Recycling Potential of Brewer’s Spent Grains for Circular Biorefineries

Brewer ’ sspentgrain(BSG)isthemajorby-productofthebrewing industry. BSG is principally composed of carbohydrates and proteins, with substantial amount of lipids. Presently, BSG usage is restricted to low-grade applications such as ruminant feed or landfills. The high volume, nutrient-rich composition, low cost ( V 35/ton),abundance,andaroundtheyearavailability,makesita promising and renewable feedstock for biorefinery development. The current review begins with beer production process, where BSGisproduced.Further,itappraisesemergingbiotechnological advancements and green processes targeting BSG valorisation ensuring maximal resource recovery. Particularly, it illustrates diverse marketable products obtained by repurposing carbohydrate and protein fraction of BSG using either isolated or cascading approach. We believe that this review will encourage more research groups to work on developing innovative technologies for integrated and holistic valorisation of BSG. Inclusive efforts towards reduced water consumption and waste mini-misation is further advocated, which are presently primary challenges associated with beer industry. It will leave a significant imprint on environmental sustainability and pave a way for developing circular bio-based economy.


Introduction
Brewer's spent grain (BSG) is an abundant, highly nutritious by-product of the brewing industry, holding special significance by virtue of its specific biochemical composition.This lignocellulosic material accounts for 85% (w/w) of the total by-products generated in beer production and is formed from grains leftover after the mashing and filtration process [1].Statistically, every litre of beer produced, generates w0.2 kg of BSG.Considering this data, in the year 2021, 37.2 million tonnes of BSG was produced globally from 1.86 billion hectolitres beer, demonstrating the abundance of this valuable bioresource [2].Currently, BSG is primarily used as a low-value feed ingredient suitable for ruminants; or otherwise ends up in landfill posing a potential environmental threat [3].However, BSG is emerging as an ingredient in functional foods, owing to relatively high levels of dietary fibers in the form of b-glucan and arabinoxylan, proteins, and the presence of polyphenols (such as hydroxy-cinnamic acids) which display high antioxidant or other nutritional properties.All these components are considered to offer potential health benefits [4,5].Furthermore, depolymerisation of the carbohydrate fraction produce simple sugars, which are precursors for the production of several industrially important products using either chemical or biochemical routes.However, the high degree of biomass recalcitrance, along with limited shelf life and increased risk of susceptibility to microbial attack due to high moisture content act as major roadblocks in harnessing the true potential of BSG.
These opportunities have shifted the attitude towards BSG, and researchers are adopting a holistic approach and targeting maximum resource recovery, thereby showcasing its value as biorefinery feedstock [6,7].This review begins with brief description on how this renewable waste originates and its distinct chemical composition.From the perspective of emerging global interests in implementing the concept of sustainable circular economy, the review then considers technological developments in the past five years where inexpensive carbon-rich BSG has been explored for the production of marketable and bio-based materials including green fuels and chemicals.Particularly, the review highlights the green approaches used for waste mining from BSG, which targets either recovery of its carbohydrate fraction or protein or have fractionated each of these valuable biologically important compounds in a cascading mode and attempted their valorisation.Furthermore, the existing challenges of beer industry are showcased and thrust on developing end-to end processes are discussed, which can leave everlasting impact on environmental sustainability besides developing bio-based economy.
A brief overview of the brewing process and chemical composition of BSG Traditionally beer is fermented from malted barley.However, as beer styles and demand changed and, in recent times the use of other grains such as maize, wheat, and oats as adjuncts has escalated, thereby changing the chemical makeup of BSG.A typical process of beer production is outlined in Figure 1, showing various stages such as malting, milling, mashing, brewing, and fermentation [8].
