Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes

Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes

6.43 Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes F Molinari and D Romano, University of Milano, Milan, Ital...

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6.43 Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes F Molinari and D Romano, University of Milano, Milan, Italy R Villa, Cranfield University, Cranfield, UK J Clark, University of York, York, UK © 2011 Elsevier B.V. All rights reserved.

6.43.1 Introduction 6.43.2 Extraction and Extraction Techniques 6.43.3 Modification of Carbohydrates 6.43.4 Modification of Lipids (Oils and Fats) and Glycerol 6.43.5 Modification of Proteins 6.43.6 Modification of Phenol Derivatives 6.43.7 Production of D-Glucurono-γ-Lactone from Corn Wastes – A Case Study 6.43.8 Conclusions Acknowledgments References

Glossary biocatalysis The use of biological catalysts (biocatalysts) to perform transformations on organic compounds. Enzymes can be used as catalysts within the whole cell or used as isolated proteins. biorefinery A facility that integrates biomass conversion processes and equipment to produce fuels, power, heat, and fine chemicals from biomass. The biorefinery concept is analogous to today’s petroleum refinery,

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which produces multiple fuels and products from fossil fuel. fine chemicals Pure, single, chemical substances that are commercially produced with chemical reactions for use in highly specialized applications. green chemistry A set of principles and practices involving the use of renewable resources, lowenvironmental-impact chemical manufacturing, and safer and more sustainable products.

6.43.1 Introduction The future success of many biorefineries is likely to depend on obtaining fuel, chemical, and material value from biomass [1]. Agro-food byproducts, residues, and wastes provide a renewable resource not only for energy/fuels and bulk products, but also for the extraction/ preparation of fine chemicals. Molecules with high- or mid-added values can be directly extracted from crude residues or can be prepared after chemical transformations [2]. Biocatalysis is a valuable alternative for the production of chemicals from biomass: Biotransformations are performed under mild conditions and can be highly selective, being therefore suitable for the modifications of mixtures of polyfunctional molecules, such as saccharides, lipids, peptides, proteins, and other naturally occurring molecules [3]. The use of lowenvironmental-impact technologies will help ensure that those products are genuinely and verifiably green and sustainable as it is likely to be required from new and future standards imposed by the major trading regions including the European Union (EU) and the US. Biocatalysis is currently employed for the production of pharmaceuticals or their intermediates (e.g., antibiotics, statins, and enantiomerically pure building blocks), fine chemicals (e.g., amino acids and vitamins), and food products (e.g., sweeteners, lipids, and nutraceuticals) [3]. Stereoselectivity is a key issue in most of the bioprocesses developed for transformations of organic molecules. Processes based on biocatalysis can further broaden their applicability and meet criteria of sustainability if efficiently employed for the transformation of cheaply available agro-food wastes and surplus, such as lignocelluloses, starch, molasses, cheap proteins, and lipids. The application of biocatalysis on an industrial scale is still limited, but the growing importance of enantioselectivity has turned it into a complementary tool for synthetic chemistry. The use of biocatalysis is on the verge of significant growth also due to the impressive advances achieved in the last few years in the fields of protein engineering, bioprocess engineering, and molecular biology. The traditional limitations of biotransformations (low stability, low productivity, low reproducibility, and low activity in organic solvents) can be mostly overcome now by an integrated use of methods such as protein engineering (notably, directed evolution), enzyme and cell immobilization, protein overexpression, membrane reactors, and metabolic engineering. The major limitation of new biocatalytical applications is the time needed to set up a cost-convenient process more than the possibility to achieve it. This article complements the information contained in article 3.28, its focus being on biomass waste and residue biotransformation. Biocatalysis is mostly feasible if a cost-effective extractive method is available for the recovery of molecules from the biomass or its wastes.

