Key findings:

The study found that adding black cumin oil to yogurt significantly affected pH and water holding capacity but not viscosity. Higher concentrations of black cumin oil decreased the pH, viscosity, and water holding capacity of the yogurt. Optimal concentrations should be carefully considered to balance these effects.

What is known and what is new?

Yogurt is traditionally made through bacterial fermentation, and black cumin oil has anti-allergic, anti-tumor, antibacterial, and anti-inflammatory properties. This study reveals that adding black cumin oil to yogurt significantly impacts pH and water holding capacity, with higher concentrations decreasing these parameters, while viscosity remains unaffected.

What is the implication, and what should change now?

Incorporating black cumin oil into yogurt can alter its pH and water holding capacity, potentially affecting its texture and shelf life. Producers should optimize the concentration of black cumin oil in yogurt to achieve desired health benefits without compromising product quality, possibly focusing on lower concentrations to maintain optimal pH and water holding capacity.

The milk processing industry is one of the food manufacturing sectors that contribute industrial waste in the form of sludge originating from the wastewater treatment process. Increasing processed milk production affects increasing the volume of wastewater produced. An average of 2.5 – 3.0 liters of wastewater is produced from one liter in the dairy industry process [1]. If the volume of waste water is not handled properly, it will cause environmental pollution. DWS has characteristics that can burden the environment, namely COD 61,000 mg/L, BOD5 20,000 mg/L, crude protein 34.98%, lactose 4.1%, crude fiber 9.77%, calcium 2.33%, phosphorus 1.05 %, and also a high oil and grease content, namely 11.04% [2,3]. Handling dairy industry wastewater generally uses a series of processes combining physics, chemistry and biology. This process aims to reduce various dissolved organic materials and other organic compounds with the help of anaerobic bacteria, then through filtration it helps absorb various remaining chemicals and separates water from solids. Solids sourced from this treatment are known as Dairy Wastewater Solids (DWS). 

             DWS contains sufficient organic material as a source of nutrition for indigenous microorganisms. Several types of bacteria are found in DWS such as Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Streptococcus faecalis, Enterobacter, Lactobacillus delbrueckii, Staphylococcus aureus, Enterococcus hirae and also several types of yeast such as Candida, Saccharomyces and Cryptococcus [4,5].

             The nitrogen (N) content in milk sludge is quite high, which plays a good role in the growth of decomposer microorganisms that will break down organic materials into inorganic compounds for plants, however, the C/N ratio of milk sludge is still low for optimal microorganism metabolism so a mixture of other materials is needed as a carbon source. (C) including rice straw [6,7]. Rice straw is an agricultural waste with a fairly high carbon content, so it acts as a mixture of milk sludge to obtain a C/N ratio that is suitable for the growth of indigenous microorganisms in DWS so that it can be converted into organic fertilizer. The process of converting DWS into organic fertilizer includes an initial decomposition stage and continues with vermicomposting. Each stage of the process cannot be separated from the role of indigenous microbes as decomposers and earthworms as detritivores which break down organic material into simple compounds that are available as plant nutrients (7,19,20). Vermicomposting is a composting process that involves earthworms as detritivores which will continue the bioconversion process carried out first by microorganisms so that the composting process runs faster [8,9]. 

             Initial decomposition or pre-composting is the stage of decomposing organic material by utilizing controlled microbial activity to convert complex compounds into simpler compounds in warm, humid, and aerobic environmental conditions [10,11].  Pre-composting is a term used in vermicomposting that means the initial active phase of composting when the pile temperature exceeds 55° C (131° F) for a minimum of three days. If these conditions are met, parasites, pathogens, and weed seeds will be destroyed and a large amount of heat energy will be removed from the raw material mixture [12,13]. Before the active phase of composting is complete (14 to 21 days), the composted material is put into the vermicompost system for the ripening process.

             The microbes that play a dominant role in the initial decomposition process are bacteria. Bacteria act as decomposing microbes which first degrade organic materials, synthesize nitrogen compounds, dissolve phosphates, and have better diversity than other microbes. Bacteria that grow at mesophilic temperatures include Flavobacterium spp., Streptomyces spp., Pseudomonas spp., Bacillus spp., Achromobacterspp., Clostridium spp. Meanwhile, thermophilic temperatures include Thermus spp., Streptomyces spp., Micropolyspora spp., Thermomonospora spp., Bacillus spp., Thermoactinomyces spp. [14].

