Converting organic waste in to value-added products using microbial consortia -

Converting organic waste in to value-added products using microbial consortia

With a focus on the bioconversion of organic waste into value-added products, municipal vegetable waste was fractionated into liquid and solid part. The solid fraction was subjected to biodegradation in the presence of different microbial consortia and different enzymes were extracted. The remaining solid stuff was converted into animal feed, followed by the evaluation of its microbiological and chemical indices. Liquid fraction separated from waste was used as a crude medium to grow different bio-fertilizer strains. Among the three consortia, consortium C (Aspergillus terreus and Myrothecium verrucaria) showed the maximum production of amylase, cellulase, xylanase and pectinase after 4 days. Results of the microbiological and chemical parameters of the animal feed prepared from the fermented vegetable waste showed that the various parameters were found to be within the permissible limit. Thus, the present study demonstrated the bioconversion of vegetable waste into crude enzymes, animal feed, compost, and liquid biofertilizer.

Materials and methods

Isolation and identification bacteria for isolation of the bacteria. One gram of soil sample was added into the tube containing 10 mL of sterile distilled water followed by thoroughly mixing and dilution up to 10−6. 100 µL of the diluted samples were spread on sterile Nutrient agar (Hi-Media, India) followed by overnight incubation at 37 ◦C. Individual unique colonies were purified using sub-culturing. Bacterial isolates were screened for various enzyme productions by allowing them to grow on plates containing minimal salt medium along with different substrates as described below.

Revival of fungal cultures

Fungal isolates i.e. Acremonium implicatum, Phanerochaete chrysosporium, Hanseniaspora guilliermondii and Talaromyces pinophilus were revived from the Bank A Bug (BAB), an in house microbial repository of GBRC. Cultures were streaked on the Potato Dextrose Agar (PDA) (Hi-Media) plates, followed by incubation at 28 ◦C for 3–7 days. A. terreus and M. verrucaria were isolated from the cow dung. For this, 10 gm of cow dung sample was collected in 50 mL sterile Falcon tube from a sub-surface of the dung pit. One gm of dung sample was used for serial dilution in phosphate buffer (pH 7) up to dilution 10−6 and from this 10 µL was spread on PDA The plates (were incubated at 25 ◦C (± 2 ◦C) for 3–7 days for observable fungal growth. Individual unique colonies were purified and were screened for various enzyme production, followed by their identification by sequencing of 18S rRNA gene.

Screening of bacterial and fungal cultures for the production of enzymes total 95 bacterial isolates were screened for their potential to utilize various substrates viz. gelatin, starch, xylan birchwood, cellulose, lignin, and pectin. Single bacterial colony was pointed on the plates containing 0.5% substrate in minimal salt medium. For fungal cultures, a plate containing fungal growth was chunked using a 8 mm cup borer and placed on the respective substrate plates. Plates containing bacterial cultures were incubated overnight at 37 ◦C. The plates containing fungal cultures were incubated at 28 ◦C for 72 h. Post incubation, the zone of hydrolysis was detected after flooding the plates with mercuric chloride solution for protease, and iodine solution for amylase, cellulase, pectinase, and xylanase.

The potential bacterial and fungal isolates were tested for their compatibility with each other to develop different microbial consortia. Cross streak method and disc method were used to check the compatibility between bacterial strains. In a cross streak method, different permutations and combinations of bacterial strains were streaked horizontally and vertically on a nutrient agar plate followed by incubation at 30–35 ◦C for 24 h. In the disc method, disc was dipped into the 24 h old culture of the isolate and then placed on the nutrient agar plate containing the target organism followed by incubation at 30–35 C for 24 h. For fungal compatibility test, eight mm mycelial discs from seven day old cultures of fungi were placed at opposite ends on PDA plates followed by the incubation at room temperature for 5 days.

