This was a part of project under RARA Biotech in collaboration with Department of Biotechnology, Himalayan WhiteHouse International College, Purbanchal University. Three students Aayushi Sharma, Abhibind Khaniya and Sanjog Raj Silwal have submitted their work in Jan, 2020 as their Bachelor’s Thesis award degree under supervision of Ms. Sumeena Karki (Biotechnologist, Founder/CEO) and co-supervision of Ms. Apshara Parajuli (Biotechnologist, Scientific Researcher) from RARA Biotech. Mr. Nagendra Awasthi, Ph.D Fellow, Deakin University supported as external supervisor for this project.
Study on Black Soldier fly is not new in case of Nepal. Studies have found that local un-named companies have been supplying eggs of BSF for decomposing degradable waste in home-made compost bin. However, in more transparent way rearing of BSF for commercial feed production has already started. The project started under supervision of Mr. Mahabir Pun where two of the students are doing their thesis from Rampur Campus, Chitwan. They have imported BSF from Indonesia from a commercial company and have been doing research from past 3 years in validating rearing of BSF in Nepal’s environment in cost effective way using different solid as well as fecal waste. BSF used by them are African species.
RARA Biotech is the first company to start research in Nepalese Black Soldier fly. Objective of our research was to use BSF specific mitochondrial COI gene for the identification of Nepalese Black Soldier Fly and to compare the protein content of different BSF larvae. Through genetic identification we could open scenario of using our own species of BSF for waste management. By comparing the protein content in different feed conditions, we could know whether they are suitable to be used as feed for poultry and fish farms or not.
As there were various commercially available Black soldier fly for waste management but there were no data on the Nepalese Black soldier fly used in waste management. So, in this study we aimed to genetically identify the Nepalese BSFL and explore its protein content on the basis of substrate used which can be used for waste management and as a feed. For the genetic identification, the genomic DNA of wild type BSF was extracted by PCI method and then Polymerase Chain Reaction (PCR) amplification of BSF mtCOI gene was done by using BSF mt COI gene primer.
Hermetia illucens in Biotechnology
Management of solid waste in the developing countries remains a challenging and neglected vital concern (Nguyen et al., 2015). The rapid increase in the waste production, increasing waste management cost, lack of technical expertise for proper management and negligence of the environmental issue posed burden of the waste management for government as well as society (Guerrero, Maas, & Hogland, 2013).
A large fraction of solid waste in developing countries composed of biodegradable material such as kitchen waste, agricultural waste, animal manure, and fruit and vegetable market waste. If left untreated, the valuable energy and nutrients contained in the waste results into the increase of annual greenhouse gas emissions (Skillicorn, Zheng, & Morselli, 2013) and a leaching of nutrients into water bodies, causing eutrophication (Sharpley et al., 1994). Over and above this, there is a risk of spreading the pathogens contained in the organic waste.
The rapid population growth and urbanization of the Kathmandu valley and other urban area of the country results increased generation of the waste. The Kathmandu valley alone generates around 500 tons of the waste per day and among that, organic waste constituted 63.22% (Dangi, Pretz, Urynowicz, Gerow, & Reddy, 2011). However, almost all of the organic solid waste is still not recycled and generally remains in the waste stream, i.e. it is either dumped in the landfills or remains uncollected on the street resulting in public health issues too (Hoornweg & Bhada-Tata, 2018). Most of the organic waste can be recycled through composting – turning waste materials into a rich soil supplement which is practiced by few private sectors but not the government. By composting, not only can we help to reduce the amount of waste that goes into landfill but you can also help to reduce contamination and greenhouse gasses (Couth & Trois, 2011; Rodic, 2010).
Black soldier fly larvae are considered as feeding on different variety of organic wastes. It has already used in small scale waste management work by using substrate such as manure, rice straw, food waste, fecal sludge etc (C. Lalander et al., 2013; Sheppard et al., 2002). All these capacities of BSFL help in the reduction of organic waste. BSFL is used to convert organic waste into plant fertilizer, as compost residue (C. H. Lalander et al., 2014).
BSFL meal and oil is already considered as alternative to fish meal and fish oil to feed carnivorous fish, due to its high content of protein and fat (Kroeckel et al., 2012). It is also used as poultry feed as alternative to maize or soy-based feed. It naturally colonizes and breakdown poultry manure which help to reduce pollution (Devic & Maquart, 2015; Erickson et al., 2004).
Black soldier fly larval biomass obtained from the larvae reared on solid residue fraction have potential to produce biodiesel (Zheng, Li, Zhang, & Yu, 2012).