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6.43.2 Extraction and Extraction Techniques The extraction of valuable chemicals from biomass could potentially form the initial processing step of many future biorefineries. Although this already represents the first stage of all current oil-bearing crop-processing plants (e.g., rapeseed crushing and solvent extraction) and green biorefineries (e.g., grass pressing), this concept could be extended to the extraction of high-value chemicals from the surface of many types of biomass [4]. Wax products with numerous applications can be extracted from crops and other byproducts including wheat and barley straws, timber residues, and grasses including Miscanthus, using supercritical carbon dioxide (ScCO2) – a green chemical technology that allows the production of products with no solvent residues (Figure 1) [5]. Although biochemical conversion of the bulk biomass can be expected to continue to be the dominant technology in many biorefineries, controlled thermochemical conversion is also starting to play an important role. Pyrolysis is the thermal degradation of biomass in the absence of oxygen [6]. This thermochemical process yields liquid (bio-oil), solid (char), and gaseous products; the relative quantities depend on the method of pyrolysis and operating parameters such as temperature, rate of heating, pressure, and residence time. Currently, most of the industrial interest in pyrolysis focuses on fast pyrolysis (rapid heating rates and very short contact time for biomass (0.5–2 s)), as this results in a large amount of bio-oil (or pyrolysis oil), which can not only be directly used as boiler fuel (or even fired in certain kind of engines) but also be further converted into advanced fuels and added-value chemicals [7]. However, as with biomass gasification, pyrolysis requires a dry feed (water content below 10%). An extra energy-intensive predrying step is therefore needed if wet biomass has to be employed. One potential greener alternative to conventional pretreat­ ment methods, which has been successfully applied in other areas on a large scale and proven to be energy efficient, is microwave irradiation. Microwave heating has recently been used to dry a range of forestry and agricultural residues such as pine-wood sawdust, peanut shell, and maize stalk and is apparently more efficient than heated air drying [8]. More significantly, microwaves are being increasingly used to pyrolyze biomass [9]. This green technology provides direct and uniform heating of the sample compared with conventional thermal sources, which, in turn, allows better control over the heating process and potentially significant energy savings (Figure 2).

Personal care products

Ca. 1−5% Insect repellants Biomass

Wax products

ScCO2 extraction

Healthcare products

- Strawboard - Garden mulch - Pulp and paper - Fermentation Lignocellulose

- Co-firing

Figure 1 Use of benign supercritical carbon dioxide to extract valuable chemical products from biomass.

Microwave Conventional

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Figure 2 Comparison of microwave and conventional heating of biomass.

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Low-temperature activation of biomass can lead to remarkably high-quality oils: by combining continuous extraction with microwave irradiation, in continuous reactors, an aqueous phase containing much of the acids produced along with other problematic components can be continuously separated, leaving the oils cleaner, less acidic, and with lower quantities of other contaminants, such as alkali metals [10]. These oils have great potential as feedstock for making chemical products as well as for blending into transport fuels. Microwave and ultrasound are also additional extraction technologies with great potential for energy efficiency. On a laboratory scale, both methods are now widely accepted as greener alternatives to conventional heating as a result of specific and localized heating effects [11]. There is also strong belief in the ability of microwaves to do more than simply heat a substance – more specific activations of polarizable components is likely and may be exploitable for future chemical production. In addition, a number of companies are now selling large-scale, and, sometimes, continuous equipment, which could be taken to full-scale production by biorefineries. This green technology is faster and leads to less degradation than traditional solvent extraction (i.e., paler, purer, and safer products). Solvent selection is here, again, extremely important as microwave absorbance varies greatly from one solvent to another, for example, supercritical water is totally transparent to microwave while ethanol is highly microwave absorbing [12]. Interestingly, recent studies have also demonstrated that ultrasound can be used to significantly improve extraction rate and yield in ScCO2, and, thus, potentially improve both the energy efficiency and economics of supercritical processes [13]. Solvent-based extractions must be based on environmentally friendly solvents if the final product is to be classifiable as green and sustainable. Many of the organic solvents currently used for extractions are intrinsically hazardous. It is of paramount importance that we seek to avoid solvents with poor environmental, health, and safety properties and actively look for greener alternatives. A number of useful solvent-selection tools are now available to measure solvent greenness and applicability [14]. Most published solvent-selection tools evaluate solvents against key criteria such as health, safety, handling, and recoverability, and, increasingly, environmental impact. They do not, however, take into account the origin of the solvent, that is, whether it is petro- or bio-based. Most organic solvents commonly used for extraction are derived from petroleum and, as such, are not truly sustainable; we need to make better use of the bioresource-derived solvents, which are becoming increasingly available. These include bioethanol, ethyl lactate, 2-methyltetrahydrofuran, isosorbide esters, and γ-valerolactone. Some of these offer new advantages compared to their closest petro-analogs; 2-methyltetrahydrofuran, for example, is safer than tetrahydrofuran, as it is less likely to form peroxides. As energy is such an important issue, we also need to make sure that we employ energy-efficient technologies to extract the chemicals of interest from biomass. ScCO2 is recognized as a green solvent, as it is safe (nonflammable and nontoxic), available as an industrial byproduct (e.g., from fermentation), and gives no solvent residues. It also enables the product extracted to be classified as natural. However, it has substantial energy requirements, which may outweigh, or at least reduce, the green benefits of the solvent. It is possible that its use for the production of many chemicals may be limited to locations close to sources of renewable energy (e.g., the Icelandic Biomass Company, IBC) [15]. There is, however, enormous economy of scale, and very large ScCO2 extractors are likely to be economically viable.