Fungal cultures used to prepare consortia were grown in potato dextrose broth (PDB). For this, fungal cultures i.e. A. terreus, A. implicatum and P. chrysosporium were inoculated 5 days before setting up the experiment and H. guilliermondii and T. pinophilus were inoculated 3 days before setting up the experiment. The flasks were incubated at 28 ◦C under shaking condition at 120 rpm. After incubation, the medium containing fungal mass was filtered using autoclaved muslin cloth under laminar flow. The medium was discarded and fungal mass was collected in a sterile container. To prepare consortia, each fungal culture was used in equal amounts i.e.1:1 ratio. Similarly, bacterial cultures were inoculated into the Nutrient Broth, and the flasks were incubated overnight at 37 ◦C in shaking condition at 120 rpm. For consortia preparation, the OD600 was set at 1.0 and each bacterial culture was used in equal amounts. On the basis of compatibility tests, three consortia were prepared. Consortium A was the combination of T. pinophilus, A.implicatum, H. guilliermondii, P. chrysosporium, A. terreus. Consortium B was the combination of Bacillus haynessi, Bacillus subtilis, Bacillus licheniformis, A. implicatum, P. chrysosporium, and Consortium C was prepared by using two fungal cultures i.e. A. terreus and M.verrucaria.

Collection of organic waste and its processing solid part separated after crushing was mixed with the 5% w/w coco peat to reduce the moisture content. Organic waste degradation potential of each consortia was evaluated by inoculating 5% (w/w) each consortium individually in a duplicate experimental setup. Each consortium was added in a container containing waste, mixed evenly manually and transferred to the pots labelled accordingly (i.e. Consortium A, Consortium B, Consortium C). A pot containing 5 kg waste, without any consortium was set as control. All the pots were kept at room temperature and various parameters such as pH, temperature, enzyme activity were measured periodically at a constant interval till 10 days. Based on the results of enzyme activity assays (data not shown), enzyme activity was observed to be highest on the day 4, therefore, finally, the same experiment was set again and enzyme activity assays were performed on Day 0 and Day 4. After 4 days, the degraded organic was used to prepare animal feed.

Temperature changes during composting were recorded using a thermometer, by placing the probe 5 to 7 cm deep into the compost. pH was determined using pH meter by withdrawing the sample followed by mixing with distilled water. Changes in the MC were determined using a moisture analyser (Sartorius) by placing 1 gm of sample on the surface of the analyser plate.

Sample preparation for enzyme activity

A 5 gm sample from each pot was collected and mixed with 50 mL of phosphate buffer saline (PBS) (pH 7) in a falcon tube. Samples were mixed using rotary mixture (Rotospin) for 15 min, and then centrifuged at 4000 rpm for 20 min. The supernatants were used to evaluate total protein and enzyme activity. In the same way, the supernatant from 5 gm of sample was used for ammonium sulphate precipitation up to 80% saturation. The mixture was kept overnight at 4 ◦C, then centrifuged at 10 000 rpm for 15 min under cooling condition. The pellet was dissolved into 5 mL PBS with pH 7.

Protein quantification by Bradford assay method. DNS method was used to determine the activity of amylase, cellulase, pectinase and xylanase with 1% substrate prepared in a 50 mM phosphate buffer (pH 7). Starch, carboxymethyl cellulose, pectin and xylan were used for amylase, cellulase, pectinase and xylanase, respectively. Reducing sugar (Glucose) standard curve was used to calculate the amount of glucose released. One unit of enzyme activity is the amount of 1 µmol of substrate converted into product per minute under assay conditions (U/mL/min).

The feed for the animal from the degraded waste was prepared. In brief, the samples collected during the organic waste degradation experiment were subjected to drying at 70 ◦C until they dry properly. After proper drying, 1 gm of sample was mixed with 100 mL of distilled water and filtered using muslin cloth and the filtrate was used for the assessment of microbial load in the samples.

Three-tube most probable number (MPN) method was used for evaluation of microbiological indices including total coliforms, and total aerobic count. Presence of Staphylococcus aureus and Salmonella  was checked using Mannitol salt agar and Salmonella-Shigella agar ( Proximate and heavy metals analysis of the samples collected from organic waste degradation experiment. The samples were analysed for total organic carbon (TOC), total Kjeldahl nitrogen (TKN), total organic matter, ash, and moisture (on dry basis). Moreover, concentration of heavy metals i.e. zinc, lead, cadmium, copper, nickel, chromium in the samples was also measured from the same samples.

Use of vegetable waste liquid juice for liquid bio-fertilizer. Three bacterial strains (BAB 1980, BAB 267, BAB 662) from Bank A Bug repository of GBRC, were studied for their potential as bio-fertilizer by evaluating their effect on the seed germination and growth of Baheda plant (Terminalia bellirica). These strains were able to induce the earliest signs of germination in the Baheda plant.