             Various composting methods have been researched, one of which is vermicomposting. Vermicomposting is composting with the help of earthworm organisms, one of which is Eiseniafetida. Several species of earthworms are capable of consuming various kinds of organic waste such as animal waste, sewage sludge, agricultural residues, domestic waste, and the like. Earthworms will digest insoluble nutrients in waste such as nitrogen, potassium, and phosphorus, into soluble nutrients that can be used as plant fertilizer. During the composting process, organic material changes and the growth of various functional microbes that play a role in ecological function occurs. Soil beneficial bacteria such as nitrogen (N) and phosphate (P) fixing bacteria are expected to be present in organic fertilizers. This research aims to describe the role of microorganisms and earthworms in each phase of DWS vermicomposting. 

Pre-Composting

Prepare materials for initial decomposition, namely milk mud and rice straw, then air them for 15 days to reduce water and ammonia levels to prevent rotting. Calculate the mass of the compost mixture with a C/N ratio of 25 to determine the ratio of the media mixture. Mix the milk mud and rice straw according to the calculated level and put it in a compost bag with a volume of 50 liters for the solid fermentation process. Then incubate for 7 days aerobically. 

Medium Preparation 

Weigh NA (bacteria) and MEA (yeast) according to the label, then put each into a beaker. Dissolve each medium with distilled water up to 1 liter and put it in a Schott bottle. Heat the media using a hot plate stirrer. Sterilize using an autoclave to a temperature of 121°C for 15 minutes with a pressure of 1 atm. Put 10-15 ml of each medium into the petri dish when the medium has started to cool.

Total Bacteria Population Count

Take a 10gram sample from the compost bag, and then grind it using a mortar. Put it in an Erlenmeyer flask and add 90 ml of NaCl solution, then homogenize it. Transfer 1 ml of the suspension with a 1000 µl micropipette into 9 ml of NaCl solution to obtain a 10-2 dilution and homogenize using a vortex, then take 1 ml of the 10-2 dilution and transfer it to the 10-3 dilution, using the same steps to make a 10-2 dilution. 4 to 10-10. Dilutions up to 10-10 were obtained from pre-study. Take 1 ml from the 10-10 dilution using a 1000 µl micropipette for bacterial inoculation, then put it in a petri dish. The cup containing the bacterial suspension, then using the pour plate method, includes NA media as a nutrient for bacterial growth. Place the cup containing the bacterial suspension and media, then let it sit until the media freezes. Incubate at 37℃ for 1 x 24 hours by turning it upside down. Count the number of bacterial colonies growing in a petri dish. The number of bacterial colonies counted was between 30-300 colonies. Next, the bacteria that grow in the media are calculated using the following formula [14]:

N                             : Number of bacterial colonies per gram (CFU/gram)

∑ Coloni                : Number of bacterial colonies on the plate  (30-300)

Vermicomposting 

After the pre-composting process for 7 days, Eiseniafetida earthworms were planted in media with a stocking density according to the treatment (P1 = 1 kilogram/m3, P2 = 2 kilograms/m3, P3 = 3 kilograms/m3). The vermicomposting process is carried out for 14 days with regular inspection and aeration. Harvesting is done using manual methods. The material containing the worms is dumped in a pile on a flat surface above a light source, because the worms move quickly to avoid the light, they can be easily identified, picked, and collected in a container. After the earthworms and the growth medium were separated, each was weighed. 

Soil Beneficial Bacteria (Nitrogen-fixing Bacteria and Phosphate Solubilizing Bacteria) Count

Put 90 ml of physiological NaCl into an Erlenmeyer 250 and 9 ml into a test tube. Dilute to 10-6 then plant in Jensen and Pikovskaya media and incubate at 37°C for 48 hours.  Calculating the NFB and PSB population uses the same formula as calculating the total bacterial population.

Data Analysis

Bacterial populations in the initial decomposition process increase in earthworm biomass and vermicomposting production, N-fixing and P-fixing bacterial populations in vermicomposting were analyzed using ANOVA and Tukey's advanced test with a confidence level of 95% SPSS 26 (2022). 

 Pre-Composting Temperature

The temperature during composting reflects the ongoing activity of microorganisms. The temperature began to increase after incubation for 24 hours (day 1) then the temperature gradually decreased until the 7th day of incubation (Fig. 1). At the start of incubation, the pile temperature follows room temperature, namely 25^oC. Sometimes the microbes need time to adapt to the composting media so that the manifestation is not yet an increase in temperature at the start of composting. Based on the microbial growth curve, the microbial growth phase begins with the adaptation phase. Temperature increases as microbial activity increases in breaking down organic materials into simpler compounds. During this process, microbes will grow, develop, and release enzymes. The types of microbes that grow will follow the environmental temperature so that at the beginning of the pre-composting process at a temperature of 25^oC, microbial growth is dominated by mesophilic bacteria, then when the temperature increases to 59.42^oC, the dominance will change where the role of microbes will be replaced by thermophilic bacteria. When the temperature starts to decrease to 32.67^oC, mesophilic microbes will dominate again (Fig. 1). Several studies report temperatures during pre-composting ranging from 18-60^oC [15-17].