6.43.3 Modification of Carbohydrates Carbohydrates are ideal substrates for biocatalysis. Due to their structure, it is difficult to perform selective transformations using chemical reactions. Different classes of enzymes, mostly glycosyl hydrolases, oxidoreductases, and isomerases, can be used for the modification of sugars [16, 17]. Glycosyl hydrolases are a huge group of fascinating asymmetric biocatalysts widespread in nature. Some of them have also been extensively used for practical purposes. The enzymatic hydrolysis of poly- and oligosaccharides is not only mild and regioselective, but also highly enantioselective. The hydrolysis of glycosides occurs by retaining or inverting the stereocenter involved in the hydrolysis, but always with high stereoselectivity. One of the most common biotransformations of agro-food waste is the hydrolysis of cellulose and starch catalyzed by different hydrolases, producing glucose, disaccharides (cellobiose and maltose), and oligosaccharides (dextrins and cyclodextrins) [18]. Enzymatic hydrolysis of cellulose and starch is used in the fermentation industry to release more easily fermentable sugars, especially for bioethanol, biobutanol, and lactic acid production. The hydrolysis of starch is also employed for the production of glucose, mostly used as a substrate for the production of highfructose corn syrup (HFCS). Fructose is a better sweetener than glucose, giving a lower glycemic response. Isomerization of glucose into fructose is catalyzed by bacterial xylose isomerases from different sources, mainly actinomycetes. The reaction is equilibrium-driven and satisfactory conversions can be obtained only by using relatively high temperatures (55–60 °C). A few technical hurdles have been overcome for scaling up the bioprocess to industrial scale: Thermostability of the enzymes’ binding ability toward magnesium for avoiding the necessity of removing the ion in the downstream process, reaction rates, and setup of bioreactors with immobilized biocatalysts. Screening, protein engineering, and efficient procedures of immo­ bilization have been developed by different companies for obtaining stable biocatalysts employable on industrial scale [19]. Other sweeteners have been obtained by the biocatalytical modification of largely available substrates from agro-food wastes and surplus, such as sucrose from molasses. Large amounts of molasses, which can be directly commercialized or further transformed, are now available as a consequence of improved technologies for their concentration and partial purification. Fructooligosaccharides (FOSs) consist of a mixture of fructose oligomers with one or more fructose units bound to the β-2,1

