Assessment of Alternative Media Viability for Cell Growth Phase in the Lab-Scale Xanthan Pruni Production—Part I

Abstract

Xanthan is a highly relevant commercial microbial biopolymer. Its production occurs in two steps: the bacterium is cultivated in a nitrogen-rich medium for cell multiplication, and the obtained biomass is used as an inoculum for the polymer production phase. Different media compositions for cell growth were investigated, seeking to reduce or replace the peptone used in the standard medium. Peptone (P), yeast extract (YE), and rice parboiling water (RPW) concentration combinations were tested in cultivating Xanthomonas arboricola pv. pruni 101. A CRD 2³ design, performed in a shaker, was used to assess the effects of independent variables on xanthan pruni microbial growth, N consumption, yield, viscosity, pseudoplasticity, and xanthan mineral content. After 24 h an increase in N was observed, without any significant impact on cell growth. Xanthan yield increased as a result of the alternative treatments, with P and YE influencing positively. However, T1, with the lowest levels of P, YE, and RPW increased viscosity and pseudoplasticity of xanthan pruni. RPW increased phosphorus, silicon, calcium, and magnesium, and P and YE increased potassium. These results indicate that partial replacement of P by RPW and YE is an economically viable and sustainable approach for the xanthan pruni production.

1. Introduction

The properties of natural polymers such as biocompatibility, biodegradability, and renewability are attracting growing attention to these products. Among these polymers, xanthan, an extracellular biopolymer produced by bacteria of the Xanthomonas gender is highlighted. Due to its rheological and structural properties, xanthan is widely employed in the food, pharmaceutical, cosmetic, paintings, and petroleum industries [1]. The global demand for this product is registering constant growth. According to Global Market Insights, the xanthan gum market could reach USD 1.2b by 2030, at a composite annual growth rate (CAGR) of 5.71%. Brazil also exhibits impressive consumption, attaining 30 thousand tons a year, supplied exclusively by international industries [2,3].

For the Xanthomonas cultivation and production, carbon sources are used as substrates, while sources of nitrogen and minerals are employed as nutrients [4]. The high costs associated with substrates constitute a process limitation. To minimize such costs, agro-industrial wastes are being studied as alternative substrate sources, such as water obtained from oil production [5], pineapple wastes [6], rice hulls [7], rice hulls [8], beet molasses [9], bread wastes [10], orange peels [11], and wine industry waste [12]. The development of sustainable, low-cost solutions for obtaining xanthan can significantly increase market opportunities [6].

Rice (Oryza sativa L.) is one of the most consumed staple foods, parboiled rice being one of its most consumed forms in Brazil. The rice parboiling process involves a hydrothermal treatment in which the hulled grain is immersed in drinking water to soak at temperatures above 58 °C, followed by partial or total gelatinization and further drying [13]. In the rice parboiling process, the grain’s outer mineral layers, especially in the bran and aleurone layer, migrate towards the interior of the grain, and a portion of these RPW minerals are primarily determined by process parameters temperature, time, and operating conditions [14]. At the end of the process, the wastewater generated, containing high phosphorus and nitrogen, causes high biochemical oxygen demand, requiring suitable treatment before disposal. The composition of this nutrient-rich water represents a significant challenge for the rice industries, especially in view of the complexity of phosphorus and nitrogen management, which can cause environmental harm if not duly mitigated [15,16].

Until now, no strategies have been identified aiming at redirecting RPW waste to other industries, adding value to another product. RPW study was pioneered by our research group in the X. arboricola pv. pruni (ex X. campestris pv. pruni) production phase for obtaining xanthan, directly influencing the characteristics of the obtained gum [17]. Besides minimizing the environmental impacts, the use of this waste as substrate could contribute to xanthan cost reduction, making viable domestic production. Investment in xanthan domestic production brings economic benefits, such as a reduction in dependence on imports and job creation, in addition to promoting environmental sustainability by reducing impact through the use of industrial effluents as substrate [4]. Therefore, the scientific and technological advancement in xanthan production in Brazil is crucial for the development of this industry in our country, rendering it competitive in the global biopolymers market.

