foods Article Conversion of Exhausted Sugar Beet Pulp into Fermentable Sugars from a Biorefinery Approach Cristina Marzo , Ana Belén Díaz * , Ildefonso Caro and Ana Blandino Department of Chemical Engineering and Food Technology, Faculty of Sciences, IVAGRO, University of Cádiz, Campus Universitario de Puerto Real, 11510 Puerto Real, Spain; [email protected] (C.M.); [email protected] (I.C.); [email protected] (A.B.) * Correspondence: [email protected] Received: 29 August 2020; Accepted: 21 September 2020; Published: 24 September 2020 Abstract: In this study, the production of a hydrolysate rich in fermentable sugars, which could be used as a generic microbial culture medium, was carried out by using exhausted sugar beet pulp pellets (ESBPPs) as raw material. For this purpose, the hydrolysis was performed through the direct addition of the fermented ESBPPs obtained by fungal solid-state fermentation (SSF) as an enzyme source. By directly using this fermented solid, the stages for enzyme extraction and purification were avoided. The effects of temperature, fermented to fresh solid ratio, supplementation of fermented ESBPP with commercial cellulase, and the use of high-solid fed-batch enzymatic hydrolysis were studied to obtain the maximum reducing sugar (RS) concentration and productivity. The highest RS concentration and productivity, 127.3 g L 1 and 24.3 g L 1 h 1 respectively, were obtained at 50 C · − · − · − ◦ and with an initial supplementation of 2.17 U of Celluclast® per gram of dried solid in fed-batch mode. This process was carried out with a liquid to solid ratio of 4.3 mL g 1 solid, by adding 15 g · − of fermented solid and 13.75 g of fresh solid at the beginning of the hydrolysis, and then the same amount of fresh solid 3 times every 2.5 h. By this procedure, ESBPP can be used to produce a generic microbial feedstock, which contains a high concentration of monosaccharides. Keywords: enzymatic hydrolysis; solid-state fermentation; sugar beet; sugars hydrolysate; generic microbial feedstock 1. Introduction The production of high-value-added products, such as organic acids, vitamins, enzymes, bulk chemicals, biofuels, etc. through biotechnological processes using agro-industrial lignocellulosic residues (wheat straw, sugarcane bagasse, sweet sorghum bagasse, etc.) as raw material has become more interesting over the years [1]. They do not compete with food supply, they are generated in big volumes, and they are a sustainable and abundant renewable resource [2,3]. For the conversion of lignocellulose to fermentable sugars, a hydrolysis step is required. This step can be carried out chemically or enzymatically, although the latter shows the added advantage of not generating toxic compounds and being more environmentally friendly [4], while chemical hydrolysis needs several steps to completely remove the chemical from the final products. Moreover, the enzymatic process does not produce unfavorable by-products, the downstream processing is simpler, and corrosion of equipment is avoided [5]. However, the commercial and industrial application of enzymes is limited given their expensive production process and poor stability [6]. Enzyme costs contribute significantly to the economic viability of transforming lignocellulose into bioproducts of interest for the energy industry and the chemical, food, and materials sector. As a result, biomass hydrolysis continues to be a bottleneck of the overall process. Therefore, different strategies, Foods 2020, 9, 1351; doi:10.3390/foods9101351 www.mdpi.com/journal/foods Foods 2020, 9, x 1351 FOR PEER REVIEW 2 of 14 strategies, such as the seeking of new sources of enzymes or different hydrolysis technologies have beensuch investigated as the seeking to make of new the sourcesprocess more of enzymes cost effective or diff [7].erent hydrolysis technologies have been investigatedOne of the to makemost interesting the process ways more for cost the eff conversionective [7]. of cellulose and hemicellulose into reducing sugarsOne for of the the production most interesting of different ways forproducts the conversion is the use of of cellulose cellulolytic and hemicelluloseenzyme cocktails into of reducing fungal originsugars [8]. for This the productionfact is the consequence of different of products the capability is the useof some of cellulolytic fungi to produce enzyme hydrolytic cocktails ofenzymes fungal [9].origin Therefore, [8]. This there fact is isa great the consequence interest in identifying of the capability fungi that of secrete some fungithese toenzymes produce efficiently hydrolytic by usingenzymes cheap [9]. carbon Therefore, sources. there In isthis a greatregard, interest solid‐state in identifying fermentation fungi (SSF) that is secrete an interesting these