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position of sucrose; the main components are 1-kestose, 1-nystose, and 1-fructofuranosyl nystose; FOSs are interesting alternative sweeteners (they are calorie free, noncariogenic, and stimulate the growth of bifidobacteria) and can be produced using sucrose obtained from molasses [16]. Sucrose can be converted into FOSs by transfructosylation catalyzed by β-fructofuranosidases (FFases, EC 3.2.1.26) and/or fructosyltransferases (FTases, EC 2.4.1.9). Although FFases and FTases are widespread in the plant world, the industrial enzymes are generally microbial, mostly from fungi (Aspergillus, Penicillium, and Aureobasidium spp.). FFases are able to catalyze the hydrolysis of FOS and, under thermodynamically favorable conditions, the reaction can be reversed furnishing FOSs, while FTAses should possess only transfructosylating activity. Maximal FOSs production depends on the relative rates of transfructosylation/hydrolysis reactions; the use of an immobilized enzyme from A. aculeatus allowed for a 61.5% (w/w) conversion of FOSs starting from 630 g l−1 of sucrose, with a ratio of kestose/nystose/neokestose of 6.2/3.7/0.1. Similar results have been reported with other microbial enzymes, indicating that the thermodynamic of the reaction plays a crucial role. Palatinose (also known as Palatinit®, or isolmaltulose, or isomalt) is a sweetener with low insulin stimulation and low cariogenic potential. It naturally occurs in low amounts in honey and sugarcane extract and can be obtained by the action of a glucosylmutase on sucrose (Figure 3) (see Reference 17 and references therein). A stable and enantioselective glucosylmutase has been found as cell-bound enzyme in Protaminobacter rubrum. A process has been set up by Südzucker AG (Germany) which employs immobilized whole cells of P. rubrum with 65% yields and 99% chemical purity of palatinose recovered after selective crystallization. The controlled enzymatic hydrolysis of starch produces commercially valuable compounds, such as maltodextrins, branched dextrins, and cyclodextrins, depending on the enzymes employed. Maltodextrins are food ingredients composed of a mixture of oligosaccharides classified by their degree of hydrolysis expressed as dextrose equivalent (DE), and traditionally produced by the action of thermostable α-amylases by controlling the hydrolysis until the desired degree is attained [20]. Maltodextrins with high DE have been found to be unpleasant to consume in large quantities because of their sweet tastes and their tendency to remain in the stomach after consumption rather than being absorbed, producing a feeling of nausea. Low-DE maltodextrins are usually more stable, less sweet, and find better applications in the food and drink industry as fat replacers, lyoprotectants, flavor carriers, bulking or gelling agents, thickeners in dairy products, and so on. Low-DE maltodextrins are produced by waxy corn, potato and tapioca starch, and, more recently, from cereal starch. Recently, Syral developed a process based on the use of a branching enzyme from Rhodothermus sp., which catalyzes the formation of new 1,6-α-linkage forming maltodextrins with high proportion of DE 8; the final product is particularly suited for many food applications in the food industry. Exaki Glico Co. developed an industrial process for the production of branched cyclic dextrin (Cluster Dextrin®) from amylopectin using branching enzymes (BEs, 1,4-α-D-glucans, EC 2.4.1.18). BEs are glucan transferases responsible for the synthesis of α-1,6-glucosidic bonds in starch and glycogen in vivo; a thermostable BE from Bacillus stearothermophilus has been used industrially for starch processing to synthesize Cluster Dextrins®, a new type of dextrin highly soluble in water and highly stable during storage [21]. Cluster Dextrins® has a narrow molecular-weight distribution and relatively long side chains compared with conventional dextrins and can be used as a food ingredient for many applications, including reduction of acidity, a spray-drying aid, and an alternative sweetener in sport drinks. Cyclodextrins (CDs) are produced from starch by biotransformation (Figure 4). Since the first report of a Bacillus macerans strain capable of producing CDs from starch, many bacterial cyclomaltodextrin glucanotransferases (CGTases) have been purified and characterized, with similarity in their amino acid sequences ranging from 47% to 99% [22]. The catalytic mechanism of CGTase is an α-retaining double-displacement mechanism, which resembles that of glycosyl hydrolases, but, in this case, a carbohydrate acts as the nucleophile instead of water. The main problems encountered in the development of the bioprocess have been mostly the formation of CDs as a mixture of α-, β-, and γ-cyclic oligosaccharides and the enzymatic inhibition caused by increasing concentrations of CDs in the reaction mixture. Both the problems have been circumvented by using adsorption columns, which remove the CDs produced. The separation is achieved by selective adsorption of the different CDs on chitosan beads with suitable ligands (α-CDs are selectively adsorbed on stearic acid-derivatized chitosan, while β-CDs selectively interact with chitosan treated with cyclohexanpropana­ mide-caproic acid).

Figure 3 Biotransformation of sucrose into palatinose.

Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes

( )n

( )n−6

Glycosyltransferase

+

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OH

OH

OH

O

Starch

O O HO

HO OH

OH

OH O HO HO

O OH

HO

OH

HO

OH O HO

O OH

OH HO O

OH

Figure 4 Biotransformation of starch into cyclodextrins.

