Microbial Exploitation of Biofertilizer, Pullulan and Biochar from Floral Waste

  

    
Serial    No 
    
Source Microorganisms 
    
Pullulan Concentration 
  
  

    
1 
    
Aureobasidium pullulans ATCC42023
    
3.21±0.35 g/L 
  
  

    
2 
    
Aureobasidium pullulans IMI145194
    
13g/L 
  
  

    
3 
    
Aureobasidium pullulans HP-2001
    
5.5g/L 
  
  

    
4 
    
Aureobasidium pullulans FB-1
    
44.7g/L 
  
  

    
5 
    
Aureobasidium pullulans P 56
    
31.3g/L 
  
  

    
6 
    
Aureobasidium pullulans NCIM    976
    
23.6g/L 
  
  

    
7 
    
Aureobasidium pullulans HP-2001
    
11.49g/L 
  
  

    
8 
    
Aureobasidium pullulans ATCC201253
    
23.1g/L 
  
  

    
9 
    
Aureobasidium pullulans SZU1001
    
25.6g/L 
  
  

    
10 
    
Aureobasidium pullulans LDT-1
    
31.25g/L 
  
  

    
11 
    
Rhodotorula bacarum
    
59.0g/L 
  
  

    
12 
    
Pullularia pullulans
    
70.0g/L 
  
  

    
13 
    
Aureobasidium pullulans SK    1002
    
30.28g/L 
  
  

    
14 
    
Aureobasidium pullulans CJ001
    
26.13g/L 
  
  

    
15 
    
Aureobasidium pullulans RBF4A3
    
70.43g/L 
  
  

    
16 
    
Aureobasidium pullulans NPM2
    
25.1g/L 
  
  

    
17 
    
Aureobasidium pullulans FB-1
    
23.1±0.02 g/L 
  
  

    
18 
    
Aureobasidium pullulans RG-5
    
37.1±1.0 g/L 
  

Table 1: Pullulan Production Efficiency of Some Reported Strains [11].

Floral waste degradation is a prolonged process compared to kitchen waste degradation [1]. By keeping this in mind, we have attempted to develop consortia to degrade the floral waste. The exploitation of the metabolic adaptability of microorganisms is advantageous in biological waste treatment. This led to a shortening of the time for the production of pullulan and Biochar.

Materials and Methods

Biofertilizer production

Biofertilizer production is a needy and valuable product for the cultivation of crops in recent times.

Sample collection and screening of microbes: Floral waste enriched soil samples were collected from the temple area. 10gm of soil sample were taken into 250ml of the conical flask and put on 90ml of sterile distilled water from sample soil. The flask was agitated for 10min on a rotary shaker. 1mL suspension was added to ml of via and shaken for 2min. Serial dilution technique was done if this suspension was expanded on flower waste agar plates up to 10-7 an aliquot (0.1ml). Incubation of plates performed for 24hrs at 37°C. Different characteristics of colonies after incubation were detected, such as shape, size, elevation, surface, margin, colour, odour, pigmentation, etc. and gram’s staining. Eight bacterial colonies were selected for flower waste degradation from all bacterial colonies grown on medium [2,3].

Development of Consortium: Different combinations of eight bacterial isolates were produced and utilized for biofertilizer preparation. Among these, the combination giving rapid degradation of flower wastes was selected for the consortium preparation. An optimal growth from 24hrs old bacterial culture of different organisms in a fixed variety was inoculated in minimal broth containing flower waste. The broth was incubated at 370C for 48hrs. After incubation, this broth was used as a consortium, and then 20% (v/w) of this consortium as inoculum was added to the flower waste [2,4].

Designing of degradation chamber: All organic material wastes available in the form of flowers were collected. Crushing of flower waste done by a crusher machine before being piled. Small size compost material is delicate and decomposes fastly. All the ingredients are mixed all together. These grind particles mix with a small amount of soil and blend the mixture equivalently. Two glass chambers with dimension 10.5×10×5.5cm were selected. In the first layer of coconut coir of height 2 cmat the bottom of the chambersaerobic conditions conserved. The coconut coir layer was covered by garden soil (2cm). The third layer was prepared by using flower waste inoculated with a 20% consortium. Alternative layers of soil and flower wastes inoculated with consortium were given as above. The second chamber was used as a control chamber using flower waste without consortium by following the exact procedure [5,6]. After absolute degradation of flower wastes, the degraded material is utilized as a biofertilizer.

Chemical analysis: Degradation completed then samples testing was done for pH, total minerals and N, P, K content. The total nitrogen, phosphorus and potassium (NPK) in keeping to the content of the following method of samples were investigated; using the macro Kjeldahl system; nitrogen was set on by the acid combustion elemental analysis method [7]. The phosphorus, potassium, and micronutrients were processed using the acid digestion method and investigated spectrophotometrically using the EPA technique [7]. The pH value was estimated in a 5-fold dilution of distilled water equilibrated with the sample for an hour with a pH meter. C% and N% was analyzed by the APHA method (APHA, 2005).