The first step of brewing involves soaking of barley in warm water (steeping) before being allowed to rest and partially germinate.This process activates starch degrading enzymes within barley to produce metabolisable sugars, which yeast can metabolise.The barley is then kiln-dried.The combined step of steeping, germination and kilning is termed as malting.The dried malted barley is then subjected to roller or hammer mill to produce a coarse powder known as grist.If other grains are being used in addition to barley, they are usually added either directly before or directly after the mill, depending on the character required.The grist is soaked again in warm water in a process known as mashing.This releases soluble, fermentable sugars into the liquid.The residual barley solids (the spent grains) are removed, usually by filtration, to leave a liquid rich in soluble sugars (wort).The wort is then boiled for sterilisation prior to fermentation, causing protein coagulation and its precipitation, which may otherwise negatively affect the flavour or appearance of the beer.These precipitates (known as trub) are removed in a whirlpool and the cooled wort is transferred to a fermenter, where yeast ferments sugars to produce ethanol and other volatile organic compounds, conveying desired beer flavor profile.The 'green' beer from the finished fermentation can then be matured in various ways.Thus, BSG is the waste produced during the intermediate step of beer production.
Based on the compositional analysis, BSG essentially comprises of 60%e70% fibre and 15%e30% protein [9].The detailed constituents of BSG as depicted in Figure 2, reveals its high nutritive nature and heterogeneous distribution of functional organic carbon compounds [10].Considering its diverse chemical composition, recent research has primarily focused on either fractionating the key components in their pure form or hydrolysing its constituents followed by transformation to commercially viable products.Whilst some approaches chose to focus on carbohydrate or protein as their primary targets, others have attempted holistic utilisation of both key fractions of the biomass.

Carbohydrate fractionation of BSG and its valorisation
Being a rich source of the structural and storage polysaccharides, cellulose, hemicellulose, and starch, BSG serves as an inexpensive and efficient feedstock for the production of a plethora of bio-based energy and nonenergy products.Some of the commercially scalable products, produced from its carbohydrate fraction are shown in Table 1, covering the salient features of each process.
One area of research draws special attention to Table 1, where the researchers demonstrated the accumulation of > 100 g/L 2,3-butanediol (BDO) from the hydrolysed glucan fraction of BSG [17].The group further conducted a detailed techno-economic analysis of the process, where they redirected the fermentation residue and the acid hydrolysate (which is rich in soluble sugars) for biogas production [23].Process integration using pinch technology reduced the hot and cold utility consumption by 34 and 18%, respectively.A technoeconomic assessment (TEA) revealed that the plant size critically affected the minimum selling price (MSP) of BDO.This price reduced significantly from 3.36 USD/kg to 2 USD/kg when the BSG processing capacity was raised from 100 to 2000 MT/day and BDO titre was assumed to be 100 g/L [23].
Complete valorisation of storage and structural carbohydrate fraction of BSG to ethanol was earlier attempted by Rojas-Chamarro et al. [24].The authors developed an optimised dilute acid pretreatment strategy (12.5% solids; 1% H 2 SO 4 ; 130 C; 26 min) that recovered 94% carbohydrates in the resultant solid residue.Simultaneous saccharification and fermentation was performed using Cellic CTec3 and Scheffersomyces stipitis.Parallely, the acid hydrolysate comprising of variegated sugars and inhibitors was also fermented to produce ethanol using recombinant E.coli strain.The authors demonstrated production of 18.1 kg ethanol from 100 kg BSG [24].Along similar lines, glucose-rich enzymatic hydrolysate of BSG was explored for BDO, lactic acid (LA) and ethanol production by Bacillus polymyxa DSM 742.The said process produced 15.43, 1.22, and 2.61 g/L of LA, BDO, and ethanol, respectively.However, when the same organism was chosen for the valorisation of detoxified acid hydrolysate rich in pentose sugars in a stirred tank bioreactor, LA was the major product (30.07g/L) with minor quantities of acetate (3.96 g/L) [25].[28].Later, when three scenarios were evaluated for TEA of BSG valorisation, the most favourable scenario was the coproduction of xylitol and XOS > XOS > xylitol [29].
Likewise, the hemicellulosic liquor fraction of BSG was evaluated for xylitol and ethanol production using S. stipitis and Pachysolen tannophilus.It was found that the former yeast produced better ethanol while the latter strain produced high xylitol under oxygen-limited conditions [30].
