Eugéne van Rensburg

High-solids simultaneous saccharification and co-fermentation (SScF) of paper wastes were compared in lab-scale fed-batch (vertical) and batch (horizontal) bioreactor cultures. Fermentation of untreated label backing (LB), multilayer paper-based packaging (ML) and potato paper sacks (PS) paper wastes were optimised for solid loading to minimise the exogenous enzyme dosage with advanced consolidated bioprocessing (CBP) yeast strains, to achieve maximum technical performances in terms of ethanol concentration, yield and enzyme efficiency. Using cellulase-producing CBP yeast (Cellusec ^® 2.0, 3.3 or 4.0) improved the enzyme efficiency by 1.5-fold at a final solid loading of 20% (w/w dry) compared to a higher solid loading of 30% (w/w) in fed-batch SScF. At 20% (w/w) solid loading and 2 FPU/g ds, enzyme efficiencies of 0.109, 0.078 and 0.067 g ethanol/FPU were recorded with corresponding ethanol titres of 59.3, 45.2 and 38.2 g/L and ethanol yields of 58.5, 43.9 and 57.1% of the theoretical maximum for LB, PS and ML, respectively. Whereas batch SScF at 30% (w/w) solid loading and 2 FPU/g ds achieved higher ethanol titres of 65.6, 50.7 and 54.3 g/L, the corresponding ethanol yields and enzyme efficiencies were markedly lower at 36.7, 28.0 and 52.1% of the theoretical maximum and 0.069, 0.050 and 0.062 g ethanol/FPU for LB, PS and ML, respectively. Fed-batch SScF in a vertical bioreactor at 20% (w/w) final solid loading was, therefore, the preferred configuration due to more favourable enzyme efficiencies, ethanol yields and enzyme cost.

Enzymes can play an important role in environmentally friendly processing of seaweed biomass, and there is a rich diversity of microbes that may harbour seaweed specific polysaccharide-lyases. In the context of alginate bearing brown seaweed, wild-type alginate lyase (AL) producing microbes would be useful for alginate-related bioprocesses. Eight microorganisms capable of extracellular AL production were isolated and identified up to genus level using 16S and ITS sequencing. Seven of these microbes are bacteria of the genera, Bacillus sp (4/8), Pantoea sp. (1/8), Lysinibacillus sp. (1/8), Stappia sp. (1/8), and a yeast of the genus Rhodotorula sp. (1/8). Through employing a screening approach in different media, isolates were evaluated for their capacity to produce AL, and three isolates were selected from the genera Bacillus sp., Lysinibacillus sp., and Rhodotorula sp. for displaying high relative AL activity among the candidate isolates. In addition, a preliminary medium was identified which contained two nitrogen sources (peptone and yeast extract) at a ratio of 5:1 and the highest salt concentration of all media tested. This medium may be utilised as a starting point for development of similar bioprocesses in the future. Importantly, the Rhodotorula sp. isolated in this study potentially marks the first report of these yeasts displaying AL activity, rendering this a valuable strain for future kelp valorisation practices. Moreover, this study identified three microbes which were confirmed to produce high levels of AL: two bacterial strains (a Bacillus sp., which is of a genus that is widely employed in bioprocessing, and a lesser known Lysinibacillus sp.), and a Rhodotorula sp. yeast, which is of a versatile genera that could also produce other valuable products.

Purpose: Brewers’ spent grain (BSG) is a major processing residue from the beer brewing industry, often applied for low-value animal feed or, process energy, or otherwise disposed. The processing of BSG into xylooligosaccharides (XOS), a proven prebiotic with application in the food, beverage and health industries, can be achieved through selective solubilisation/hydrolysis of its hemicellulose content. Methods: In this study, the sequential processing of BSG to maximise the yield of XOS was achieved by screw-pressing to remove a protein-rich press liquid fraction, followed by steam explosion pretreatment prior to enzymatic hydrolysis of the remaining hemicellulose in the feedstock. Results: A combined XOS yield of 37% (kg XOS/kg hemicellulose) was achieved from the steam explosion liquor (two-thirds) and the enzymatic hydrolysis of pretreated solids (one-third). The crude, longer-chain XOS products were subsequently depolymerised through an additional enzymatic hydrolysis step, to achieve the desired XOS molecules with degrees of polymerisation between 2 and 3, resulting in an overall XOS yield of 16%. Conclusion: The overall XOS yield was increased by adding enzymatic hydrolysis of the solids fraction to the XOS production process, however, the findings of the study presented a potential trade-off between the increase in XOS yield and the cost of the increased enzyme requirements. Graphical

Bioaugmentation of anaerobic digestion (AD) systems is considered a cost-effective and environmentally friendly strategy to combat incomplete digestion of recalcitrant lignocellulosic substrates. This study investigated the lowest microbial inoculum size required for once-off bioaugmentation of AD cultures to enhance biomethane yield and process performance. The batch, laboratory-scale anaerobic co-digestion was carried out using pretreated corn stover (PCS) and food waste (FW), with cellulolytic Bacillus subtilis, Serratia marcescens and Bacillus licheniformis. The bioaugmentation screening was accomplished through a stepwise increase in the microbial loading using an initial standardised concentration of 0.4 × 10 ¹¹ colony-forming units (CFU)/mL within the system. Bioaugmentation decreased the digestion time by up to 11 days. The inoculation of B. subtilis at a microbial concentration of 20 × 10 ¹¹ CFU/mL (4.85 g DCW/L) improved the biomethane yield by 34% compared to the unaugmented control and produced 525 NmL CH ₄ /gVS. Additionally, S. marcescens at 12 × 10 ¹¹ CFU/mL doubled the volumetric methane productivity from 0.47 ± 0.02 to 1.04 ± 0.02 mL/(mL.day) when compared to the unaugmented control. The application of Nanopore sequencing after AD, to investigate the microbial community dynamics and structure in this treatment, underlined 43.52, 7.69 and 25.26% increases in the bacterial alpha diversity, namely the Shannon-, Simpson- and Observed indices, respectively. Moreover, a high abundance of between 50 and 80% of the Firmicutes population was identified.

