According to Ibadurrohman et al., ( 2020) , the larva of the fly soldier B has an extraordinary ability of 75% in recycling biological waste, where 4 kg of waste produces 800 g of larval biomass.
During the bioconversion process carried out by the black soldier fly larvae before entering the predation stage, the black soldier fly larvae reduce the waste used as food to residues of the reduction process that can be used as organic fertilizer or compost [9].On average, the composting time of black soldier fly larvae is about 12-15 days, which is much faster than microbes or earthworms where in composting earthworms it takes 3-5 weeks [10].The compost produced from the bioconversion of black soldier fly larvae is black, finely divided and smells of soil [11].Different concentrations of growing media used in BSF larvae will produce maggots with different nutrient content.The high protein content in the maggot growing medium will have a very good effect on the protein in the maggot [12].
According to the above-mentioned problems, it is necessary to conduct further research on the effect of a mixture of dairy waste and organic waste on the weight of black soldier fly larvae.This research is also expected to be able to provide important information for the development of researchers on mixed media of milk waste and mixed media of organic waste to produce BSF larval weights through the larval bioconversion process.

1) Population and Sample
This final project research uses an experiment method with a quantitative approach.The population in this study was Black Soldier Fly Larvae from the harvest of black soldier fly larvae from the bioconversion process on milk waste and organic waste.Because this aspect of the study uses an experimental approach, random samples of black soldier fly larvae from each treatment of A1, A2, B1, B2, C1 and C2 were taken to be tested in the laboratory for protein content in them.

2) Data Collection
The data used in this final project research is primary data.The data collection method used in this study is the experiment method by conducting maggot bioconversion experiment research for 13 days to obtain weight and weight data on maggots and laboratory experiments.The interpretation in this final project research is defined as an investigation of the hypothesis by conducting scientific tests.The bioconversion process of Milk waste and organic waste was carried out in December 2022 and the protein test experiment was carried out in January 2023.
This study focused on the influence of the quantity and quality of black soldier fly larvae, especially in the parameters of wet weight, dry weight, compost reduction weight and protein content in maggots.Simultaneously, the independent variables of this experiment were the concentration of dairy waste and organic waste while the dependent variables in this study were wet weight, dry weight, and compost reduction results.
The experimental method in this study uses a randomized block design (RBD) method with nonfactorial preparation with 3 treatments, and 2 replications for treatment.With the weight of each treatment is 2 kg or 2000 grams.The 3 types of treatment in this study are distinguished as follows: Treatment A: Milk waste 100% (milk as much as 2000 ml) Treatment B: 50% milk:50 % organic waste (1000 ml milk: 1000 gr organic waste) Treatment C: 70% milk:30 % organic waste (1400 ml milk: 600 gr organic waste)

3) Wet and Dry Weight Measurement
On the 18th day after the harvest period, the first parameter to be measured is wet and dry weight.The 18 DOL larvae will be separated with the remaining bioconversion compost residues, the BSF larvae are separated by sifting, after the maggots have been separated from the growing medium then weighed using digital scales.Then the result of the wet weight is recorded its results.

4) Residual Compost Measurement
Waste Reduction Index (WRI) is a method to determine and measure the waste reduction index in larvae.If the value of the Waste Reduction Index in the larvae is high, it indicates a high level of ability in the larvae to reduce waste.To find the WRI value calculated using a method that refers to the equation (Diener, 2009) [13]:

𝐷 = 𝑊 − 𝑅 𝑊
Information: W = Total waste amount (gr) t = total time the larvae in eating waste (days) R = Total waste residue after a certain time (gr) D = Total waste reduction (gr) WRI = Waste Reduction Index [14] 5) Protein Quality Test Content testing.Measurement of protein content in BSF larval samples for each treatment was carried out in the Environmental Engineering laboratory, President University.Testing on this protein test was carried out by the Kjeldahl method referring to the AOAC 2001 [35].The protein content test process in this method includes 3 stages consisting of the digestion process, distillation process and titration process.The step steps on each sub parameter will be explained as follows.
1. Put in 1 gram of sample into Kjeldahl flask.2. Add 7 grams of K2SO4 and 0.8 gr of CuSO4 to the Kjeldahl flask. 100 %