BSG-derived sugar hydrolysates have also been explored for the cultivation of oleaginous microbes, as their lipid composition is typically suitable for conversion to biodiesel.For instance, recently, Rhodosporidium toruloides was grown on BSG hydrolysate (w72 g/L of monomeric sugars) generated by sequential organosolv process and enzymatic saccharification [31].A maximum biomass of 18.44 AE 0.96 g/L was accumulated on a dry weight basis containing 56.45 AE 0.76% lipids.In another study, the suitability of sugar-rich BSG-derived acid hydrolysate was assessed for the cultivation of Trichosporonoides spathulata JU4-57, Rhodotorula mucilaginosa G43 and Yarrowia lipolytica TISTR 5151.T. spathulata emerged as the best oleaginous yeast and produced 62.9 mg lipid/g substrate [32].Further isolated study confirmed that the unsaturated fatty acids constituted 78.4% of the total lipids and were predominant in oleic acid, followed by linoleic acid and palmitic acid [33].
Unlike more conventional products discussed in the preceding sections, recently BSG was simultaneously evaluated for lignocellulolytic enzyme production and its viscozyme L treated sugar-rich fraction was assessed for polyhydroxyalkanoate production.Three fungal strains (Aspergillus niger, Thermoascus aurantiacus, and Trichoderma ressei) were grown on BSG using solid state fermentation (SSF) which displayed high xylanase activities with side activity of cellulases.Additionally, Cupriavidus necator produced a maximum of 9.0 AE 0.44 mg of PHA/g BSG, demonstrating the feasibility of BSG for the production of multiple bioproducts [34].In a later work, the same group showed high polyhydroxy-3-butyrate (yield: 23 AE 1 mg PHA/g BSG) by Burkholderia cepacia when grown on enzymatically hydrolysed BSG under submerged conditions [35].Similarly, T. reesei strain 938 (CBS 836.91) was grown on BSG as solid substrate for lignocellulolytic enzyme production.Later the same enzyme cocktail, liberated 193.35 mg sugars/g dry BSG from BSG pretreated with H 2 O 2 [36].

Protein fractionation of BSG and its valorisation
Proteins constitute a significant fraction of BSG and are mainly present as hordien, glutelin, globulin, and albumin.These proteins offer numerous health benefits (eg.anti-inflammatory, anti-oxidant properties) over and above simple nutritional value, if extracted in a suitable fashion [37].
In a recent study, combinatorial protease pretreatment with 0.5% protamex and 0.1% flavourozyme (50 C; pH 8.5; 3h) resulted in the production of protein hydrolysates (PH).In-depth techno-functional analysis revealed that these PH's had high protein content (31.4%).Further they showed high oxygen radical absorbance capacity (ORAC), and antioxidant properties (ABTS assay), with enhanced water solubility index, emulsifying activity, foaming capacity and stability.Additionally, the sediments from the process got enriched with Flavon-3-ols [38].In a separate study, when the same group fortified yogurt with enzymatically hydrolysed (0.5% Protamex) BSG, it not only improved the survival rate of Lactobacillus bulgaricus but increased LA production also.Further, the texture, surface and functional properties of the yoghurt enhanced significantly, especially during long-time storage [39].In yet another study BSG was fermented by Rhizopus oligosporous and later the fermented BSG was extracted with ethanolic-alkali mixture followed by protein precipitation at isoelectric point.Fermentation with R. oligosporous enriched protein from 25.2 to 34.1% [40].
The fermentation enhanced levels of essential amino acids (valine, methionine, lysine, and histidine) in the protein extract, but at the expense of proline and glutamine levels.In addition to significant improvements in foaming, emulsifying, water, and oil binding capacities, the protein displayed high anti-oxidant properties and no cytotoxic effect.The protein's application as a plant emulsifier and as an excellent substitute for egg during mayonnaise preparation was also demonstrated [40].In the same year, Alcalase pretreatment of BSG improved the antioxidant or anti-oxidative properties of hydrolysed protein and enhanced emulsion capacity by 2.4 fold (from 30% to 72%).This study claims the potential of BSGderived protein hydrolysate for food applications [41].