Significant wastes such as bagasse, molasses, and vinasses are produced during sugarcane processing. Due to their high sugar content, these wastes are commonly used as low-cost substrates for biofuel production. However, these substrates are also suitable for the microbial synthesis of high-value biochemicals like biosurfactants. Sporisorium scitamineum, a smut fungus capable of growing on sugarcane residues and producing mannosylerythritol lipids (MELs) and cellobiose lipids (CBLs), was identified as a promising candidate for valorizing sugarcane wastes. This study investigated MEL and CBL co-production from pure sugars and sugarcane molasses using an S. scitamineum strain isolated from sugarcane residues originating from KwaZulu-Natal, South Africa. Among the sugars tested, sucrose supported the highest glycolipid production, yielding 0.24 g/L MELs and 2.73 g/L CBLs. Lower titers were achieved with fructose, and no production occurred with glucose. Sugarcane molasses also proved to be an effective substrate, yielding 1.46 g/L CBLs—the highest reported titer from an industrial waste to date. However, all titers remained far below those of other glycolipids, which consistently exceed 50 g/L. Future efforts should focus on enhancing CBL production through process optimization or genetic engineering.

Purpose: Converting lignocellulosic residues, like sugarcane bagasse, into single-cell proteins (SCPs) offers a sustainable protein production method to aid in ensuring global food security. Methods: This study evaluated steam-explosion and enzymatic hydrolysis as pretreatment for sugarcane bagasse feedstock, with a subsequent investigation of SCP production from the pretreated sugarcane bagasse, individually employing six ‘generally-regarded-as-safe’ microorganisms: Bacillus subtilis , Lactobacillus delbrueckii , Streptococcus thermophilus , Fusarium venenatum , Pleurotus ostreatus and Saccharomyces cerevisiae . Microbial biomass growth and protein production were investigated under both batch as well as fed-batch fermentation conditions. Furthermore, an integrated SCP biorefinery was designed and simulated in Aspen Plus ^® , with a subsequent techno-economic analysis to determine the profitability of such a SCP biorefinery. Results: S. cerevisiae achieved the highest biomass concentration (20.37 ± 11.81 g/L) in batch culture, while S. thermophilus also exhibited some tolerance to inhibitors produced during steam explosion (1.28 ± 0.17 g/L). Pulsed fed-batch fermentations enhanced biomass growth, yielding biomass concentrations of 52.65 ± 0.80 g/L and 6.57 ± 0.09 g/L, and SCP concentrations of 24.71 ± 1.44 g/L and 4.34 ± 0.10 g/L, for S. cerevisiae and S. thermophilus , respectively. The cost of the S. cerevisiae bulk product (1.91 US$/kg) was considerably lower than that of S. thermophilus (15.51 US$/kg), further supporting its suitability for SCP co-production with bio-ethanol. Conclusion: The production of SCP from steam-exploded and enzymatically hydrolyzed sugarcane bagasse was found to be profitable, with S. cerevisiae being the preferred ‘generally-regarded-as-safe’ microorganism for this process.

Conventional sugarcane-to-ethanol conversion occurs via a series of process steps, inter alia, energy-intensive juice extraction and concentration, followed by fermentation of the extracted juice under submerged (liquid) fermentation conditions. Solid-state fermentation (SStF), occurring in the absence of free water, is a promising alternative approach, potentially offering higher product concentrations, reduced water requirements and liquid effluent from the process, and elimination of the substantial energy requirements of the juice extraction step. While SStF has been applied to various substrates, such as sweet sorghum, there is a lack of studies considering the SStF of sugarcane, which is considered a more challenging substrate. The present study investigated the SStF of whole, milled sugarcane in 3-L horizontal, rotating reactors, to assess the effect of inoculum size, mixing speed, and particle size on ethanol production. The maximum ethanol concentration and yield were 86.7 g/L and 6.15 g/100 g wet mass (90.5% of the theoretical maximum), respectively, achieved at an inoculum size of 5% (w/w), rotation speed of 5 rpm, and particle size range of 8 to 20 mm. The fermentation was scaled up to a 50 L solid-state reactor, applying intermittent mixing to obtain a similar ethanol concentration and yield of 87.5 g/L and 6.61 g/100 g wet mass, respectively.

Three unrecyclable waste-paper streams, namely label backing paper (LB), multilayer paper-based packaging (ML) and potato paper sacks (PS) were characterised as fermentation feedstocks. Industrial Saccharomyces cerevisiae strain Ethanol Red® and recombinant S. cerevisiae strains Cellusec® 2.0 and Cellusec® 3.3 engineered for both cellulase-secretion and xylose utilisation were compared. Fermentation performance using Ethanol Red® with batch simultaneous saccharification and fermentation (SSF) was higher compared to separate hydrolysis and fermentation (SHF), and pre-saccharification and SSF (PSSF). Shake flask fed-batch SSF at 30% (w/w) final solid loading produced the highest ethanol titres of 75.01, 49.9 and 82.81 g/L for untreated LB, ML and PS, respectively, and yields above 70% of the theoretical maximum, at an enzyme dosage of 10 FPU/g dry solids (ds), using Ethanol Red ® . However, Cellusec® 2.0 and 3.3 in batch SSF at a two-to-three-fold reduced exogenous enzyme requirement of 3.5 FPU/g ds, achieved ethanol titres and yields higher than Ethanol Red ® . Graphical Abstract