Fig. 1. Pre-composting temperature and pH during a 7-day incubation period

              Apart from temperature, other factors determine the decomposition process, namely pH or acidity level. Several studies report that the pH during pre-composting ranges from 4.9 to 8.3, varying depending on the materials used in composting [18,17]. During pre-composting, mesophilic phase I, thermophilic phase, and mesophilic phase II are reached. The mesophilic phase (20-45^oC) lasts very short, namely a few moments after the substrate is stacked, put into a compost bag, and then the decomposing bacteria start to work. The thermophilic phase (45-65^oC) lasts several days but gradually the temperature continues to decrease until it reaches the mesophilic phase II.

Total Population of Bacteria and Fungi

The increase in temperature values is caused by a gradual combination of microbial activities. This combination of activities is realized through certain routes: degradation of organic materials can cause an increase in temperature and microorganisms that are active in a certain temperature range will take part in composting, thereby causing a gradual increase in temperature. The total population of bacteria and fungi during the pre-composting process is presented in Table 1.

Table 1. Total Bacteria and Fungi Population on Pre-Composting

^a,bMeans with the same letter were not significantly different at a α 0.05 level.

During the thermophilic phase (day 1) the bacterial population reaches the highest value and the microbial population is influenced by the C/N ratio, a C/N ratio of 25 produces the highest total bacterial population in the thermophilic phase. However, in the mesophilic phase II the total bacterial population was relatively the same at a C/N ratio of 25 - 30 (Table 1). The C/N ratio is a description of the availability of nutrients for microbes. During composting, microbes break down organic compounds to obtain energy for metabolism and obtain nutrients to maintain their growth. However, C and N are the most important: C is used as an energy source, while N is used to build cell structures.

In contrast to bacteria, in this research the growth of fungi was relatively fluctuating and did not follow a clear pattern. However, at a C/N ratio of 30, fungal growth is higher than at C/N 25. This is due to the basic differences in nutritional requirements between bacteria and fungi. Bacteria require a smaller C/N ratio than fungi because they require nitrogen-rich food [19]. 

Beneficial Soil Bacteria

 One indicator of the quality of organic fertilizer is its content in the population of beneficial soil bacteria, including nitrogen-fixing bacteria and phosphate-solubilizing bacteria [20,21]. The population of nitrogen-fixing bacteria contained in DWS vermicompost and rice straw ranged between 5.5 x 107 - 8.7 x 107 CFU/g and phosphate solubilizing bacteria ranged between 4.4 x 107 - 8.5 x 107 (Table 2).

The vermicomposting process is a bioconversion method that relies on synergy between decomposer organisms and detritivores. The decomposer works to break down organic material externally by producing enzymes, while the detritivore will continue the work of the decomposer, namely breaking down organic compounds that have been degraded by the decomposer and then decomposing them internally by swallowing them and becoming a source of nutrition for the detritivore. The results of its decomposition will be excreted through the digestive tract and become inorganic compounds available to plants. Thus, the vermicomposting process is seen as more effective and efficient in decomposing organic materials through bioconversion [22,23]. In this research, the Eiseniafetida earthworm was added to the growth media resulting from the pre-composting process with differences in stocking density. Several research results report that a stocking density of 1.60 kg-worms/m2 (0.33 lb-worms/ft2) produces the highest bioconversion of substrate into earthworm biomass [24,2].

Earthworm stocking density is the number of earthworms spread or maintained in a certain unit area. The more earthworms that are spread within certain limits can increase the content of functional bacteria in the bioconverted organic fertilizer. A very interesting phenomenon that cannot yet be explained. In our opinion, this is related to the increasing amount of substrate or organic material consumed by earthworms as detritivores and excreted through their digestive tract. Earthworm digestion is a bioreactor that can break down organic compounds into inorganic compounds. In the earthworm's digestive system, there are several enzymes released, one of which is by bacteria in the earthworm's ecological system [18].

Table 2. Average of Nitrogen Fixing Bacteria and Phosphate Solubilizing Bacteria on vermicompost

^a,bMeans with the same letter were not significantly different at a α 0.05 level.

Indigenous microbes in the composting process work synergistically with earthworms in decomposing milk sludge organic material into compounds available for plants and providing an ecological environment for soil fertility through the availability of beneficial soil microbes, nitrogen-fixing bacteria and phosphate-solvent bacteria.

Conflict of Interest: The authors declare that they have no conflict of interest

Funding: No funding sources

Ethical approval: The study was approved by the Institutional Ethics Committee of Universitas Negeri Makassar

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