BSG fractionation in a cascading manner and valorisation
Complete fractionation of BSG has been employed by a few research groups, with intent of maximising the recovery of diverse functionally active components.For instance, Rojas-Pe ´rez et al.
[42] employed a sequential enzymatic deproteinisation followed by acid catalysed steam explosion (ACSE).ACSE hydrolysed the hemicellulosic fraction of BSG.The research reported recovery of w64% protein, with 30% and 93% of xylose and arabinose, respectively, extracted as monomers in the acid hydrolysate.Hydrolysis of the glucan-rich residue resulted in 72.2% glucose yield.This investigation opens further avenues for the valorisation of the separated BSG fractions [42].In another study, 85.65% lignin was recovered from BSG along with 90% hemicellulose removal by optimising an ethanol organosolv process.Simultaneously a highly digestible glucan-rich (59.68% cellulose yield with 37.6% purity) pulp with a low total crystallinity index (TCI) was generated.Further processing of recovered lignin reduced its yields to 58% but attained 95% purity [43].Earlier, the same group performed hydrothermal pretreatment of BSG (180 C; 30 min) leading to 83% and 48% solubilisation of starch and protein, respectively.When Neurospora intermedia was cultivated separately on the liquid and solid fraction post-pretreatment, this edible fungus accumulated 50.42 and 30.86%(w/w) protein with ethanol as a byproduct, demonstrating the feasibility of BSG for the development of a brewery-based biorefinery [44].
Earlier, He et al.
[45] took a holistic approach and in the first stage enzymatically separated the protein fraction of BSG, and later subjected the carbohydrate fraction to acid pretreatment followed by cellulase-mediated hydrolysis.Alcalase (20 mL/g) pretreatment led to the generation of hydrolysate with a protein concentration of 41.4%.On the other hand, sugars produced in Stage II were assessed for BDO production using thermophilic Bacillus licheniformis YNP5-TSU under sterile and nonsterile conditions.A maximum BDO concentration of 20.4e21 and 12.2e12.3g/L was attained from sugar-rich enzymatic hydrolysates and mixed sugars (acid and enzymatic hydrolysates) respectively, with productivity being 0.285 and 0.17 g/L/h, respectively [45].These examples elucidate the emerging trends and opportunities which are yet to be unleashed fully, targeting fractionation of important constituents of BSG and their valorisation.However, there are various processes during brewing which pose critical environmental threats and need to be simultaneously addressed.These issues are dealt in forthcoming section, citing remedies and path forward.The authors anticipate that an integrated approach can further optimise both carbon and water footprint of breweries and contribute towards sustainability development goals of nations where it is a predominant revenue generating industry.
Environmental challenges associated with brewing industry.

Current challenges and path forward
Huge water consumption, production of solid waste, waste water and greenhouse gas (GHG) emissions at various stages of brewing (Figure 3), pose severe environmental challenges as per the extensive review done by Olajire [46].Reduced water consumption and waste minimisation are presently high-priority areas for the brewing industry.Hence, a holistic approach is highly desirable and should not be underestimated.To accomplish these targets, brewing industries should accelerate their R&D efforts and work cohesively with academia to address these critical issues.A rapid upsurge in recycling, reusing or valorising their important by-product streams like "BSG" shows a clear path of biorefinery development.

Concluding remarks
The present review demonstrates the potential of BSG for biorefinery development with multiple renewable energy and non-energy products as examples.However, more focus is needed to develop a cost-competent, environmentally benign, green, and sustainable end-to end processes targeting maximum recovery of valuable carbonaceous components from side waste streams, including solids, water and gaseous streams.Since BSG accounts majorly among all the solid waste streams, its pivotal role in diversifying product portfolio, generating huge revenues and enhance profitability of brewing industries is undeniable.The work discussed above add significant value to science around BSG as the feedstock, but none standing alone offer the end-to-end processing, crucial for a commercial biorefinery.Thus, future work should be directed toward the development of pretreatment technology to ensure efficient, benign, and optimal resource recovery, robust bio-transforming strains with high TYP metrics matching industrial requirements, process intensification, and carrying out sustainability (techno-economic and environment) assessment of promising technologies.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this article.