Wet weight
The wet weight value that has been obtained after the harvest period on day 18 of each treatment is shown in Table 2. Based on the wet weight results obtained in each treatment in Table 2, it can be seen that the average wet weight of larvae is 18 DOL with various ratios of treatment media administration and observation time, it can be seen that the total and average amount of wet weight is obtained from treatment B with a combination media, which are 1000 ml of organic waste and 1000 ml of organic waste mixture.The average wet weight of maggots in the treatment averaged 315.25 gr.B closely the average black soldier fly larva in treatment C is lower than treatment B, this is due to the organic content in treatment B and treatment C which triggers the growth and weight development of black soldier fly larvae.The growth rate of larval weight is getting faster supported by the addition of UHT milk which provides additional nutrition for the development of black soldier fly larvae weight.A comparison of the initial weight of 5 DOL larvae and the final weight after harvesting 18 DOL larvae can be seen in Figure 2. From Figure 2, it is very clear the comparison of the initial weight and the final weight in each treatment.This spurs on the composition contained in the treatment media, where the media is equipped with various vegetable waste, fruit waste, and even food waste left over and texture media that is not too wet after being mixed with a mixture of organic waste spurs the growth of BSF larvae.The lowest wet weight of maggots is found in treatment A (full milk waste) with an average of 8.75 gr, this is because the milk waste media used contains high water, so that the media can have an inhabitant influence on the breeding of maggots.Of all the treatments, the results were followed by an ANOVA test to see if there were statistically significant differences between the groups.The data of the ANOVA test results on the wet weight parameter are shown in Table 3.Based on the data from the ANOVA test results shown in Table 3, it is known that the F table value is 1% < F value ≥ F table is 5% at a significance level of 1% and 5% which means that there is one or more treatments that significantly different, which is marked with the notation **, then reject H0.So it was found that there was a significant difference between treatment A, treatment B, and treatment C. Furthermore, because there was a significant difference between the three treatments, a follow-up test of the LSD test was carried out.The results of the LSD test on wet weight are shown in Table 4 below: Note: If the average value is followed by the same letter in the same column, it means that in the LSD test 5% between treatments did not differ significantly.And if the average value is followed by different letters, it means that in the LSD test 5% is significantly different.
In the 5% LSD test analysis for wet weight results, there are two treatments that have the same notation in notation b, namely Treatment B (50% milk waste & 50% organic waste) and treatment C (70% milk waste & 30% organic waste), because the average value in treatment B and C is followed by the same notation, then treatment A not significantly different on treatment C. Based on the results of the 5% LSD test on the wet weight value in practice A, the average value is followed by the notation a and the treatment B the average value is followed by the notation b.Because the average value is not followed by the same letter notation in the 5% LSD test, the A treatment is significantly different from the B treatment and the A treatment significantly different from the treatment C at the level of α = 0.05.

Dry weight
The value of the dry weight yield on those obtained after the washing process and the drying process of each treatment is shown in Table 5.Based on the dry weight results obtained in each treatment in Table 5, it can be seen that the average dry weight of larvae is 18 DOL with various ratios of treatment media administration and observation time, it can be seen that the total and average amount of wet weight is obtained from treatment B with a combination media, namely 1000 ml of organic waste and 1000 ml of organic waste mixture.The average dry weight of maggots in the treatment averaged 266.5 gr and was followed by the average C treatment of 213.65 and the smallest weight in treatment A with an average of 6.9 gr.It can be seen in the measuring data that this dry weight is the pure weight of maggot larvae because they have been washed and cleaned of the remaining feeding residues that are still attached to the body of the BSF larvae.Data on the comparison of initial weight and final weight after the harvesting process can be seen in Figure 3. Furthermore, all dry weight results in each treatment were tested with advanced ANOVA assays to see if there were significant statistical differences between treatment groups in the dry weight parameters.ANOVA test data on dry weight variables can be seen in Table 6.Based on the data from the ANOVA test results shown in table 6, it is known that the F table value is 1% < F value ≥ F table is 5% at a significance level of 1% and 5% which means that there is one or more treatments that significantly different, which is marked with the notation **, then reject H0.So it was found that there was a significant difference between treatment A, treatment B, and treatment C. Furthermore, because there was a significant difference between the three treatments, a follow-up test of the LSD test was carried out.LSD results on dry weight can be seen in Table 7. Note: If the average value is followed by the same letter in the same column, it means that in the LSD test 5% between treatments did not differ significantly.And if the average value is followed by different letters, it means that in the LSD test 5% is significantly different.
Based on the data from the 5% LSD test results for dry weight results found in table 8, there are two treatments that have the same notation in notation b, namely Treatment B (50% milk waste & 50% organic waste) and treatment C (70% milk waste & 30% organic waste), because the average values in treatments B and C are followed by the same notation, then treatment not significantly different from treatment C. Based on the results of the 5% LSD test on the dry weight value in practice A, the average value is followed by the notation a and in treatment B the average value is followed by the notation b.Because the average value is not followed by the same letter notation in the 5% LSD test, the A treatment is significantly different from the B treatment and treatment A significantly different from treatment C at level α = 0.05.

