Elucidation of New Xylose Metabolizing Pathway in Pseudomonas Gessardii VXlt-16 and its Correlation with Xylitol Production from Sugarcane Bagasse Hydrolysate

Scientific interventions have identified lignocellulosic biomass as potential raw material for various industrial processes. However toxic byproducts released during the process result in deterioration of environment to a greater extent. Microbes can utilize these wastes for production of products of commercial value like bio-fuels, protein, organic acids and xylitol. However, high production cost and astringent operating conditions have been the major bottlenecks for its commercial production. In microbes, xylose is metabolized by xylose isomerase (XI) and xylose reductase-xylitol dehydrogenase (XR-XDH) pathways, with later having ability to transform pure xylose as well as xylose rich lignocelluloses. Efforts to find hyper producer isolates for xylitol production resulted in identification of one such isolate Pseudomonas gessardii VXlt-16 (MG770460) by 16s rDNA sequencing. Statistical optimization resulted in 7.28 folds’ increase in xylitol yield with 64.76% xylose bioconversion. Conversion of xylose to xylitol even at large scale suggests the possible application of bacterial isolate for the production of this useful product at industrial scale.


Introduction
Unmanaged waste majorly comprises of organic matter as most of industries utilize some of the fraction of raw material and discarded rest Organic matter from agricultural practices and relevant industries such as paper-pulp is rich in xylan and xylose can be a potential substrate for many commercial by products including nutraceuticals, biofuels, phenols, organic acids, biopolymers, enzyme, animal feed, bio-fertilizer and even xylitol if handled scientifically for the production of commercial byproducts [1][2][3][4]. Xylitol is a 5carbon sugar alcohol having equal sweetness to table sugar but with lesser calories besides other pharmacological benefits such as anti-cariogenecity, antimicrobial and oral hygiene. It doesn't add to blood glucose pool which makes it a suitable sweetener for diabetic patients.
Moreover, its antimicrobial potential adds it application in maintaining oral hygiene and preventing demineralization of tooth by limiting the growth of oral bacteria. Its unique metabolism and pharmacological potential makes it one of the top twelve bioproducts, having high market demands [4]. Extraction from plants was the only source for xylitol initially which was replaced by more efficient processes such as chemical reduction of xylose for xylitol. Chemical catalysis operates at high temperature and pressure. However the process is efficient but requirement of high energy and extensive purification enhance the production cost [5,6]. Bio-production of xylitol may be helpful in reducing the production cost along with higher yield and efficient production. Cells can metabolize xylose by two metabolic pathways i.e. xylose isomerase (XI) pathway and xylose reductase-xylitol dehydrogenase (XR-XDH) pathway. XR-XDH pathways are common in eukaryotes such as yeast while prokaryotes including bacteria follows XI pathway mostly. However earlier researches have also enlisted some of the bacteria such as Cellulomonas, Serratia, Corynebacterium [7] Bacillus and Pseudomonas [8] for XR-XDH pathway and xylitol production. Xylose reductase is the key enzyme for xylitol production therefore purified enzyme can also be used for xylose bioconversion specifically but it requires purified substrate which also adds cost to pure substrate. Moreover, cells are able to produce number of enzymes which can degrade polymeric biomolecules such as carbohydrates into monomers and further used for energy generation. To overcome the drawbacks and limitation of chemical catalysis and enzymatic conversion, continuous efforts have been made to find some robust hyperproducer microorganism for the bio-transformation of xylose into xylitol [9][10][11]. Pseudomonas is one of the most prominent strains among bacterial kingdom. Some of the species of Pseudomonas have been reported for xylitol production [8] but no literature is available for xylose reductase pathway and xylitol production from fluorescent Pseudomonas and it is possibly the first report for describing the XR-XDH pathway and xylitol production from Pseudomonas gessardii. To enhance the product yield, growth conditions can be optimized either by one variable at a time (OVAT) approach or by statistical modeling like response surface methodology. Statistical modeling is preferred as it comprises the interactions of all the factors to maximize the production of desired products [12,13]. Therefore, the current investigation was to enhance the production of xylose reductase from Pseudomonas gessardii VXlt-16 and establish the relation between XR activity and xylitol field.