enzymes option, becauseefficiently heterogeneous by using cheap solids, carbon such sources. as agro In this‐food regard, industry solid-state wastes fermentation and agricultural (SSF) residues, is an interesting which areoption, low‐ becausevalue and heterogeneous abundant raw solids, materials, such ascan agro-food be used industryas a solid, wastes support, and carbon agricultural source residues, and as inducerswhich are for low-value fungal growth and abundant and enzyme raw production materials, [10]. can beSeveral used agri as a‐food solid, residues, support, such carbon as soybean source meal,and as sunflower, inducers for or fungalwheat growthwaste, have and enzymebeen converted production into [ 10hydrolysates]. Several agri-food by using residues, crude enzymes such as producedsoybean meal, by the sunflower, solid‐state or fermentation wheat waste, of havethese been solid converted wastes. Other into authors hydrolysates have even by using employed crude mixturesenzymes producedof residues, by such the solid-state as sugarcane fermentation bagasse ofand these soybean solid wastes.hull or Otherfood and authors bakery have waste. even Afterward,employed mixtures fermented of residues,solids were such used as sugarcaneas a source bagasse of enzymes and soybeanto obtain hull fermentation or food and feedstocks bakery waste. [11– 15].Afterward, fermented solids were used as a source of enzymes to obtain fermentation feedstocks [11–15]. Taking into account the above considerations, the main goal of this study is the production of a hydrolysate rich in fermentable sugars, which could could be be used used as as a a generic generic microbial microbial culture culture medium, medium, using exhausted sugar beet pulp pellets (ESBPPs) as raw material. For this purpose, the hydrolysis was performed through the direct addition of the fermented biomass obtained by SSF as an enzyme source. In this way,way, the the cost cost of of the the process process could could be be reduced reduced as as enzyme enzyme extraction extraction and and purification purification are arenot not required. required. Figure Figure1 illustrates 1 illustrates the the di ff differenceserences between between the the conventional conventional enzymatic enzymatic hydrolysis, hydrolysis, in inwhich which enzymes enzymes produced produced through through solid-state solid‐state fermentation fermentation are extractedare extracted and lyophilizedand lyophilized before before their theiruse (Figure use (Figure1A) and 1A) the and one the employed one employed in this in study this study (Figure (Figure1B). 1B). ESBPPs are the solid obtained after industrial sugar extraction by diffusion diffusion from sugar beets and they areare normally normally dehydrated, dehydrated, granulated, granulated, and and sold sold for for animal animal feeding. feeding. In this In this work, work, firstly,ESBPPs firstly, ESBPPs were wereused asused a substrate as a substrate to produce to hydrolyticproduce hydrolytic enzymes using enzymesAspergillus using awamoriAspergillussolid-state awamori fermentation. solid‐state fermentation.This fermented This solid, fermented containing solid, the containing hydrolytic the enzyme hydrolytic cocktail enzyme secreted cocktail by secreted the fungus by the during fungus its duringgrowth, its was growth, used for was the hydrolysisused for the of fresh hydrolysis ESBPPs. of Finally, fresh monosaccharideESBPPs. Finally, concentration monosaccharide and concentrationcarbon to nitrogen and (Ccarbon/N) ratio to nitrogen in hydrolysates (C/N) ratio were in measured hydrolysates to evaluate were theirmeasured potential to evaluate as a microbial their potentialculture medium. as a microbial culture medium. Figure 1. SchemesSchemes of of the the enzymatic enzymatic hydrolysis hydrolysis of of exhausted exhausted sugar sugar beet beet pulp pulp pellets pellets (ESBPPs) (ESBPPs) by crude enzyme extracts obtained obtained by by solid-state solid‐state fermentation fermentation (SSF) (SSF) (A ()A and) and by by the the addition addition of fermented of fermented solid solid (B). (B). 2. Materials and Methods 2. Materials and Methods 2.1. Raw Material 2.1. RawFresh Material ESBPPs were obtained from AB Azucarera Iberia (AB Sugar—ABF Group, Jerez de la Frontera, AndalucFreshía, ESBPPs Spain). Sampleswere obtained were collected from AB and Azucarera stored at 4Iberia◦C until (AB use. Sugar—ABF Detergent fiberGroup, analysis Jerez ofde the la Frontera, Andalucía, Spain). Samples were collected and stored at 4 °C until use. Detergent fiber analysis of the solid showed that they are mainly composed of pectin (41.13%), cellulose (25.18%), Foods 2020, 9, 1351 3 of 14 solid showed that they are mainly composed of pectin (41.13%), cellulose (25.18%),