Reduction Rate
The residual value of waste reduction results in the Maggot bioconversion process in each treatment is shown in Table 8.As shown in Table 8, after the results of residual compost residue were weighed during the mass harvest period for treatment A at repetitions 1 and 2 was the largest compared to treatment B and C, namely treatment A had a residual residue weight of 805.7 gr in replication 1 and residue of 924.5 gr in replication 2, this was due to the small weight produced in treatment A so that the ability of black soldier fly larvae lowers in reducing food or media in treatment A. Another fact in the field at the time of the experiment was that there were many black soldier fly larvae that died and only some lived and thrived until harvest day.The results of the waste reduction index in each treatment can be seen in Figure 4.The highest Waste Reduction Index value is in treatment C, which is 4.96% where treatment C consists of 1400 ml of milk waste and 600 gr of organic waste mixture.The WRI value obtained in this study is above the WRI value obtained (Aulia Arief Nursaid) which uses fruit waste bait.The type of growing media feed or feed media given affects the ability of black soldier fly larvae to consume bait or feed media given.

Protein Test
The nitrogen test results in each treatment are shown in Table 9.Table 9 showing that the highest protein content results were obtained from treatment B where there was an average protein content of 33.5%, while in treatment C there was an average crude protein content of 29.59% and it can be seen that the lowest protein content was obtained from treatment A with an average protein content of 18.34%.One of the factors causing the difference in some of the average crude protein content in the sample is the vulnerable harvest period and the drying process of maggot larvae with the study time in the laboratory.Then from each treatment for the test results on crude protein, the results were followed by the ANOVA test to see if there were statistically significant differences between the protein test results in each treatment.Based on the data of the Anova test results shown in Table 10, it is known that the F table value is 1% < F value ≥ F table is 5% at a significance level of 1% and 5% which means that there is one or more very noticeably different treatments, which are marked with the notation **, then reject H0.So it was found that there was a significant difference between the protein test results in treatment A, treatment B, and treatment C. Furthermore, because there were significant differences between the protein test results in the three treatments, a follow-up LSD test was carried out.The results of LSD pada protein test results can be seen in Table 11.Note: If the average value is followed by the same letter in the same column, it means that in the LSD test 5% between treatments did not differ significantly.And if the average value is followed by different letters, it means that in the LSD test 5% is significantly different.
Based on the 5% LSD test result data for the protein test results found in Table 11, there is no treatment that has the same notation in treatments A, B and C. Because the average values in treatments A, B and C are followed by different notations, then that protein test in each treatment, both treatments A, B and C, differs significantly at the level of α = 0.05.This statistical test shows that the hypothesis of the ratio of the growth medium of Black Soldier Fly larvae affects protein yield.
Treatment B had the highest crude protein content of 33.5% and was followed by treatment C with an average protein content of 29.59%, and the smallest protein content in treatment A of 18.34%.The results of laboratory tests show that treatment B is a treatment that has results that match the general criteria of crude protein content in maggots.For this reason, it is necessary to do uji continued statistics, the value of P(T<=t) one-tail will be used to check the results of the t-test.Based on the results of the t-test in Table 12 indicates that the value of P(T<=t) one-tail in treatment A and treatment B is smaller than the alpha value (α = 0.05).Since the alpha value is less than the alpha value or less than the alpha value, the null hypothesis is rejected and the hypothesis a (Ha) is accepted in treatments A and B.Where the average crude protein is less than the standard of general crude protein found in maggots.As for treatment C because it has a one-tail P(T<=t) result greater than the alpha value (α = 0.05) then the null hypothesis is accepted, the hypothesis a is rejected, it means the result the protein in treatment A is more than or equal to the general standard crude protein content in maggots.