Hydrolysate preparation for xylitol production
Sugarcane bagasse hydrolysate was generated by combining dilute acid and steam blast treatment using 0.3% HCl, at 121°C for 4 cycles of 20 min each. After this, sample was cooled and settled down to separate liquid fraction. Liquid extract was analyzed for the sugar content, which was further used for fermentation.

Statistical modeling for batch fermentation of sugarcane hydrolysates
Statistical model was designed with six production parameters including hydrolysate concentration, inoculum size, pH, production time, temperature and rpm using Taguchi Orthogonal Array (TOA) approach.

Scale-up: Batch fermentation
For higher xylitol yield, 1500 mL of sugarcane bagasse hydrolysate was used for fermentation in 2L fermenter (New Brunswick Scientific, USA). For xylitol production kinetics, XR, XDH activities and xylitol yield were quantified after every 4 h till 96 h, as discussed in earlier sections.

X-ray Diffraction (XRD)
Every material has its own X-ray diffraction pattern due to interaction between X-ray and atoms/crystals. It covers almost all kind of interactions like elastic, inelastic scattering, photoelectric absorption and pair production [16]. X-ray diffraction pattern composed of peaks at different angles which aid in elucidation of crystal lattice structures. XRD pattern of xylitol crystals were prepared by using X-ray diffraction instrument at Rigaku MiniFlex 600; Central Instrumentation Facility 'CIF'-Jiwaji University, Gwalior. A thin film of purified and dried xylitol crystals was prepared and scan at the rate of 10 min −1 in the wavelength range of 2-90°. CuKα radiation source of 40 kV was used as source of X-ray.

Fourier-Transform Infrared Spectroscope (FTIR)
Functional groups and chemical bonds in a compound own a distinct transmission/adsorption behavior for infrared radiation. Spectrum profiling can be very helpful in qualitative estimation of samples' purity [17]. In addition, the FTIR profile may also help in estimating the damages and changes associated with heating and chemical exposure. To confirm the functional moieties and bonds in sample, purified xylitol crystals were analyzed between wavenumber of 450-4000 cm -1 in transmission mode. The spectrum was prepared with FTIR spectrometer at IIT Roorkee Uttarakhand.

Product recovery
Xylitol was crystallized from the fermentation broth after removal of solid particulate matter and other unwanted microbial byproducts. To remove the particulate matter, fermented broth was centrifuged at 10000 rpm for 10 minutes at 4°C. Supernatant was detoxified with activated charcoal followed by ion exchange resins [18]. The decolorizing efficiency was determined as follows (1): where η is decolorization ratio; A0 is absorbance of fermentation broth before decolorization and A is absorbance after decolorization Detoxified broth was concentrated at 40±5°C under vacuum till supersaturation of xylitol solution. Concentrated broth was seeded with 1% xylitol crystals and kept at low temperature for up to 24-48 h to crystallize the xylitol by nucleation. Recovered crystals were washed with petroleum ether and acetone to remove residual impurities.

Analytical methods
Xylitol concentration in fermentation broth was determined by HPLC (Shimadzu HPLC 20 A unit) with refractive index detector using acetonitrile: water (70:30) as eluent at 40°C.
Xylose and xylitol (Alfa Assar US) of high purity was used as standard.

Reuse of activated carbon
Xylitol production from hydrolysate and its purification holds relies on activated carbon for detoxification and clarification but its one-time use imparts substantial burden on product cost as well as on environment. Physical and chemical methods were employed for carbon regeneration and each sample was evaluated for restoration of detoxification strategy. Along with that selected method was used for successive cycles of regeneration to determine the regeneration potential of spent carbon. Spent carbon was regenerated by washing with acetic acid [19], chromic acid and distilled water. Spent carbon was mixed with acetic acid/chromic acid/distilled water (1:4) at 30 o C for 30 min and filtered. The process was followed by washing with distilled water for 4-5 time to attain neutral pH. Virgin, spent and regenerated carbon were characterized for their iodine number (D28 Committee, n.d.), broth decolorizing power and removal efficiency for dye (methylene blue), furfurals and phenols.

Statistical modeling for xylitol production at pre-bench scale
Statistical model, designed with Taguchi Orthogonal Array (TOA) approach considering six production parameters offered twenty-five combinations. XR activity and xylitol yield from provided combinations were calculated by the following equation (6)  The purpose of the correlation study was to identify the inherent factor responsible for maximum xylitol yield. The key findings were determined by measuring enzyme activities of both the enzyme (XR and XDH) for XR-XDH pathway (Fig. 2). Results suggested that the ratio between XR to XDH tend to favor higher xylitol yield instead of XR alone. This fact also supports the benefit of using whole cell catalysis over enzymatic transformation.