Lactose is largely available as cheap substrate, being the major component of whey, and it has been estimated that more than 1 billion ton (dry weight) of lactose is discarded yearly from dairy industries. Different biotransformations have been studied for a commercially significant valorization of lactose, such as its hydrolysis [23], and its conversion into galacto-oligosaccharides [24] or lactobionic acid (oxidation) [25]; other bioconversions of lactose have been devised, but not developed yet, such as its reduction to lactitol and its direct isomerization to lactulose. Most of these transformations are hardly feasible on a large scale by conventional chemical means.

6.43.4 Modification of Lipids (Oils and Fats) and Glycerol The global production of oils and fats from renewable (agri-food) sources reached 160 million tonnes in 2008–09. This figure represented an increase of approximately 3% over the previous year, mainly due to biodiesel production [26]. Approximately 15% of the total production is currently used by the chemical and biotechnology industries for further conversion to green chemical products [27, 28]. Different classes of enzymes, such as lipases, esterases, phospholipases, lipooxygenases, and monooxygenases, can be used for the selective transformation of oils and fats. A number of these applications are industrially exploited using either commercially available enzymes or whole cell microbial systems. In addition, intermediate products derived from oil purification processes can be used to extract byproducts such as tocopherols or sterols. For example, tocopherols and sterols can be extracted from soybean-oil steamer distillate, and from lecithin after degumming, or from crude tall oil [29]. These products are added as dietary supplements to food preparations, and are often the basis for manufacturing nutraceutical or functional food. Oil hydration is a crucial transformation as long as it is regio- and stereoselective. A Pseudomonas species, isolated from fatcontaining material, has been observed to hydrate oleic acid to (R)-10-hydroxystearic acid with a yield of 14% [30]. This hydration ability was also observed in other bacterial genera, yet, low yields and protein availability have limited the exploitation of this reaction. The production of dicarboxylic acids via microbial ω-oxidation of fatty acids is also of interest. For example, oleic acid has been converted into the corresponding dicarboxylic acid using Candida tropicalis, with yields up to 50%. Similarly, hexadecanedioic acid has been produced from palmitic acid in batch fermentation with a yield of 35% [31]. Lipases are the most exploited catalysts for fat and oil transformations in the food, cosmetic, pharmaceutical, leather, and detergent industries [28, 31]. They can modify ester bonds and also catalyze nonnatural reactions including the transformation of hydrophilic polyoil and amine-based compounds, the peroxidation of fatty acids, and the production of polyesters. Lipases do not require cofactors, are easily immobilized on different carrier materials, and have a high degree of activity and stability even in nonaqueous media. They can also be chemo-, regio-, and stereoselective. All these properties are potentially useful for biotechnological applications. Lipases have been extensively used for the transesterification of triacylglycerols. One of the most exploited reactions is the industrial production of cocoa butter equivalents. Cocoa butter is mainly composed of 2-oleyl glycerides of palmitic and stearic acids (P/S-O-P/S). Cheap commercial oils, which have triglycerides with oleic acid in the sn2 position, can be converted to cocoa butter equivalent. Palm oil is a low-cost fat with a chemical composition similar to cocoa butter. Its mid-fraction is rich in palmitic–oleic–palmitic (POP) triacylglycerol and can be converted into cocoa butter by acidolysis catalyzed by chemo­ and regiospecific lipases able to introduce stearic acid in the sn1 and sn3 positions. The biotransformation can be carried out in solvent-free medium, where palm oil is used in large excess [28, 31, 32].