Xanthan pruni, a polymer analogous to xanthan and produced by X. arboricola pv. pruni, differs from commercial xanthan in its chemical composition. Studies have demonstrated that xanthan pruni contains rhamnose in its structure, likely incorporated in the side chains, which distinguishes it from conventional xanthan [18]. Studies on X. arboricola pv. pruni remain limited, and there is a lack of information regarding the impact of various alternative media on the production of xanthan pruni. Perez et al. (2020) [19] evaluated the supplementation of the medium with yeast extracts and observed a significant increase in biopolymer production. In the study by Moreira (2023) [20], culture media containing different minerals were used to produce xanthan pruni. Among the components, the use of RPW stood out, as well as carbon sources such as sucrose and cellulose, and nitrogen sources such as peptone and mineral salts. Given the scarcity of studies addressing different medium formulations to produce xanthan pruni, this work aims to contribute to the advancement of this topic by evaluating the feasibility of using alternative nutrient sources and their impacts on the production and characteristics of the biopolymer.

The aim of this study was to investigate the effects of rice parboiling water (RPW) use combined with peptone and yeast extract as N sources in culture media for Xanthomonas arboricola pv. pruni cell growth (the 101 strain being used as a model) and the production of its biopolymer, referred to in this study as xanthan pruni. The viability of these alternative media was examined by comparing cell growth, xanthan pruni yield, and its rheological properties with those obtained from conventional culture media. Besides, the main compounds present in parboiling water were identified to check if they influence xanthan synthesis. This is meant to contribute to the development of more sustainable, economically viable processes, by exploring alternative sources of substrates and assessing the effectiveness of different culture media components.

2. Materials and Methods

2.1. Rice Parboiling Wastewater Characterization

RPW was collected from the Nelson Wendt company in Pelotas, RS, Brazil, from the outlet tube during the discharge process into the effluent collection tank. The rice immersed in the tanks was a varietal mixture (2022 harvest). After collection, RPW samples were stored in plastic bottles and frozen at −18 °C for further characterization and utilization. The pH value [21], reducing sugars [22], and nitrogen content (Urea kit CE ref. 27—Labtest^®, Delta, BC, Canada) were determined. Mineral characterization was performed with the aid of a MIP OES (Agilent Technologies, model Agilent 4200, Melbourne, Australia) spectrometer [23] (

Table 1. Rice parboiling process wastewater characterization.

Parameters
pH	4.6 ± 0.2
Reducing Sugars	3.40 ± 0.16
Nitrogen (mg/dL)	140.00 ± 1.12
Phosphorus (mg/L)	304.96 ± 9.90
Zinc (mg/L)	1.53 ± 0.05
Iron (mg/L)	9.36 ± 0.33
Silicon (mg/L)	39.43 ± 10.26
Calcium (mg/L)	32.80 ± 0.41
Potassium (mg/L)	4411.96 ± 196.21
Magnesium (mg/L)	169.23 ± 8.37
Manganese (mg/L)	43.17 ± 1.37
Sodium (mg/L)	21.86 ± 1.88

).

2.2. Inoculum Preparation

The X. arboricola pv. pruni 101 bacterium was kept in agar SPA [24] and cultivated in a liquid medium with different peptone (Himedia^®, Kennett Square, PA, USA) concentrations, yeast extract (Procelys by Lesaffre^®, Marcq-en-Barœul, France), and RPW, according to a central composite design 2³ [24]. Factors were codified in three levels: low (−1), medium (0), and high (+1), with the central point representing the repetitions [25] (

Table 2. Matrix of the complete factorial design 2³, with 3 central points for defining the peptone, yeast extract, and RPW concentrations for X. arboricola pv. pruni 101 inocula production.

