CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) Microbial Xylitol Production from Corn Cobs Xylitol, a five-carbon sugar

alcohol, has attracted much attention because of its use as a natural food sweetener, a dental caries reducer, and a sugar substitute for diabetics. Xylitol has found many applications in the food and drink industries. Conventionally, xylitol is produced via chemical reduction of wood hydrolysates. This process is energy intensive and not environmental friendly. Therefore alternative routes have been researched; one of them is going to be described in this paper. The alternative route being described here uses an agricultural residue, i.e. corn cob, via fermentation using various microorganisms. [Key words: microbial, xylitol, corn cobs]

Introduction Since the 1960, xylitol (a sugar alcohol or polyol) has been used as a food additive and sweetening agent. It is produced commercially and is used in some food because of a number of advantageous natural properties. It is naturally found in many fruit and vegetables [1, 2], and it therefore has always been a natural component of human diet (although it is usually in a very small amount, i.e. < 1%). Man also produces a small amount of xylitol (5-15 g/day) during normal carbohydrate metabolism in the liver [3]. Xylitol, C5H12O5, is a five carbon polyol (pentitol) and is the sweetest of all of the polyols [4]. By far xylitol is the only polyol to exhibit a sweetness intensity equivalent to that of sucrose; xylitol is equisweet to sucrose at concentrations of 10% solids, and is reported to be 20% sweeter than sucrose at a concentration of 20% [5]. Xylitol has a lower caloric value compared to other sugars, i.e. approximately half that of glucose (2.4 kcal/g) [6]. The relatively low caloric value puts xylitol into the group of sweeteners that are safe for diabetics. Following the ingestion of xylitol, the blood glucose and serum insulin responses are significantly lower than those following glucose or sucrose ingestion [7]. This finding is linked to xylitols relatively poor absorption and metabolism.

The most significant property of xylitol, however, is its anticariogenic property as a sweetener, i.e. xylitol can promote oral health and prevent caries. Hence the bulk of xylitol production is used in various food products such as chewing gum, sweets, soft drinks and ice cream. It gives a pleasant cool and fresh sensation due to its high negative heat of solution. Increases in public awareness of health issues also ensured that substances like xylitol are attracting growing attention [8]. Production of xylitol Although xylitol is found naturally in certain fruits and vegetables, its concentration is so low (< 1%) that producing xylitol by extraction from these natural sources is difficult and not economical [6]. Hence, much research has been focused on the production of xylitol by chemical or biological means. Xylitol is produced by reducing xylose, which is derived mainly from hemicellulose hydrolysates of wood or other xylose rich materials. Hemicellulose is a heterogeneous polymer of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids. These sugars are released when hemicellulose is hydrolyzed. There are a number of ways to hydrolyze hemicellulose, i.e. using acid (concentrated [9] or dilute acid [10]), alkaline [11], SO2 [12], hydrogen [13] peroxide , steam explosion (autohydrolysis) [14], ammonia fiber explosion 1

CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) (AFEX) [15], wet-oxidation [16], lime [17], liquid hot water [18], CO2 explosion [19], and organic solvent treatments [20]. The reduction of xylose to xylitol can be done chemically or biologically. In a chemical reduction, xylose is reduced to xylitol using Ni/Al2O3 (Raney-Nickel catalyst). Biologically, xylose is fermented to xylitol by various microorganisms. Chemical versus biological process On an industrial scale, xylitol is currently produced by chemical reduction of xylose derived from wood hydrolysates. Xylose is chemically hydrogenated to xylitol using Ni/Al2O3 catalyst. The productivity of xylitol in a continuously operating apparatus, with recycling of solvents and reagents, can be as high 75 kg h-1. The conventional process of xylitol production includes four main steps: acid hydrolysis of biomass, purification of the hydrolysate to either a pure xylose solution or a pure crystalline xylose, hydrogenation of the xylose to xylitol, and crystallization of the xylitol [21]. The purification of the xylose is a critical step in the conventional process. The hemicellulose hydrolysate contains appreciable amounts of polymers of other sugars, salts, degradation products (furfural and hydroxymethyl furfural), and also color. The salts, degradation products and color can poison the catalyst, while the contaminating sugars can complicate crystallization and purification of xylose. Ion exchange chromatography is employed to remove salts and degradation products, and activated carbon is used to remove color [22]. Because of the high purity of xylose required, the chemical process is expensive. Moreover, the chemical process requires high temperature (80-140oC), high pressure (up to 50 atm), and expensive catalyst, making operational costs relatively high compared to that of biological process. According to Kim et al. (US Patent No. 5998181), the yield of xylitol in the chemical process is only 50-60%, while the biological process can give a yield from 80 to 97%. The high pollution levels and waste generated from the chemical process also adds to the drawbacks of the chemical process compared to the biological one. Pretreatment of hemicellulose Hemicellulose is usually found together with cellulose and lignin, and together they comprise lignocellulosic biomass. Lignocellulosic biomass includes various agricultural residues (straws, hulls, stems, stalks, etc), deciduous and coniferous woods, municipal solid wastes, waste from the pulp and paper industry, and herbaceous energy crops. The compositions of these materials vary. The major component is cellulose (35-50%), hemicellulose (20-35%) and lignin (10-25%) [23]. The pretreatment of hemicellulose is crucial before chemical reduction or fermentation. Pretreatment of hemicellulose comprises of hydrolysis of lignocellulosic biomass and treatment of the hydrolysate to purify the xylose solution. Various pretreatment options are available to fractionate, solubilize, hydrolyze and separate cellulose, hemicellulose, and lignin components. In each option, the biomass is reduced in size and its physical structure is opened. The use of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar constituents is well known and effective [24, 25]. Two categories of dilute acid hydrolysis are used: high temperature (>160oC) continuous-flow for low loading (5-10%, w/w) and low temperature (<160oC) batch process for high solids loading (10-40% w/w) [26]. Dilute acid pretreatment at high temperature usually hydrolyzes hemicellulose to its sugars (xylose, arabinose and other sugars), which are water soluble. Hemicellulose, being an open structure, facilitates diffusions of acid into the polymer and speeds up its hydrolysis. 2

CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) The residue contains cellulose and often much of the lignin. The lignin can be extracted with solvents such as ethanol, butanol, or formic acid. Further hydrolysis can release watersoluble sugars from cellulose. However, further hydrolysis tends to generate phenolic compounds from lignin degradation, furan derivatives (furfural and hydroxymethyl furfural) from sugar degradation, and aliphatic acids (acetic acid, formic acid, and levulinic acid). These compounds are considered to be fermentation inhibitors [27].; The hydrolysate is then subjected to ion exchange chromatography to remove salts and degradation products, and activated carbon to remove color. To obtain higher xylitol productivity, treated hydrolysates are concentrated by vacuum evaporation to provide higher initial concentration of xylose [28]. The significance of using corn cobs Corn cob is an agricultural lignocellulosic material that can be fractionated to separate cellulose, hemicellulose and lignin in separate streams. As an agricultural residue, corn cob promises to be a cheap feedstock for xylitol production; corn cob has a hemicellulose content as high as 41.4% dry matter [29]. Among various agricultural residues, corn cobs are regarded as promising agricultural resource for microbial xylitol production because corn is widely cultivated, and corn cobs are rich in hemicellulose but are not effectively utilized [30] . Microbial production of xylitol Production of xylitol by fermentation is becoming more attractive because of the problems associated with its chemical production. Although microorganisms would assimilate and ferment glucose more readily than they would xylose, there are bacteria, yeasts and fungi capable of assimilating and fermenting xylose to xylitol, ethanol and other compounds [31]. 1. Yeasts Many yeasts and mycelia fungi possess NADPH-dependent xylose reductase (EC 1.1.1.21), which catalyzes the reduction of xylose to xylitol as a first step in xylose metabolism [32]. Xylitol can be subsequently oxidized to xylulose by the action of xylitol dehydrogenase, which preferentially uses NAD as an acceptor [33]. In xylose-fermenting yeasts, the initial reactions of xylose metabolism appear to be rate-limiting [34]. This causes various degrees of xylitol accumulation in the medium, depending on the culture condition and the yeast strain used. Transient oxygen limitation would cause a surplus of NADH, which would in turn result in xylitol accumulation [35]. Some natural xylosefermenting yeasts known to produce xylitol are: Candida boidini, Candida guillermondii, Candida tropicalis, Candida parapsilosis, and Debaryomuces hansenii [36]. Winkelhausen and Kuzmanova [22] concluded that the best xylitol producers belong to the genus Candida. Figure 1 shows schematic representation of Dxylose metabolism in yeasts. 2. Bacteria A few bacteria such as Corynebacterium sp. [37], Enterobacter [38] liquefaciens , and Mycobacterium smegmatis [39] have been reported to produce xylitol. The first two bacteria use D-xylose as a substrate and produce xylitol, while for the last one the substrate was D-xylulose (D-xylose isomerized by commercially immobilized D-xylose isomerase). Izumori and Tuzaki [39] found that M. smegmatis (either as washed or as immobilized cells) converts xylulose to xylitol at the same rate, with a yield of 70%. They also studied the conversion of D-xylose to xylitol using commercial D-xylose isomerase from Bacillus coagulans and immobilized M. smegmatis. From 10 g of D-xylose only 4 g of xylitol can be obtained. These yields are relatively small, 3

CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) so xylitol-producing bacteria did not attract researchers interest. Process conditions affecting biological production of xylitol For industrial operations, it is very important to know what parameters would affect a certain process. Knowing these parameters and their effects, optimization of the process conditions can be done and the production rate and yield can be maximized. 1. Effect of xylose concentration A higher initial concentration of xylose (substrate) favors the production of xylitol in osmophilic microorganisms. Generally, in a batch process, an increase in the initial sugar concentration would lead to an increase in production rate and yield, if the microorganism is able to tolerate high concentration of sugar and hence higher osmotic pressure. When Horitsu et al. [43] increased the xylose concentration in C. tropicalis culture from 100 to 150 g/liter, sufficient cell growth was obtained at higher aeration rate (400 ml/min) by 90% O2. Xylitol production rate was increased from 1.78 g/liter/h to 2.44 g/liter/h. In increased concentration of xylose, with appropriately high aeration rate, the concentration of cell is increased and, consequently, the production of xylitol is also increased. Meyrial et al. [44] studied the substrate tolerance of Candida guillermondii by varying the initial xylose concentration, starting from 10 g/liter to 300 g/liter. The yield of xylitol is found to increase with the increase in initial concentration, with the highest yield being 0.75 g/g at 300 g/liter of initial xylitol (the theoretical yield is 82.6%). However, the rate of xylitol production only increased up to 200 g/liter of xylose, where the specific productivity was 2.4 times higher than that observed at 10 g/liter xylose. Cell growth was found to be inhibited at high concentration of xylose; the highest value of specific growth (i.e. 0.11/h) was observed at 20 and 50 g/liter xylose.

Figure 1. Schematic representation of D-xylose metabolism in yeasts [22].

3. Fungi Conversion of D-xylose to xylitol in a fermentation system using a fungal culture Petromyces albertensis has been reported by Dahiya [40]. After 10 days of cultivation, 100 g of D-xylose supplemented with 1% v/v methanol gave a xylitol yield of 39.8 g/L and D-xylulose of 2.8 g/L. 4. Recombinant In 1991, Hallborn et al. [41] combines the xylose reductase (XR) gene from Pichia stipitis CBS 6954 into Saccharomyces cerevisiae. This recombinant cell gives over 95% conversion of xylose into xylitol. 5. Mixed culture Nishio et al. [42] reported xylitol production by immobilized cells of Candida pelliculosa and Methanobacterium spp. A steady-state continuous production of xylitol in a column reactor packed with the immobilized cells is operated stably for two weeks. A conversion of almost 100% was reached after 33 hours of incubation using immobilized metanogen and C. pelliculosa with a ratio of 1:2.

CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) 2. Effect of aeration rate For effective xylitol production, rapid accumulation of microbial cells in the culture medium is to be achieved. This can be done by aerating the medium, and increasing the dissolved oxygen level [43]. However, because xylitol is produced under anaerobic (or microaerophilic or semi-aerobic), aerating the medium through all periods of cultivation would lead to a production of D-xylulose instead. Therefore, the higher level of dissolved oxygen is to be maintained only at the earlier phase of cultivation and thereafter it should decrease due to respiration of the microorganisms. According to Horitsu et al. [43], C. tropicalis would accumulate oxygen under oxygen limited conditions because at low dissolved oxygen level there are relatively high levels of NADPH and NADH in cell, leading to reduction of xylose and, subsequently, accumulation of xylitol in medium. 3. Effect of nutrition Various media have been used to culture xylitol-producing yeasts, and some generalizations can be made: (i) For some yeasts, yeast extract is important for xylitol production. (ii) For other yeasts, yeast extract has no significant effect on xylitol formation. These yeasts prefer urea or urea and Casamino acids. Some yeast are found to be categorized in both group by different researchers. In both cases, nitrogen source is important in xylitol production by yeast. In S. cerevisiae, the pentose-phosphate pathway is regulated by nitrogen, and ammonium salts have been found to stimulate the oxidative pentose phosphate pathway. Horitsu et al. [43] found that yeast extract as nitrogen source is important in xylitol production in C. tropicalis, while Onishi et al. [44] found that polyol formation is higher in medium with lower nitrogen level. Lee et al. [45], found that biotin level affects the selectivity of xylitol over ethanol. In cultures of Pachysolen tannophylus, high level of biotin favors ethanol production while in cultures of Candida guilermondii, high level of biotin favors xylitol production. 4. Effect of presence of other sugars in the medium Glucose is shown to inhibit xylose utilization in Candida and [46] Schizosaccharomyces . After a short period of time, the inhibition effect on xylose reached its maximum. The ability to convert xylose was, however, quickly restored once the glucose concentration was low. This indicated that catabolite repression was not the regulatory mechanism; inhibition was in main control of xylose transport in the presence of glucose. A short transition period between glucose and xylose utilization also suggests that inhibition was imposed by intracellular concentration of glucose or its catabolite. Candida guillermondii, characterized by its high potential for the production of xylitol from xylose-rice materials, are able to ferment non-xylose sugars as well. Meyrial et al. [44] found that glucose, mannose, galactose and arabinose are fermented rapidly only for growth and ethanol productions; their corresponding polyols are not found in the fermentation broth. 5. Effect of pH and temperature The optimal temperature for xylitol in yeasts was shown to be 30oC. Small temperature variations above this temperature do not affect xylitol production in Candida tropicalis significantly; the xylitol yield was, for most part, temperature independent when the cultivation was done in a temperature between 30-37oC. However, once the temperature is higher than 37oC, the yield decreased significantly [46]. The effect of temperature on selectivity (xylitol over ethanol) was investigated by du Preez et al. [47]. It was found that at higher temperatures, production of xylitol was favored over that of ethanol; the xylitol production of C. shehatae increased by 5

CN 5172 - Biochemical Engineering Novi Wijaya (A0068355X) 8-fold as the temperature was increased from 22 to 36oC. P. stipitis produced xylitol at 36oC, but did not produce any detectable amount of xylitol at lower temperatures. The pH exerted little effect of yield. C. tropicalis was reported to be not very sensitive to pH, and maintained maximum xylitol yield at a pH as low as 2.5 [46]. Most yeasts are cultivated at pH values between 4 and 6. And although pH has little effect, if uncontrolled, pH will drop during fermentation. Therefore, the initial pH value in uncontrolled fermentations is set higher than that of controlled fermentations. The optimal initial pH for C. boidinii was 7 [48] for uncontrolled fermentations, while for controlled conditions, a pH of 5.5 was better [49]. Downstream processing In a microbial production system, the downstream processing is usually much more expensive than the upstream. For microbial xylitol production from corn cobs, the fermentation broth would contain cells, volatile and nonvolatile compounds (VCs and NVCs) [50]. VCs include solvents and other compounds generated in the chemical processing of the raw material. NVCs are xylitol, unfermented sugars (xylose and arabinose), arabitol, proteins, inorganic compounds (measured as ashes), and others (mainly acid-soluble lignin, uronic acid, and extractives). Rivas et al. [50], introduced a downstream scheme where the fermentation broth is subjected to charcoal adsorption to remove color. The processed stream was vacuum evaporated to produce a concentrated liquor, which is then precipitated with ethanol. The xylitol in the resulting liquid stream accounted for 92.6% of xylitol present in the fermentation broth. This liquid stream is concentrated and precipitated again. The resulting liquid stream contains concentrating again then crystallized. After washing and drying, the resulting xylitol crystal is regularly shaped, well-formed, homogenous, and containing 98.9% wt of xylitol. Conclusions To produce xylitol by extraction from natural sources would be difficult and uneconomical, mostly because xylitol only occurs in low concentration (<1%). Xylitol can be produced via chemical or microbial process. On industrial scale, xylitol is being produced through chemical reduction of xylose derived from hemicellulosic hydrolysates (mostly birchwood). The intensive purification and separation that is required to remove other by products from xylose and xylitol solution makes the process relatively expensive. The yield of xylitol is only about 50-60%. Fermentative and biocatalytic processes attracted attention because it does not require pure xylose as substrate (mixed sugars can be used as substrate, so the separation of xylose from other sugars in hemicellulose hydrolysate, which is difficult, is not necessary), and it gives a reasonably high yield (up to 90%). For the industrial production of xylitol, fermentation conditions still need to be optimized. This can be done either by kinetic approach with a model, of by applying statistical methods (experimental design).

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