Efficacy of biofertilizers: The potential for the biofertilizers was carried out for several days. The soil for this experiment was garden soil having neutral pH and moisture content. A local species of wheat, green gram and Jowar were utilized as test plants. The experimental design was a completely randomized design with two replicates. Two sets of pots were developed for each test plant, one using prepared biofertilizer and another without using biofertilizer as control. Then fix the measured amount of good qualities seeds of all test plants were planted in corresponding pots. The pots were then watered daily for seven days. During harvesting, the plants were cautiously uprooted from each pot, and the plant height was recorded [8].

Pullulan production

The sample collection process is the same as biofertilizer production, but after having bacterial strains, further processing is done by solid-state fermentation diluted with deionized water. After fermentation, we get biomass, which is separated by Centrifugation or Filtration. After centrifugation, we get biomass and supernatant, activated charcoals or alcohols separated biomass as waste and supernatant with melanin with a combination of salts. Now the supernatant was precipitated and purified by Ultracentrifugation or Ion-exchange Chromatography. Finally, we get a purified Pullulan [9].

Biochar production

Sample collection was done, and after that, segregation was performed, which form different combinations of agitated piles using CCD-RSM (Composite central design- Response surface methodology). There is a decrease in size in the initial days of composting; to optimize the properties, we measure various parameters like pH, temperature, electrical conductivity, total organic carbon and nitrogen, nutrients, and C/N ratio. After optimization, we get a final compost, i.e., Biochar (rich in carbon).

Application of Biofertilizers, Pullulan and Biochar: Various applications are employed for small and large-scale industries and utilized as a good source of commercialization.

• Biofertilizers are extensively used in Agriculture markets, as they are eco-friendly and can be easily replaced by harmful chemical fertilizers.

• Pullulan is mainly used in food, pharmaceuticals, biomedical industries.

• Biochar has multifunctional values that include the soil amendment to improve soil health,

• Nutrient and microbial carrier,

• Immobilizing agent for remediation of toxic metals and organic contaminants in soil and water,

• The catalyst for industrial applications, porous material for mitigating greenhouse gas emissions and odorous compounds,

• Nutrient intake efficiency and Feed supplement to improve animal health consequently, the productivity [10].

Conclusion

The floral waste acted as a hub for the bacterial consortium and was used effectively by several preliminary techniques to produce biomaterials like biofertilizers, biopolymer (Pullulan), Biochar. Further, some parameters were analyzed to check the optimization studies of biomaterials. Validation experiments were performed and the results obtained showed the productions of multiple biomaterials viz; biofertilizers, pullulan and Biochar.

Acknowledgement

The authors want to express their gratitude to Dr. G. Viswanathan, Chancellor, VIT University, Vellore, for his constant encouragement

References

1. Jadhav AR, Chitanand MP, & Shete HG. Flower waste degradation using microbial consortium. IOSR Journal of Agriculture and Veterinary Science. 2013; 3: 1-63.
2. Navarrete-Bolanos JL, Serrato-Joya O, Botello-Alvarez E, Jimenez-Islas H, Cardenas-Manriquez M, Conde-Barajas, et al. Analyzing microbial consortia for biotechnological processes design. Communicating Current Research and Educational Topics and Trends in Applied Microbiology. 2007; 1: 437- 449.
3. Jelin J, Selvakumar TA, Dhanarajan MS. Phytological Analysis for Designing A Microbial Consortium To Enhance Plant Growth. International Journal of Chem Tech Research. 2013; 5: 1370-1375.
4. Pindi PK. Liquid Microbial Consortium-A Potential Tool for Sustainable Soil Health. J Biofertil Biopestici. 2012; 3: 124.
5. Borate A, Vamsi KK, Jhadav A, Khairnar Y, Gupta N, Trivedi S, et al. Biofertilizers: A novel tool for agriculture. International Journal of Microbiology Research. 2009; 1: 23.
6. Gurav MV, & Pathade GR. Production of vermicompost from temple waste (Nirmalya): A case study. Universal Journal of Environmental Research and Technology. 2011; 1: 182-192.
7. Tandon HLS. In: Tandon HLS (Eds.). Methods of analysis of soils, plants, waters and fertilizers. Fertilizer Development and Consultation Organization, New Delhi, India. 1993; 36-48.
8. Siti ZH, Awad HM, Sheikh I, Siti H, Mohamad RS, & Ramlan A. Agriculture wastes conversion for biofertilizer production using beneficial microorganisms for sustainable agriculture applications. Malaysian Journal of Microbiology. 2013; 9: 60-67.
9. Singh RS, Saini GK & Kennedy JF. Pullulan: microbial sources, production and applications. Carbohydrate polymers. 2008; 73: 515-531.
10. Bolan N, Hoang SA, Beiyuan J, Gupta S, Hou D, Karakoti A, et al. Multifunctional applications of Biochar beyond carbon storage. International Materials Reviews. 2021: 1-51.
11. Mishra B, & Suneetha V. Biosynthesis and hyperproduction of pullulan by a newly isolated strain of Aspergillus japonicus-VIT-SB1. World Journal of Microbiology and Biotechnology. 2014; 30: 2045-2052.