Semi-pilot production of xanthan gum using nejayote as culture medium substrate 3

15 Background: Xanthan gum is an industrialized polysaccharide produced by Xanthomonas 16 genus. Alternative carbon sources for Xanthomonas culturing may help reducing its 17 production cost. Nejayote , a residue from maize nixtamalization process, is an alternative 18 culture medium substrate to produce xanthan. In this study, industrial and semi-industrial 19 nejayote alone or in combination with supplements, were tested for xanthan production by 20 Xantomonas campestris (1 × 10 8 cells/mL inoculum), using YGC medium as control, in 21 100 mL and 1 L volumes. Cellular growth was determined by the colorimetric MTT 22 reduction assay (OD 540 ). Xanthan pyruvate and acetyl groups from nejayote plus 23 supplement cultures in the bioreactor were evaluated (OD 570 ). 24 Results: Industrial nejayote steadily produced up to 4 g/L xanthan, as compared with YGC 25 medium control, which increased its production over time up to 9.3 g/L at 96 h. Cellular 26 activity assay revealed that the highest values after 24 h (3.88 and 2.71 OD 540 for YGC and 27 industrial nejayote , respectively). Nejayote supplemented with MgSO 4 •7 H 2 O resulted in 28 the highest xanthan production (10.8 g/L), but low cell growth (3.6 and 1.82 OD 540 for 29 YGC and nejayote plus supplement, respectively), after 96 h of culture. Furthermore, gum 30 yield reached up to 6 g/L and 1.9 OD 540, after 96 h of nejayote plus supplement culture, 31 using a 14 L bioreactor. Xanthan pyruvate and acetyl groups from nejayote plus supplement 32 cultures in the bioreactor resulted in similar amounts (0.107 and 0.130 OD 570 , respectively), 33 compared with a commercial biopolymer (0.148 and 0.127 OD 570 respectively).

supplemented with nitrogen sources, was reported to be a suitable substrate for xanthan  At industrial level, nixtamalization is a process in which corn kernels are cooked in 77 a saturated calcium hydroxide solution and after wet grinding, they are hydrated to extract 78 starches. Next, they are cooked under alkaline treatment (0.6% to 1.2% calcium hydroxide 79 with regard to corn weight), during a period of 50 to 70 min at 90-95 °C, followed by a disposal costs, as well as environmental contamination, and potential legal penalties 97 (Rosentrater, 2006).  hydrogen (bioH2) production in a discontinuous reactor from the co-digestion of nejayote, 109 evaluating the effects of different proportions of residues, reporting that the microbial 110 6 structure of the inoculum allowed adaptation for feeding on substrates. Based on this and 111 the different protocols to treat nejayote, the use of this residual effluent to reduce 112 contamination levels and obtain a value-added product, such as xanthan gum, should be 113 evaluated.

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Nejayote represents a potential culture medium substrate to be used in xanthan 115 production, based on X. campestris metabolic rate. It then becomes an alternative for 116 reducing production costs by optimizing this bioprocess and facilitating waste re-117 incorporation into soil or use in other processes, because of the metabolic modifications 118 made by this microorganism. Therefore, the aim of the present study was to determine 119 feasibility and optimization of xanthan gum production up to semi-pilot level, using 120 nejayote as substrate. Similar xanthan production was observed at 24 h of culture in nejayote and YGC 128 broth. However, gum generated in nejayote-based medium did not increase over time, 129 obtaining results not greater than 4 g/L at the end of the test. In contrast, gum production in 130 YGC broth continued to increase during 96 h up to 9.3 g/L (Fig. 1). Cellular activity using 131 nejayote as culture medium substrate, showed an exponential rise during the first 24 h, 132 which decreased over time, as compared with that using YGC medium, maintaining activity 133 during the incubation time. After 24 h of culture, the highest ODs were 3,884 and 2,713 by 134 control medium and industrial nejayote, respectively (Fig. 1). reading at 600 r/min, respectively. Nevertheless, particle sizes did not satisfy the standards, 142 since their dimensions were below the required specifications (Table 1). After evaluating 100 mL of medium at 24 h of culture, production was higher in the 147 nejayote medium (2.56 g/L yield) alone or supplemented with calcium carbonate (4.20 g/L 148 yield) and hepta-hydrated magnesium sulfate (3.66 g/L yield), than that of YGC medium (2 149 g/L yield), maintaining similar production values until the end of the fermentation time, 150 whereas bacteria kept an exponential increase in YGC medium ( Fig. 2A) CaCO3, yeast extract + MgSO4 • 7 H2O, and yeast extract + CaCO3 + MgSO4 • 7 H2O did 154 not produce gum ( Fig. 2A). The treatment that showed the best results in terms of 155 biopolymer production was nejayote supplemented with CaCO3, producing 5.7 g/L after 156 120 h of culture, which was lower compared with that of YGC medium (7.36 g/L).  In addition, semi-pilot (14 L) xanthan production was developed from semi-  After xanthan gum produced in nejayote was analyzed, the positive control had 182 characteristics that clearly differentiate it from the rest of the samples, such as a larger size, 183 as well as a spongier consistency and cleaner appealing (white coloration). Xanthan gum 184 produced in the semi-industrial nejayote treatments, had smaller size and were more 185 compact with harder consistency compared with control, before and after drying, in 186 addition to a darker coloration (brownish). The gum from industrial nejayote resulted in an 187 even darker coloration (light brown) and after dried, it lost its spongy characteristic and 188 resulted in an even more compact product (Table S1). CaCO3 treatment, compared with previous tests, with a production of 6.06 g/L (Fig. 3D).