Determination of per cent (%) juice recovery and pH Per cent (%) juice recovery was determined by calculating the volume of juice obtained after crushing the vegetable. pH was determined from the 10 mL of liquid sample.

Determination of reducing, non-reducing and total sugar Reducing sugar was estimated using the DNS method. Total sugar was calculated using freshly prepared Anthrone reagent. Non-reducing sugar was calculated by subtracting reducing sugar from total sugar.

Pasteurization of liquid separated from waste

Before pasteurization, the liquid was filtered using muslin cloth. The liquid was preheated at 40 ◦C followed by pasteurization of samples in a water bath at 70 ◦C for 30 min and rapid cooling at 30 ◦C. Liquid was stored at 4 ◦C until further use. Samples from the pasteurized liquid were spread on the Nutrient agar (Hi-media, India) plates to check the presence of any viable microbe.

Growth curve of bio-fertilizer strain in undefined medium (liquid separated from vegetable waste)

 To check whether bio-fertilizer strains (BAB 1980, BAB 267, BAB 662) were able to grow in juice separated from waste, they were inoculated into 500 mL of pasteurized juice, followed by incubation at 37 ◦C. Growth study was performed by withdrawing the sample after every 1 h and OD was measured at 600 nm. Simultaneously, 100 µL of sample was spread on the N-agar plates for CFU count. CFU/mL was determined by the formula: CFU/mL = Number of colonies/aliquot taken for spreading x dilution factor.

The nitrogen fixing ability of cultures, they were grown in an undefined medium and Nitrogen free Ashby’s mannitol agar. Cultures were spread on Ashby’s mannitol agar plates and incubated overnight at 37 ◦C. For quantification of ammonia from liquid bio-fertilizer, the samples were centrifuged at 12,000 rpm for 10 min, followed by the collection of the supernatant. A drop of Nessler’s reagent was added in each sample and incubated for 5 min. Readings were measured at 425 nm. The standard curve was prepared using ammonia as substrate.

To check the phosphate and potassium solubilizing potential of the cultures, they were streaked on Pikovskaya’s agar and Alexsandrow agar. A clear halo around the growth indicates the solubilization.

Results

Ninety-five bacterial cultures were isolated. Isolates were checked for their potential to produce amylase, cellulase, protease, pectinase, and xylanase which are reported to be effective for organic waste degradation. Amongst the bacterial isolates, B.# subtilis showed maximum cellulase activity i.e. 6.4 cm zone of hydrolysis on cellulose containing plate. Among the fugal isolates, T. pinophillus and M. verrucarria showed 2.5 cm zone of hydrolysis on cellulose containing medium, which was the highest amongst the fungal isolates. Pectinase activity was observed highest with B. licheniformis i.e. 3.5 cm zone on pectin agar plate, followed by A. terrus (3.0 cm) and M. verrucaria (3.0 cm). Results of compatibility studies between the bacterial strains, B. haynessi, B. subtilis and B. licheniformis were found to be compatible with each other. Among the fungi, Talaromyces pinophilus, A. implicatum, H. guilliermondii, P. chrysosporium, and A. terreus were found to be compatible with each other.

Moisture is a crucial environmental factor which can affect the biodegradation kinetics through changes in oxygen diffusion, and microbial growth rates. The optimum moisture content can vary for different compost mixtures and it ranges from ∼50%–70%. Initial moisture content in the vegetable waste in this study was around 78%. The moisture content was found to be reduced in control as well as test pots, except consortium A. In total, moisture content was found to be reduced by 13% in control, whereas in experimental pots it was found to be in the range of ∼20%–24%. Reduction in moisture content indicates increase in microbial activities.

Temperature is one of the important parameters during the composting process as it represents the metabolic activities of the microbial community during the biodegradation. In 10 days experiment, maximum temperature noted was 41.5 ◦C in Consortium A. An average 40 ◦C temperature was maintained in the experiment till the end of the 4th day, and then it started to decrease. Degradation in composting occurs quickly at 40–60 ◦C, which lasts for a period of time depending on the size and the components of the system. Highest temperature 42 ◦C was noticed in Consortium C.

