Bioconversion of lignocellulosic biomass by the black soldier fly in combination with solid state fermentation for biofuel and larval biomass production
The digestion of wheat dried distiller’s grains with solubles (DDGS) by the black soldier fly larvae (BSFL) was investigated. DDGS is a high carbohydrate, high protein by-product of grain ethanol fermentation. Around ten percent of the dry weight of DDGS is cellulose, and a further five percent is starch which together represents a large fraction of glucans that escaped the ethanol fermentation process. Digestion by BSFL was expected to result in the degradation of DDGS constituent matrices, particularly fiber, which would increase the recovery of glucose from DDGS to be used in further bioethanol fermentation. Additionally, proteins and digestible carbohydrates could be recovered as BSFL biomass. We considered two studies based on this hypothesis: enzyme and microbial investigation in BSFL culture; and microbiological aids in the BSFL degradation of DDGS. Secretion of enzymes from the BSFL into their feed bed can potentially contribute to the insects DDGS digestive capabilities along with symbiotic bacteria associated with the BSFL. The nutrient composition of the BSFL's diet may impact their digestive capabilities. To investigate this hypothesis, and specifically the effect of the fiber content of the feed, we first analyzed enzyme activities in feed beds which varied in cellulose content using food commodities (corn meal, DDGS, and whole milk powder) containing complex matrices to evaluate the effect of cellulose content directly. Enzyme activities in spent larval feeds were shown to decrease as the cellulose content of the feed increased. From these BSFL spent feeds, bacteria associated with BSFL were isolated and included Lactobacillus plantarum, Sphingomonas melonis, Psuedomonas sp., and Klebsiella pneumoniae. These bacteria should have originated from the digestive system of the BSFL, which were hypothesized to play a role in the BSFL’s digestive capabilities. The contribution of these isolates in enzyme activities were also analyzed, and Lactobacillus plantarum showed the greatest overall enzyme activity based on protease, cellulase, amylase, and lipase assays of the isolated bacteria. Because the digestive capabilities of BSFL were shown to decrease as the cellulose content of their feed increases in this study, we further hypothesized that fermentation of cellulosic materials prior to BSFL digestion can assist the contribution of the larvae in glucose recovery from the cellulosic materials. It is investigated whether the use of solid state fermentation (SSF) could degrade DDGS fiber matrices and improve the overall nutrient profile for BSFL culture and recovery of fermentable sugars. The following fungal strains, chosen for their cellulolytic ability: Trichoderma koningii, Aspergillus niger, Aspergillus fumigatus, and Phanerochaete chrysosporium; and Lactobacillus plantarum isolated from the BSFL, were used in SSF of DDGS, prior to larval digestion. SSF was shown to open the DDGS structure; reduce the cellulose content by around 5 percent; decrease the total carbohydrate content by up to 13 percent; and increase the protein concentration by up to 8 percent. While all fungal SSF treatments showed positive changes to DDGS nutrient composition, SSF using Aspergillus niger had the greatest effect on DDGS, reducing the carbohydrate content by 13 percent, and increasing the protein content by 8 percent. Following this analysis, we investigated whether fermented DDGS was suitable for larval feeding, and the effect that BSFL digestion had on the fermentable sugar recovery from DDGS. In order to analyze the susceptibility of DDGS cellulose into glucose, a two-step hydrolysis assay was employed. Dilute acid hydrolysis was utilized as an initial hydrolysis, which hydrolyzed primarily amorphous cellulose along with starches and bound glucose into monomeric glucose. Then, the solid matter from dilute acid hydrolysis was subjected to enzymatic hydrolysis as the second step of sugar liberation, where cellulases hydrolyzed the remaining crystalline cellulose into monomeric glucose. Digestion of fermented DDGS by BSFL reduced the dry matter of the substrate and incorporated it into a high protein (37% dwb), high fat (23% dwb) larval biomass. Considering DDGS contains very low fat (2%), the significant fat recovery/yields can be achieved though SSF-BSFL treatment. When untreated DDGS was given to BSFL, the larvae incorporated 43.7 % of dry mass of DDGS; whereas SSF DDGS was incorporated up to 80.4% after the digestion by a combination of SSF and BSFL culture. The relative carbohydrate contents of the substrates were increased during larval digestion which, along with opening of the DDGS constituent matrices during treatments, allowed for increased glucose liberation from the substrates in spent feeds. Optimization of hydrolysis conditions, including higher temperatures, pressure, and substrate loading concentration, further provided large increases in total glucose yields compared to unfermented DDGS (8 % of dry weight of DDGS carbohydrates), particularly for Lactobacillus plantarum SSF (16% of dry weight of DDGS carbohydrates)