Treat.	Codified Levels			Real Levels
	x1	x2	x3	x1	x2	x3
1	−1	−1	−1	1	1	20
2	+1	−1	−1	5	1	20
3	−1	+1	−1	1	5	20
4	+1	+1	−1	5	5	20
5	−1	−1	+1	1	1	80
6	+1	−1	+1	5	1	80
7	−1	+1	+1	1	5	80
8	+1	+1	+1	5	5	80
9	0	0	0	3	3	50
10	0	0	0	3	3	50
11	0	0	0	3	3	50

x1 = peptone (g/L), x2 = yeast extract (g/L), and x3 = RPW (%); Treat. = treatment.

).

All the treatments were diluted in a solution of (g/L): 20 sucrose, 0.5 dibasic potassium phosphate, and 0.25 heptahydrate magnesium sulfate (Synth^®, Diadema, Brazil). SPA standard medium [24] was utilized as a positive control, the composition of which is in (g/L): 5 of peptone, added to the previous solution. Cultivation was conducted in a 250 mL Erlenmeyer flask containing 40 mL of cultivation medium and 10 mL of bacterium suspension (9.14 UFC/mL), in SPA medium, incubated at 28 °C, 150 rpm for 24 h in an orbital shaker (B. Braun Biotech, Certomat BS-1, Melsungen, Germany). Initial and final bacterial concentrations were assessed by means of serial dilutions and plating in agar SPA medium, the colonies counting being expressed in UFC/mL.

2.3. Xanthan Pruni Production

The bioprocess occurred in 250 mL Erlenmeyers flasks containing 45 mL of MPII production medium [17] and 5 mL inoculum (Section 2.2) in an orbital shaker (B. Braun Biotech, Certomat BS-1, Melsungen, Germany) at 28 °C and 200 rpm for 72 h. Biopolymer recovery was performed with ethyl alcohol 96° GL (4:1 v/v), followed by oven-drying (CE-220-81, Cielenlab, Campinas, Brazil) at 56 °C to constant weight. The yield was determined by gravimetry and expressed in g/L.

2.4. Nitrogen

Residual nitrogen content (mg/L) was quantified in two steps with the aid of the Urea Kit CE (Labtest^®, Richmond, BC, Canada, ref. 27). Broth samples were collected at the 0 and 24 h times, centrifuged at 10,000 rpm x for 10 min at 4 °C in a refrigerated centrifuge (Electron Corporation^®, Sorvall-Thermo, EUA, Tokyo, Japan). The supernatant was analyzed in a UV-Vis spectrophotometer (Shimadzu, UV-1900i, Kyoto, Japan) at 500 nm. Nitrogen content calculation followed the standard method (urea 70 mg/L and sodium azide 7.7 mmol/L), according to Equation (1):

$$\mathrm{Residual} \mathrm{Nitrogen} (\mathrm{m}\mathrm{g}/\mathrm{L})={\displaystyle \frac{Absorbance of Sample }{Absorbance of Standard }}\times 70\times 10$$

(1)

2.5. Viscosity

Rheological properties of xanthan aqueous solutions were assessed with the aid of a rheometer (RheoStress 600, model RS150, Haake^®, Vreden, Germany). 1% (m/v) Xanthan aqueous solutions were prepared, agitated for 2 h, and stored at 4 °C for 24 h. Rheometric analysis was performed at 25 °C, with a cone and plate geometry (C60/2° Ti sensor; 0.105 mm interval).

2.6. Minerals

Mineral analysis was performed according to the method described by Rosa et al. (2016) [26]. At first, xanthan gum samples were treated by acidic digestion with the aid of an open system in a digesting block. Approximately 100 mg of the samples were weighed and transferred to glass digesting tubes, to which 5 mL of concentrated nitric acid (HNO₃) was added. The mixture was heated in a digester at 220 °C under reflux for 3 h. The resulting solutions were diluted in ultrapure water and analyzed in triplicate with the aid of a microwave-induced plasma optical emission spectrometer (MIP OES) (Agilent Technologies, model Agilent 4200, Mulgrave, Australia).