Table 1
Commercially important and valorised products from carbohydrate fraction of BSG.
The present review already demonstrates the rationale repurposing of BSG-derived carbohydrates and proteins to numerous product such as ethanol, butanol, biomethane, xylitol, BDO, fertilisers, enzymes, prebiotics (XOS, ArXOS), functional foods and nutritional supplements.It further illustrates cases where spent yeast has been successfully used as an excellent substitute of yeast extract during microbial fermentation.This organic nitrogen source, rich in water soluble vitamins can be used for food fortification as well.Side by side the breweries should identify areas where water recycling/reuse is possible.They should strive for reducing the water footprint during the overall process of beer production.Recovery of gases such as Brewers' spent grain liquor as a feedstock for lactate production with Lactobacillus delbrueckii subsp.lactis.Eng Life Sci 2020, 20:168-180.https://doi.org/10.1002/elsc.201900143.It is the first kind of its article where the liquor rich in glucose, maltose and its oligomers is separated from wet Brewer's spent grains and valorised to lactic acid using autolysed yeast obtained from beer production.High titre of lactic acid is demonstrated with high productivity, using Lactobacillus delbrueckii strain.Food Chem X 2022, 100184:13.https://doi.org/10.1016/j.fochx.2021.100184.In this article a novel concept of improving properties of BSG protein was conceived by growing Rhizopus fungus on Brewer's spent grains.Protein extracted from fermented BSG displayed improved functional properties with no cytotoxic effect.Further its use as a potential substitute of egg is demonstrated in mayonnaise preparation.41.Vieira MC, BrandelliA, Thys RCS: Evaluation of the technological functional properties and antioxidant activity of protein hydrolysate obtained from brewers' spent grain.J Food Process Preserv 2022, 46, p.e16638.https://doi.org/10.1111/jfpp.16638.42 * .Rojas-Pérez LC, Narváez-Rincón PC, Ballesteros I: improving sugar extraction from brewers' spent grain using sequential deproteinization and acid-catalyzed steam explosion in a biorefinery context.Biomass Bioenergy 2022, 106389:159.https://doi.org/10.1016/j.biombioe.2022.106389.This recent article demonstrates effective fractionation of Brewer's spent grains.Enzyme mediated deproteinisation led to recovery of 63.9% protein.Later optimized acid pretreatment hydrolysed the hemicellulosic fraction retaining 30% and 93% xylose and arabinose fraction.The acid pretreated solid residue on enzymatic saccharification results in 72.2% glucose yields.43.Parchami M, Agnihotri S, Taherzadeh MJ: Aqueous ethanol organosolv process for the valorization of Brewer's spent grain (BSG).Bioresour Technol 2022, 127764:362.https:// doi.org/10.1016/j.biortech.2022.127764.44.Parchami M, Ferreira JA, Taherzadeh MJ: Starch and protein recovery from brewer's spent grain using hydrothermal pretreatment and their conversion to edible filamentous fungi-A brewery biorefinery concept.Bioresour Technol 2021, 125409:337.https://doi.org/10.1016/j.biortech.2021.125409.Ohair JA, Jin Q, Xu Z, Wu J, Huang H: Co-production of protein hydrolysates and 2, 3-butanediol from brewer's spent grain.ACS Sustainable Chem Eng 2021, 9:15166-15174.https://doi.org/10.1021/acssuschemeng.1c04162.This article took a holistic approach, targeting both the carbohydrate and protein fraction of BSG.If Alcalase assisted hydrolysis yielded 437.6g protein hydrolysate from one kg BSG, sugar rich hydrolysate was fermented to 126.3g 2,3 butanediol from one kg BSG using thermophilic and alkalophilic Bacillus.