The Wild types BSFL which feed on natural wastes were collected from the field of Balambu (Kathmandu valley). The Commercial BSFL which were reared in household wastes were collected from Kapurdhara (Kathmandu valley).
Findings from our study:
In this study, there were two types of black soldier fly one is from commercial one and they are reared in household organic waste and another one is collected from Balambu area which feed upon natural waste. Based on the morphology the collected insect was found to be Black soldier fly which was confirmed by NARC, Khumaltar.
Amplification of BSF mt COI by Polymerase Chain Reaction (PCR)
For the genetic identification, the genomic DNA of wild type BSF was extracted by PCI method and then Polymerase Chain Reaction (PCR) amplification of BSF_mt_COI gene was done by using BSF mt COI gene primer. The band was observed around 759 bp which indicates the presence of BSF specific mt DNA COI gene and can be said that it is black soldier fly (Photograph 3).
The total protein content was measured by BioRad DC™ Protein Assay Kit. The molecular weight distribution of proteins was characterized using SDS-PAGE. For the total protein estimation, protein was extracted from 4 types of sample (active larvae, dormant larvae, Nepali black soldier fly and Nepali BSF larvae) by using RIPA buffer. Among all the active BSF larvae contains highest amount of protein ie: 17.5% and Nepali BSF larvae contains 10.55%. In SDS, the clear multiple bands with no any protein degradation were observed in both samples and the most intensive band <75 kDa was observed in both fly and larvae protein. We have found that, in household waste feed BSF larvae the protein concentration was found to be higher than in BSF feed on natural waste. From the result it was found that the protein concentration is high in case of active larvae (17.5%) which feed upon household waste and less in case of BSF fly found in natural environment. In case of active larvae, the approximate data showed that with 1gm of larvae it will give 145mg of protein. Due to loss of some protein while extraction and the recovery of the supernatant the protein production was obtained upto 18% which is less with the result of (Shumo et al., 2019) in which it showed that dry weight of Black Soldier Fly larvae (BSFL) contain up to 50% crude protein(CP) but the protein was extracted in dry basis there (Photograph 4).
Conclusion and recommendation:
Increase in world population has resulted demand for animal proteins beyond supply and organic waste generation. However black soldier fly larvae (BSFL) bioconversion technology that uses organic waste to produce a nutritive biomass rich in proteins, offers a potential alternative solution for this nutrition scarcity and waste management. The Black soldier fly larvae have a wide range of applications ranging from those that benefit the environment (waste management, reduction of bad odors, landfill usage, greenhouse gas emissions, and environmental degradation) to food security benefits, making one of the most beneficial insects. Our sample were mostly collected from wild so to be used as animal feed and get results according to commercial one it needs to be reared in artificial environment providing high feed. Sample needs to be collected in larger amount before performing experiments.
This work aimed to characterize the Nepalese black soldier fly and explore its properties. From this project it was found that Nepali BSF larvae of wild type also consisted of high amount of protein. So, it can be used as alternative to fish feed and poultry feed. It is cost effective than other alternative feed as their rearing strategies does not need any high cost equipment and it can grow and develop even when fed plant – based waste streams. Various researches should be conducted regarding the feeding and rearing strategy of BSF fly in Nepal for suitable and cost-effective larvae media. As no any molecular and proteomic analysis was done before in Nepalese Black Soldier Fly, our study tried to explore its protein content on the basis of substrate used. Thus, with the effective substrate feed system and controlled rearing strategy, Nepalese Black soldier fly can also be taken as a good candidate for waste management and protein feed production as other commercial BSF larvae.
- Swiss-Nepal Technology Transfer Workshop 2018. Mrs. Bimala Dhakal and Mr. Nagendra Awasthi presented and won second title under project name Black worm.
- Top 24 finalist in Smart Urban Technology Challenge organized by Kathmandu Municipality, Ministry of Nepal.
Our three students Aayushi Sharma, Abhibind Khaniya and Sanjog Raj Silwal participated in this challenge. Ayushi is explaining project to the jury along with another team member.
If you are using our information for any use, citation under RARA Biotech is mandatory. For reference, thesis entitled "GENETIC IDENTIFICATION OF NEPALESE BLACK SOLDIER FLY(BSF) AND COMPARISION OF PROTEIN CONTENT OF BSF IN DIFFERENT FEED CONDITIONS" is available in Library of Department of Biotechnology, Himalayan WhiteHouse International College, Purbanchal University.