The initial pH of the samples was around 6. Microorganisms present in the composting process are metabolically active in the range of pH 5.5-8.0. Organic acids are formed during the initial stages of decomposition, and this acidic condition is favourable for breakdown of lignin and cellulose.

Total protein content was measured during composting on Day 0 and 4. With the progression of the experiment, total protein content was found to be reduced in all the test pots as well as control. Organisms involved in degradation consume sugars, proteins and amino acids.

In the present study, amylase production was compared on Day 0 and Day 4. At the beginning of the experiment, samples collected from all the pots showed ∼0.02 U/mL of amylase activity, whereas, after the 4 days, amylase activity was increased in all the pots including control. Highest amylase production was observed in the samples treated by consortium C (0.50 U/mL).

Cellulases are widely used in pulp, paper, textile, food and agriculture industries. In this study, at the beginning of the experiment, samples from all the pots were showing cellulase activity ∼0.03 U/mL. After the 4 days, consortia A, B and C showed 0.32, 0.33 and 0.50 U/mL cellulase activity,respectively, which was significantly higher than control. Again, similar to Amylase activity, consortium C showed significantly higher activity than all the other samples. In total, days and consortia (independent variables) have statistically significant effects on cellulase (dependent variable). The cellulase activity of Control was significantly different from activity of Consortia A, B and C.

Pectinase is also widely used in industry, but, its application is limited due to the high production cost of especially the nutrient medium required for the growth of the organism. Therefore, the use of alternative cheap substrates for pectinase production is necessary. Hence, in present study, used organic waste as a substrate for the production of pectinase. On day 4, Consortia A, B and C showed 0.25, 0.37 and 0.35 U/mL pectinase activity respectively, whereas in control it was 0.29 U/mL. Thus conclude that, organic vegetative waste can serve as a potential substrate for the large scale pectinase production. However, optimization is required for the process upscaling and downstream processing especially, enzyme purification.

Xylanase production was ∼0.05 U/mL, which was increased in all the pots including control. Highest xylanase production 1.11 U/mL was observed in consortium C. In the present study, among the three consortia, consortium C showed higher enzyme production as compared to consortia A and B. Similar studies have been reported by various researchers.

In order to use the fermented product of the organic vegetable waste as an animal feed, it must be free from the pathogens and simultaneously the cumulative microbial load should be within the permissible limit. Total count and total aerobic count can be used as the indicators to check the quality of the product in the context of microbiological safety.

Proximate analysis of animal feed is important as it determines the nutritive value of the feed. Control and test samples were subjected to proximate analysis which includes tests for the quantitative analysis of total potassium, total phosphorus, C:N ratio, total organic matter. Total organic matter and total organic carbon were highest in consortium C after 7 and 10 days. Total fibre content was found to be lowest with consortium B after 7 and 10 days. Results showed that the interaction of composting time and consortia had a significant effect on total phosphorus content. In the presence of consortium C, total phosphorus content was found to be increased by 31.84%.

Conclusion

From this study, it can be concluded that Consortium C which is comprise of two fungi (A. terreus and M. verrucaria) had the highest capability to produce industrially important enzymes as compared to others. The same consortium also showed the lowest protein content after the 4 days, indicating the highest degrading capability of the organisms present in Consortium C. The results of the proximate analysis and microbial indices suggest that the animal feed prepared from the vegetable waste can be a good alternative and cheap source of nutrition for the animals. A liquid separated from waste can be used as a nutrition medium to grow biofertilizer strains, resulting in liquid biofertilizer, which can be used in agriculture practices. Further, the C:N < 20:1, increased amount of potassium, nitrogen, phosphorus in compost indicate that it can be also used to increase the soil fertility. Further, the average concentrations of heavy metals in the compost was found to be within the permissible limit of FCO standard., Further, the method to evaluate the different aspects of the compost prepared by solid state fermentation for the use in animal feed can be developed.

Reference:

Dantroliya, S., Joshi, C., Mohapatra, A., Shah, D., Bhargava, P., Bhanushali, S., Pandit, R., Joshi, C. and Joshi, M., 2022. Creating wealth from waste: An approach for converting organic waste in to value-added products using microbial consortia. Environmental Technology & Innovation, 25, p.102092.