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The chemo- and regioselective hydrolysis of triacylglycerols has been exploited for the enrichment of specific fatty acids, such as polyunsaturated fatty acids (PUFAs) from fish oils. The possibility of using lipases in low-water environments has enabled their use in the production of structured triglycerides (sTAGs) by enzymatic inter- and transesterifications. sTAGs are important compounds for a range of applications in human nutrition. For example, sTAG containing long polyunsaturated acids in the sn1 and sn3 positions are used to treat patients with pancreatic insufficiency and to give rapid energy supply. sTAG is also exploited in the manufacture of Betapol® and used for infant nutrition. This sTAG contains oleic acid in the sn1 and sn3 positions and palmitic acid in the sn2 position. It is derived from tripalmitin via alcoholysis with ethanol, using a lipase from Rhizopus oryzae, which is able to remove only the fatty acids in positions 1 and 3. The resulting 2-mono-palmitin (95%) can be esterified with oleic acid, using the Rhizomucor miehei lipase in organic solvent (Figure 5) [28, 31, 32]. Lipases can also be used on an industrial scale to produce simple esters, such as cetyl ricinoleate and myristyl myristate, which are used in cosmetic applications [33]. Alkylpolyglucosides are alternative nonionic surfactants and emulsifiers derived from lipase-catalyzed synthesis of carbohydrate esters of fatty acids. Extensive work and comprehensive reviews on the production and applications of other microbially catalyzed biosurfactants can also be found in the literature [34]. Polyesters based on fatty acids are potentially useful as lubricants or in cosmetic products and can be synthesized using lipase-catalyzed processes. Commercially available phospolipases are used for degumming crude oils and for the modification of food lecithins, and specifically structured phospholipids, used for human health, are also produced using a commercially available phospholipase [35]. Lipases (e.g., from P. cepacia and C. antarctica) can also be employed for the production of biodiesel by interesterification of triacylglycerols (mostly from soy oil) with MeOH [36, 37]. Although the process is well understood, the enzymatic production of biodiesel is still not cost-effective. One of the problems is linked to the toxicity of the methanol for the enzyme, which can be overcome by sequential methanol addition; this method has become widely employed [38]. The use of waste fats and oils is limited by the high quantity of free fatty acids, which makes the use of conventional catalysts too costly, while the combined use of two enzymes (Lipozyme TL IM and Novozym 435) allowed for a 95% transesterification under optimal conditions [39]. The application of lipases should also allow the synthesis of biodiesel from raw materials such as animal fats, recycled oils from restaurants, and waste products and materials that cannot be used for human consumption. The European production of biodiesel in 2008 reached about 8 million tonnes. As biodiesel production results in about 10% glycerol byproduct, this implies that approximately 800 000 tonnes of glycerol is produced every year. The glycerol produced during the manufacture of biodiesel has different degrees of purity, depending on the process conditions and the starting feedstock material. This is particularly true for glycerol derived from used vegetable oils or fats. This has a lower market price than glycerol from virgin oil because it is more difficult to transform both chemically and biologically, making its use particularly challenging. The glycerol glut has stimulated research into new value-added derivatives, using either traditional chemical or biotechnological processes [40, 41]. Dow has recently announced a plan to build large production units for the chemical manufacture of 1,2-propanediol (PD) and epichlorohydrin from glycerol in China, while a similar plant opened in Germany in 2006 [42]. In microbial cells, glycerol is formed as an intermediate in both aerobic and anaerobic catabolism of lipids and glucose. Notwithstanding this, the commercial conversion of glycerol (when used as the sole carbon source) into value-added compounds is limited to a few microbial genera and a few chemical building blocks (1,3-PD and dihydroxyacetone). Glycerol is also used as substrate for industrial fermentations aimed at the production of citric acid, succinic acid, and propionic acid. Dihydroxyacetone is produced industrially by Merck KGaA through the selective microbial oxidation of the 2-OH group of glycerol with Gluconobacter oxydans [43]. It is mainly used as a chemical intermediate and as a tanning agent in the cosmetic industry. The process is characterized by a strong substrate and product inhibition. These limitations have been overcome successfully on a lab scale using semi-continuous batch-fed fermentation and/or immobilized cells [44]. 1,3-PD is an emerging commodity chemical, which is used as a building block for polyester synthesis such as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) fibers for textiles. Strains of Clostridium, Klebsiella, and Citrobacter are able to produce 1,3-PD from glycerol, with C. butyricum generally showing the highest productivities. The biosynthesis consists of a dehydration step to 3-hydroxypropanal, followed by a reduction to the diol. Concentrations of 100 g l−1 of 1,3-PD can be obtained using crude glycerol as a feedstock with wild-type strains of Clostridium in an integrated fermentation process [45]. Nevertheless, today, the majority of the biologically produced 1,3-PD is derived from sugar. Both Shell and DuPont have developed commercially viable polymers called Corterra® and Sorona®, respectively, obtained from 1,3-PD. However, only the DuPont process is microbially mediated and 1,3-PD is obtained by fermentation from glucose using a genetically engineered E. coli strain [46].

