Evaluation of chemical and biological refinement strategies for transforming brewer’s spent grain into a high-quality protein source for fish feed

Aquaculture is the major supplier of fish for human consumption. An expanding human
population will lead to intensification of fish farming as well as production of aquafeeds
in the coming years. Fishmeal and soybean meal constitute the most common protein
sources in aquafeeds due to their high nutritional value. Yet, increasing demands and
competition for resources have driven up the prices of these protein sources which
have heightened a need to implement sustainable alternatives. Brewer’s spent grain
(BSG) is the most abundant by-product from beer production. Due to its low market value, immense availability throughout the year and relatively high protein content (20-30% DM), BSG represents a potential protein source for aquafeeds. Inclusion of unrefined BSG in aquafeeds is hindered due to its high levels of lignin, cellulose and hemicellulose which are antinutritional factors (ANFs) for fish. Furthermore, the protein content of unrefined BSG is too low to meet the protein requirement of most fish species (25-50%). The main objective of this PhD project was to evaluate different refinement strategies for BSG that may allow for its incorporation in aquafeeds. The PhD project consisted of three parts: 1) protein extraction, 2) delignification by solid-state fermentation (SSF) and 3) valorization of cellulose and hemicellulose using a combination of hydrolysis and submerged fermentation.

Part I intended to optimize an alkaline process for extracting proteins from BSG. For this purpose, several parameters were evaluated including temperature (30, 45, 60°C), duration (30, 60, 120, 180 min), isolation mode (precipitation vs freeze-drying), and repeated extraction (single vs triple). The following conditions led to the highest protein content (55% DM) but, unfortunately, the lowest recovery (~10%): 60°C, 30 min, precipitation and single extraction. Conversely, freeze-drying combined with triple extraction maximized the protein recovery (~45%) but did not enrich the protein content of isolates (~25% DM).

Part II explored the potential of utilizing SSF to delignify BSG using the fungus Phanerochaete chrysosporium. In an attempt to enhance delignification, P. chrysosporium was cultured on solid BSG substrates supplemented with veratryl alcohol, MnO₂ and excess trace elements. The obtained results indicated that delignification occurred at a slow pace (0.75%/day) and that chitin accumulated during cultivation. Surprisingly, no differences were found between the supplemented substrates and the control (no supplementation).

Part III had a dual purpose. First, it aimed to hydrolyse cellulose and hemicellulose into fermentable sugars, thereby forming a liquid BSG hydrolysate for fermentation. Second, the sugar-containing BSG hydrolysate was utilized as a substrate for production of free amino acids and single cell protein (SCP) by submerged fermentation. Initially, different microorganisms were screened with respect to their ability to grow and produce amino acids in BSG-based media. This screening resulted in selection of two microorganisms: Saccharomyces cerevisiae and Corynebacterium glutamicum. C. glutamicum was the most promising as it produced several amino acids in shake flasks and bioreactor including alanine (Ala), proline (Pro), valine (Val) and glycine (Gly). The amino acid composition and protein content were determined for S. cerevisiae biomass to assess its potential as a protein source. This analysis showed a relatively high protein content (~38% DM) and an amino acid profile resembling that of fishmeal except for deficiencies in methionine (Met) and histidine (His).

In conclusion, this PhD project provided vital insight into various refinement strategies of BSG. Low efficiencies were a general limitation of all evaluated processes and therefore further process optimization should be the focus of future research.