Batch fermentation for xylitol production
Once the production conditions for xylitol were optimized using TOA scale of xylitol production was increased to 1.5L working volume in 2L fermenter (New Brunswick Fermentation broth contains unwanted microbial products and acid hydrolysis side products like furfurals, and hydroxyfurfurals. Presence of these contaminants was recorded as broth turbidity, conductivity and salt concentration. For the removal of these impurities, broth was treated with 2% activated charcoal as maximum decolorization (94.03 %) was achieved with minimum loss of xylitol (5.35±0.56 g). The AC-treatment is followed by ion exchange resins which further reduced the presence of various contaminates with 94.91% decolorization efficiency. The clarified broth was concentrated at 40°C and crystallized at -20°C which was followed by lyophilization. Light-pale coloured crystals were washed with acetone and diethyl ether to remove the undesired contaminants without affecting crystals (Fig. 3).
Purification profile showed that purification strategies have helped in removing the contaminants, which resulted in lowering the salt concentration and conductivity (Table 1).
Purification on one hand resulted in 99.68±0.67 % decolorization of fermentation broth, and removal of 79.88% ions but it also resulted in loss of 12.70% of xylitol. Finally, 48.49 g of xylitol crystals was recovered with up to 94.56% purity.  [20]. Bands between 1200-1000 cm -1 are the fingerprinting region for each polysaccharide. Band at 1125.96 cm −1 , 1095.25 and 1061.17 are attributed to tertiary, secondary and primary alcohol respectively [21]. Peak at 661.81 cm −1 represents -OH groups that are out of plane (Fig. 5).
Among screened physical and chemical methods, acetic acid was selected due to higher regeneration efficiency and thus used for successive cycle to regenerate the spent carbon.
Carbon wash from the first regeneration cycle was also evaluated for the presence of organic molecules absorbed from the broth by activated carbon. Xylose reductase is the key enzyme for xylitol production. Yeast may be efficient for xylitol production. Most of the available literature also reported Yeasts like Candida as an efficient organism for bioproduction of xylitol [4,22,23]. Bacteria are able to utilize lignocellulosic biomass and can survive under harsh conditions. Bacterial cells mostly followed XI pathway for xylose metabolism except few including Enterobacter, Corynebacterium and Serratia have been reported for xylitol production [16,24] Pseudomonas gessardii first time reported for xylitol production.
Statistical optimization of XR and xylitol production from Pseudomonas gessardii VXlt-16 (Genbank accession no: MG770460) resulted in maximum enzyme activity of 84.05 U/mg with conversion rate of 64.19%. Xylitol and xylose reductase was purified after fermentation.
Bacterial xylose reductase was of 67KDa composed of two monomeric units of 33.5 KDa.
LCB is a complex structure which can't be utilized completely by XR alone hence whole cell or multiple enzyme system is suitable for xylitol production from low cost complex substrates like lignocellulosic residues [25]. Recently Candida tropicalis CCT 1516 was used for simultaneous production of xylitol and ethanol from sisal fiber hydrolysate and 0.32 g/g xylose and 0.27 g/g consumed sugar was recorded [21]. Similar efforts have been made by using Pseudomonas CDS3, Bacillus 65S3, and Duganella 55S2, for simultaneous production of xylitol and ethanol. Out of these three strains, Pseudomonas CDS3, and Bacillus 65S3 was found superior for xylitol production with the xylitol yield of 0.98g/g and 0.82g/g respectively [8]. Earlier Candida tropicalis was used for xylose reductase production using meranti wood

Conclusion
Xylitol is a natural sweetener with high commercial value high has high market demand. Bioproduction of xylitol has immense future potential but this requires hyperproducer microbes and economically viable production process. Pseudomonas gessardii VXlt-16 (Genbank accession no: MG770460) was selected on the basis of highest XR activity 4.94 U/mg and better conversion (8.82%) of xylose to xylitol. Isolate was used in the present investigation.
Statistical approach used to enhance the XR and xylitol production resulted in 17.01-fold increase in enzyme production with 7.28 folds more xylitol yield. The potential of