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Cellular activity (OD540) using YGC medium was significantly (F4,5 = 660,080; p < 205 0.05) higher (more than double) compared with that of nejayote medium (OD540 = 3,636 206 and 1,506 respectively) (Fig. 3B). For the 100 mL volume experiment, semi-industrial 207 nejayote treatments presented the highest OD540 when the culture was ended, compared 208 with treatments supplemented with yeast extract, yeast extract + CaCO3 + MgSO4 • 7 H2O, 209 and yeast extract + CaCO3, with A570 of 3,198, 2,933, and 2,838 respectively, which were 210 significantly (F18,26 = 37,636; p < 0.05) lower than those of YGC medium (3,750) (Fig. 4A).    xanthan gum using industrial nejayote as culture medium, but its growth decreased after 24 260 h of culture (Fig. 1), probably due to a reduction in the carbon source availability or 261 completely nutrient consumption, negatively affecting its cellular activity. Nevertheless, 262 xanthan gum production in the first 24 h of culture was comparable with that of reference 263 medium (2.86 g/L versus 3 g/L in nejayote and YGC broth, respectively) (Fig. 1). Gum  This may explain why gum production in nejayote treatment supplemented with yeast 288 extract (high nitrogen concentration) in our study, did not correlate with higher bacteria 289 metabolic activity compared with reference medium (Fig. S1A).  In our study, xanthan production was scaled at a semi-pilot level, where volumes up 315 to 1 L were initially tested, in order to determine if this bioprocess could be carried out at 316 higher volumes. After scaling from 100 mL to 1 L volume, results showed higher gum 317 production by X. campestris in YGC medium using lower volume (7.36 and 3.86 g/L 318 respectively), whereas cellular activity remained equivalent. We believe such values are the 319 result of a significant increase in viscosity and decrease in the gas-liquid mass transfer rate 320 (oxygen transfer rate) , which could clearly lead to a reduction of gum 321 production.

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Nevertheless, xanthan yields from nejayote plus supplements were similar or higher 323 than nejayote alone. Nejayote supplemented with MgSO4 • 7 H2O showed the highest 324 production and was selected for the bioprocess scaling at bioreactor level, where production 325 yielded 5.53 g/L, which was lower than experiments using lower volumes. Rosalam & 326 Englan (2006) reported that different growth phases and perhaps a culture medium 327 alteration, such as a higher non-filterable solids amount, like nejayote, limited nutrients, 328 thus reducing yield, but no the produced xanthan quality.

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It was expected that a medium without all nutritional requirements such as nejayote, 340 a product with less amount of nutrients compared with controls, will be obtained. Gum 341 produced from nejayote in bioreactor showed no difference, where quality properties were 342 similar than those of commercial samples. Therefore, the present study offered an 343 alternative in terms of obtaining this biopolymer, whose yield and production from nejayote 344 are comparable with that obtained with conventional carbon sources, which meet the 345 quality required for this product´s commercialization.