Sugar beet pulp (SBP) is a common agricultural residue in different areas of the world as 20% of the world production of sugar comes from sugar beets (Stevanato et al. 2019). Sugar beets are cultivated in Europe, North and South America, Asia, and a few countries in Africa where temperate climates exist. The top producer is the Russian Federation, followed by France, the United States, Germany, and Turkey. In Africa, Egypt is the leading country in sugar beet cultivation and the 8th top producer in the world. After sugar extraction, SBP residue is rich in some carbohydrate polymers and other minor components. The chemical composition of SBP is approximately 21–30% pectin, 22–25% cellulose, 22–25% hemicelluloses, 5–10% protein, lignin < 5%, phenolics < 1%, and ash < 1% (Fishman et al. 2011; Li et al. 2014).
Indeed, the cell wall structure of sugar beet is quite unique and different from those of agricultural residues such bagasse, rice straw, etc. Most of the tissue in sugar beet is parenchymal, which is separated by vascular tissue (Dinand et al. 1999). The parenchymal tissue is characterized by only primary wall of very thin thickness (as low as 0.1 µm). The inter-vascular parenchyma consists of arrays of ovoid cells having diameters ranging from 50 to 200 µm. Other parenchymal cells also exist in the vascular tissue, so called phloem parenchyma, and characterized by very thin wall with an elongated structure, with diameter of 0.4 µm and lengths of around 100 µm. Similar to the primary walls in different plants, the thin walls of the different parenchymal cells contain loosely organized cellulose microfibrils embedded in a matrix of hemicelluloses and pectin. The cellulose microfibrils are either isolated with about 3 nm diameter or organized in thin bundles with a limited number of parallel microfibrils (Dinand et al. 1999). This loose microfibrillar organization in the matrix facilitates separation of cellulose microfibrils by mechanical action.
Regarding hemicelluloses in SBP, they consist of two main polysaccharides xylan and xyloglucan (Kato and Kobayashi, 2000). The xylan polymer is a linear xylopyranosyl backbone with side chains of pentoses and hexoses sugars such as rhamnose, arabinose, glucose, galactose, and glucouronic sugars. Xylan part represents about 89% hemicelluloses in sugar beet pulp. On the other hand, the xyloglucan polymer consists of glucosyl backbone with side chains of xylopyranosyl, galactopyranosyl -xylopyranosyl, and fucopyranosyl-galactopyranosyl- xylopyranosyl sugar residues. Pectin consists of two main structural elements, homogalacturonan (HG) and rhamnogalacturonan (RG). HG and RG elements are linked to each other's via different kinds of bonds (Vincken et al. 2003; Schols et al. 2009). The HG consists of linear chains of d-galacturonic acid units, which are partially acetyl-esterified and methyl-esterified at O-2 and/or O-3 and at O-6, respectively (Combo et al. 2013). On the other hand, RG consists of rhamnose and galacturonic acid units arranged in alternating fashion. The rhamnose units are anchored with linear galactan and branched arabinan sugars. In addition to the polysaccharides in pectin structure, protein and ferulic acid residues exist linked to the arabinan and galactan side chains of the RG elements (Levigne et al. 2002; Siew and Williams, 2008).
In such a uniquely complicated cell wall structure of SBP, hemicelluloses are linked to pectin via formation of ester bonds with the hydroxyl and carboxylic groups of the later (Fischer et al. 1994). Cellulose microfibrils on the other hand are linked to pectin in the cell wall in similar manner to that with hemicelluloses (Zykwinska et al. 2005; Zykwinska et al. 2007).
For efficient use of SBP residue, industrially valuable polymers such as pectin and cellulose should be isolated using optimized protocols. Pectin extraction from the cell wall of sugar beet requires breaking the bonds between cellulose, hemicellulases, and pectin. Although hydrolysis using mineral acids is a common industrial method for pectin extraction, other methods, such as enzymatic hydrolysis, have also been studied (Zykwinska et al. 2008; Concha et al. 2013; Babbar et al. 2016; Pacheco et al. 2019; Abou-Elseoud et al. 2021). In addition to being a more environmentally sound approach than mineral acid hydrolysis, enzymatic hydrolysis was also found to be effective in terms of pectin yield in these previous studies. After pectin extraction, the de-pectinated SBP becomes enriched with cellulose fibers, which can be used to isolate nanocellulose (cellulose nanofibers and nanocrystals), a substance known to have interesting mechanical, optical, and physical properties, making them applicable in several areas (Thomas et al. 2018). The isolation of CNFs from de-pectinated SBP has been studied in past years. In these studies, the main protocol was to first remove pectin from SBP by alkali treatment, then remove lignin by bleaching, and finally isolate CNFs from the purified pulp using high-pressure homogenizers (Leitner et al. 2007; Li et al. 2014; Pinkl et al. 2017). The use of mixtures of enzymes (pectinases, hemicellulases, amylase, and endo-glucanase) to purify the cellulose fraction from residual pectin after alkali treatment and bleaching of SBP was also studied. The purified pulp was then subjected to high-pressure homogenization by a microfluidizer to produce CNFs (Holland et al. 2019; Perzon et al. 2020). In a slightly different protocol to isolate CNFs, SBP was first treated with hot nitric acid solution three times, alkali treated three times, bleached with sodium chlorite/acetic acid, treated with ultrasonication, and finally subjected to high-pressure homogenization (Agoda-Tandjawa et al. 2010). CNFs from SBP were also isolated without the removal of pectin or other polysaccharides; only a bleaching step with sodium chlorite/acetic acid was carried out, and the CNFs were isolated by high-pressure homogenization using a microfluidizer (Hietala et al. 2017); the isolated nanofibers contained significant amounts of pectin, which gave them high re-dispersion properties in water. CNFs from SBP were also isolated without chemical treatment by direct grinding using an ultrafine grinder followed by high-pressure homogenization (Vartiainen et al. 2015). Using a different approach, SBP was subjected to steam explosion pretreatment before bleaching and ultrasonic treatment to obtain CNFs (Yang et al. 2018).
Despite successful isolation of CNFs from SBP in the abovementioned studies, full use of the major components of SBP in industry, e.g., pectin and cellulose, is necessary for economic reasons to expand the traditional use of SBP for animal feed alone. Therefore, optimized isolation of pectin before using the cellulose fraction is mandatory. In addition, because different methods can be used for pectin extraction, the effect of these methods on the properties of isolated CNFs needs to be studied.
The aim of the current work was to study the differences in properties of CNFs isolated from SBP residues by first optimizing the pectin isolation using the conventional industrially-practiced sulfuric acid hydrolysis or enzymatic treatment with a mixture of xylanase/cellulase enzymes, and then using the de-pectinated SBP to isolate the CNFs. The study also focused on the progression of fibrillation using acid- and enzymatic-hydrolyzed SBP, the re-dispersion of the dried nanofibers, and the properties of CNF films prepared by casting.