Discussion
Depending on the volume of trash and the kind of waste, the amount of protein, carbs, and fat in organic waste made up of vegetable, fruit, and food waste in treatments A, B, and C is not constant.The highest content of vitamins, proteins and carbohydrates obtained from treatment B is evident from the results of the final maggot weight obtained from treatment B which is the highest compared to treatment A and treatment C. With an average dry weight in treatment B is 266.50 gr and in treatment C is 213.65.The results of the two treatments did not differ much, but when compared with treatment A, which differed greatly from the results of treatments B, and C, it only produced an average weight of 6.90.The result of larval weight in treatment B, and C is the treatment that has the most influence relating to the development of black soldier fly larvae because the content in organic waste and milk waste is fulfilled in treatment B, and C.And treatment B has the highest weight among the three treatments driven by the waste content The organic matter contained in treatment B was more, namely as much as 1000 gr and with the condition that treatment B was not too wet and too watery when compared to treatments A and C.
Maggot weight growth in treatment B was the highest compared to other treatments, but the weight in treatment C was not much different from treatment B. With the addition of organic waste in treatments B, and C where organic waste contains a lot of organic content which triggers growth and development black soldier fly larva weight.As a result of the addition of UHT milk, which offers more nutrients for the development of black soldier fly larvae, the growth rate of maggot weight in treatments B and C was greater.The sole component of treatment A that originated from the milk content had an impact on the larvae's ultimate weight, which was very modest.Supported by the large number of maggot larvae that died in treatment A due to the conditions in treatment A which were very wet because they were only filled with milky fluids, the general nature of maggot was that it was difficult to develop and grow in very wet and runny growing media, given the findings of research based on earlier studies by Salahuddin (2021) stated that the cause of maggot death was caused by the growing media which was too wet and liquid [15].
Of the 3 parameters observed for protein content in maggot larvae, only one parameter met the standard maggot protein content in the range (30-50%) namely treatment B which was 33.3% but, in the Ttest the protein content that met was treatment C with the average protein content is 29.59%.The waste reduction index for the bioconversion process utilizing Black Soldier fly larvae or maggot larvae has a percentage of 4.96% as the speed of waste reduction; However, this enables the employment of black soldier fly larvae in bioconversion an accessible alternative processing method for recycling domestic organic waste.

Conclusion
Based on the results of the experiment and analysis, it can be concluded, from the 5% ANOVA and LSD methods, it was found that Treatment A with a composition of 100 % milk waste did not significant influence on the wet weight of black soldier fly larvae, but treatment B with a composition of 50% milk waste and 50% organic waste and C treatment with a composition of 70% milk waste and 30% organic waste significantly influence wet weight of black soldier larvae.
Considering the outcomes of the ANOVA and LSD tests methods of 5%, the composition of 100% of milk waste has no significant effect on the dry weight of black soldier fly larvae, but treatment B with a composition of 50% of milk waste and 50% of organic waste and treatment C with a composition of 70% of milk waste and 30% of organic waste has a significant influence on the dry weight of black soldier larvae.Through the calculation of the waste reduction index on the residual compost results in treatments A, B and C, it was obtained that the ability of larvae to reduce waste is highest obtained from treatment C where there is an average waste reduction index of 4.96%.
The parameters of the t-test results on the crude protein content of BSF larvae, treatment A and B are still not in accordance with the general standards of crude protein content in maggots, but the protein content in treatment C is in accordance with the general standards of crude protein content in maggots.

Acknowledgment
Praise The Lord.In the Name of Jesus Christ for His abundant grace so that the author can complete this paper and I would like to express my sincere gratitude to my lecture supervisor Yunita Ismail Masjud, for the continuous support and guidance of the final project work, for her patience, motivation, enthusiasm, and extraordinary knowledge.His guidance helped me in all my time researching and writing this paper, although they may not agree with all of the interpretations/conclusions of this paper.

Figure 1 .
Figure 1.Wet and Dry Weight Measurement Process Source: Data Processing Results, 2023 12 ml of H2SO4 solution in the fume hood.4. Carry out the digestion process in the fume hood, heat the Kjeldahl flask containing the sample using Bunsen spiritus until the sample turns green, about 1 to 1.5 hours.5. Let the Kjeldahl pumpkin stand for 20 minutes for cooling then add 50 ml of aquades.6.Put the solution mixture in a boiling flask, then add 40 ml of 40% NaOH and a few grains of boiling stones.7. Add 30 ml of 2% boric acid and 3 drops of Conway indicator to Erlenmeyer distillate.8. Distillation is completed when a distillate result of 75 ml is obtained.9. Titrate the result of the distillate solution with HCl 0.1 N until the blue color of the solution turns orange.Then record the titration results by looking at the HCl number 0.1 N used [34].Protein Content % = ( − )    14.008       1000

Figure 2 .
Figure 2. Wet Weight of BSF Larvae Source: Data Processing Results, 2023

Figure 3 .
Figure 3. Dry Weight of BSF Larvae Source: Data Processing Results, 2023

Figure 4 .
Figure 4. Waste Reduction Index for Every Treatment Source: Data Processing Results, 2023

Table 1 .
Composition of 3 Treatments with 2 Repetitions

Table 2 .
Wet Weight Results from Each Treatment

Table 3 .
ANOVA Test of for Wet Weight

Table 4 .
LSD test for Wet Weight

Table 5 .
Dry Weight Value for Every Treatment

Table 6 .
ANOVA Analysis for Dry Weight

Table 8 .
Compost Reduction Value from Maggot Bioconversion Results

Table 9 .
Protein Test Results from Each Treatment

Table 10 .
Table ANOVA for Protein Test Result

Table 11 .
LSD 5% for Protein Test Result

Table 12 .
T-Test Result for Protein