O CO P O CO P O CO

P

O CO O

OH

Rhizopus oryzae lipase

Rhizomucor miehei lipase O CO P OH

Tripalmitin Figure 5 Enzymatic transformations of tripalmitin for the production of Betapol®.

O CO P

Hexane O CO O

Betapol

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Glycerol can be used by microorganisms as a carbon source for surfactant production in two ways: As precursor of the surfactant carbohydrate backbone or as an energy source. Two major categories of glycerol-derived biosurfactants (sophor­ olipids and rhamnolipids) can be synthesized by C. bombicola and Pseudomonas spp., respectively. C. bombicola is known to produce sophorolipids from a variety of saccharic and lipidic feedstocks. Felse et al. [47] identified tallow fatty as the best substrate from industrial waste for surfactant production, reaching 120 g l−1 of sophorolipids with fed-batch cultures of C. bombicola. Ashby et al. obtained a sixfold increase to 60 g l−1in the biosurfactant yield when waste glycerol (40% glycerol) from biodiesel production was used with the same bacteria [48]. Although the main industrial application of glycerol is currently in the production of 1,3-PD and dihydroxyacetone, the cost of the substrate and an increased interest recently in using renewable resources and green chemistry make glycerol a very versatile carbon and energy source with many potential uses in the biotechnological sector. In addition, the development of new processes for converting inexpensive waste glycerol into higher-value products has the potential of making biodiesel production more sustainable and economical. Simple derivatives of glycerol can also be used as chiral multipurpose building blocks. 2,2-Dimethyl-[1, 3]-dioxolane-4-methyl acetate (also called 1,2-O-isopropylideneglycerol, IPG or solketal) is a chiral molecule easily synthesized by the reaction of glycerol with acetone; optically pure solketal can be obtained by different means, including oxidation of racemic solketal or enantio­ selective hydrolysis of its esters [17, 49].

6.43.5 Modification of Proteins Physiologically active peptides are produced from several food proteins during gastrointestinal digestion or by fermentation with lactic acid bacteria; their activity is connected to their size and amino acid sequence. Milk proteins are considered to be the most important source of bioactive peptides; milk hydrolysates, containing biopeptides, have been claimed to have many beneficial activities [50]. Biopeptides can be obtained using commercial preparations of microbial and plant proteases, whole microbial cells (in fermented milk), and enzymatic extracts. Effective angiotensin-converting enzyme (ACE)-inhibitor peptides VPP and IPP, used in the treatment of hypertension, have been produced by the fermentation of milk caseins with Lactobacillus helveticus [51]. Generally, competitive inhibitors of ACE contain hydrophobic amino acids, especially proline, at the C-terminus [50]. New proteases are needed for the setup of highly productive bioprocesses to obtain useful biopeptides from milk proteins. Natural amino acids obtained by protein hydrolysis or by fermentation can be further modified by chemoenzymatic methods to obtain molecules of commercial interest. Phenylalanine and aspartic acid can be chemically modified for produ­ cing N-protected and O-protected aminoacids (Phe-OMe and Asp-Z, respectively); Phe-OMe and Asp-Z are finally coupled using thermolysin, a bacterial neutral zinc protease, to produce Aspartame®. The equilibrium-controlled reaction has been shifted toward Aspartame® formation by precipitation of the product caused by complexation achieved using an excess of Phe-OMe [52].

6.43.6 Modification of Phenol Derivatives Lignin accounts for about 30% of the weight and 40% of the fuel value of biomass. Lignin, or lignin process residues, can always be used for process power through combustion. However, new opportunities afforded by technology development (e.g., biocatalysis) can overall lead to much higher value products. Plant cells are attractive substrates for industrial (bio)transformations; xylan, in particular, is one of the most interesting building blocks, being the major constituent of hemicellulose. Its enzymatic degradation is carried out using a cocktail of enzymes (endoxylanases, acetyl-xylan esterases, feruloyl esterases, and α-D-glucuronidases) to release sugars and hydroxycinnamic acids. A large number of hypothetical structures might be available from lignin conversion; particularly, phenol derivatives could be derived from the guaiacyl and syringyl units present in lignin [53]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is the most abundant, and it is generally found ester-linked to arabinose. Ferulic acid obtained after extraction of the lignocellulose hydrolysate is used as an antioxidant and a dietary supplement or as a precursor in the manufacture of other aromatic compounds. The production of vanillin via microbial bioconversion of substrates such as ferulic acid is a feasible alternative way of obtaining vanillin; these alternative ways have gained interest in the last few years as vanillin thus produced can be labeled as natural by the European and US legislation [54]. Ferulic acid derived from sugar beet pulp can be used as a precursor in a two-step bioprocess by employing Aspergillus niger and Pycnoporus cinnabarinus. A. niger is able to convert ferulic acid by removal of CH3COSCoA by β-oxidation (pathway in red); the formation of vanillin is only transient as it is further oxidized to vanillic acid. Vanillic acid is then reduced using a specific carboxylase reductase from P. cinnabarinus. The overall yield is 760 mg l−1 over a period of 15 days. Actinomycetes are known for their ability to efficiently convert ferulic acid into vanillin. Streptomyces setonii gave good conversions (68%) and high yields (6.4 g l−1) in the direct conversion to vanillin. Higher yields (11.5 g l−1) were obtained using strains belonging to the genus Amycolatopsis. Other bacteria also possess enzymes able to convert ferulic acid into vanillin: the genes involved in the catabolism of ferulic acid in P. fluorescens BF13 were characterized, integrated into the E. coli chromosome, and stably maintained in nonselective culture conditions (Figure 6).

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Figure 6 Bioconversion of ferulic acid by Pseudomonas fluorescens BF13.

A brilliant example of efficient recovery/biotransformation of ferulic acid from wheat bran was recently reported. Wheat bran contains a large amount of ferulic acid, which can be recovered after enzymatic hydrolysis using a hydrophobic sorbent, which also allowed an attractive vanillin molar yield from ferulic acid bioconversion (75% from 0.5 mmol l−1 ferulic acid). Moreover, the residual aqueous phase was a good growth substrate for the microorganism operating the bioconversion [55]. These bioprocesses are hardly competitive with petroleum-based technology. Volatility in petroleum feedstock prices and new lignin separation and conversion technology could have an impact on the economic viability of small-molecular-weight products from future biorefineries.

6.43.7 Production of D-Glucurono-γ-Lactone from Corn Wastes – A Case Study D-Glucurono-γ-lactone

(also named glucurono-3,6-lactone or GGL) is a commercially valuable molecule supposed to have a number of positive health effects, including promoting joint health, providing an anti-inflammatory effect for the skin, and lowering abnormally high plasma concentrations of cholesterol or triglycerides. GGL is produced by a process involving the nitric acid oxidation of starch into glucuronic acid. This process may use up to 0.5 kg of concentrated nitric acid per kg of GGL produced, with significant associated environmental and safety risks. Cargill has recently introduced a biocatalytic method for producing GGL starting from myo-inositol, a substrate easily available by hydrolysis of phytic acid, which is largely present in corn wastes (Figure 7) [56]. Myo-inositol is converted into glucuronic acid by the action of a myo-inositol oxygenase (MIO). Suitable cell-bound MIOs were found in various yeasts and moulds. MIO from Cryptococcus terreus has been overexpressed into K-12 E. coli, and whole cells of the recombinant strain are used in a highly aerated batch reactor for the conversion of myo-inositol (up to 20 g l−1) with average molar conversions of 85%.

Figure 7 Biocatalytical route to D-glucurono-γ-lactone from phytic acid.

Production of Fine Chemicals by (Bio)Transformation of Agro-Food Byproducts and Wastes

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6.43.8 Conclusions The use of biocatalysis for converting agro-food byproducts and wastes into commercially valuable molecules deserves interest and perspectives. Although the examples of biocatalytical processes applied on an industrial scale are still scarce, the impact of modern techniques (such as molecular biology, high throughput screening, protein engineering, and bioprocess engineering) can have a dramatic effect on the future development of these biotransformations. The production of optically pure molecules is one of the main issues. The so-called chiral pool can be largely increased by the use of biotransformations of agro-food wastes and surplus.

Acknowledgmentss We would like to thank the British Council for providing support with a British-Italian partnership travel grant.

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