2.7. Statistics

Experiments were performed in triplicate, and the results were expressed in averages and standard deviations. A t-test was performed to observe significance at the p ≤ 0.05 level using RStudio software (2023.06.2+524). The effects of independent variables were determined using ANOVA with Statistica 10.0^® software, with p ≤ 0.05 considered significant.

3. Results and Discussion

3.1. Cell Growth

X. arboricola pv. pruni cell concentration in the examined alternative cell growth media and in standard SPA medium is listed in

Table 3. X. arboricola pv. pruni strain 101 cell growth in media containing different peptone, yeast extract, and RPW concentrations.

Treat.	x1	x2	x3	Cell Growth (UFC/mL)
1	−1 (1)	−1 (1)	−1 (20)	9.93 ± 0.01 *
2	+1 (5)	−1 (1)	−1 (20)	9.87 ± 0.04 *
3	−1 (1)	+1 (5)	−1 (20)	9.94 ± 0.04 *
4	+1 (5)	+1 (5)	−1 (20)	9.98 ± 0.04 *
5	−1 (1)	−1 (1)	+1 (80)	9.92 ± 0.04 *
6	+1 (5)	−1 (1)	+1 (80)	9.69 ± 0.01 *
7	−1 (1)	+1 (5)	+1 (80)	9.83 ± 0.01 *
8	+1 (5)	+1 (5)	+1 (80)	9.81 ± 0.01 *
9	0 (3)	0 (3)	0 (50)	9.85 ± 0.03 *
10	0 (3)	0 (3)	0 (50)	9.78 ± 0.01 *
11	0 (3)	0 (3)	0 (50)	9.82 ± 0.01 *
SPA	5	-	-	10.04 ± 0.01

x1 = peptone (g/L), x2 = yeast extract (g·L⁻¹), and x3 = RPW (%). Samples’ asterisks (*) point to significant differences as compared to the standard (p < 0.05), according to the t-test.

. In spite of the fact that the results show significant variations statistically in cell growth among the treatments and the SPA medium as seen by the t-test, the differences are relatively small. The SPA medium led to a 10.04 UFC/mL cell concentration, while in the remaining treatments, the concentration varied between 9.69 and 9.98 UFC/mL.

Treatment 6, with 80% (v/v) RPW, led to a slightly lower result (9.69 ± 0.01 UFC/mL) as compared to treatments containing 20% (v/v) RPW (9.87 to 9.98 ± 0.04 UFC/mL). The results illustrated in Figure 1 evidence this trend. The negative signal of the RPW coefficient means that cell growth increased with a reduction in the mentioned parameter value, which may be associated with inhibitory compounds or high mineral levels in the water, especially silicon [14]. This phenomenon suggests that increased RPW concentration can have an inhibitory effect on X. arboricola pv. pruni growth.

3.2. Yield

Xanthan yield varied among treatments, with values between 7.0 and 8.9 g/L, while the standard SPA medium exhibited a yield of 6.8 g/L (

Table 4. Yield of xanthan produced by X. arboricola pv. pruni strain 101 in media containing different peptone, yeast extract, and RPW concentrations.

Treat.	x1	x2	x3	Yield (g/L)
1	−1 (1)	−1 (1)	−1(20)	7.233 ± 0.20 *
2	+1 (5)	−1 (1)	−1 (20)	7.393 ± 0.42 *
3	−1 (1)	+1 (5)	−1 (20)	7.123 ± 0.51 *
4	+1 (5)	+1 (5)	−1 (20)	7.333 ± 0.22 *
5	−1 (1)	−1 (1)	+1 (80)	7.020 ± 0.32
6	+1 (5)	−1 (1)	+1 (80)	7.533 ± 0.26 *
7	−1 (1)	+1 (5)	+1 (80)	7.343 ± 0.35 *
8	+1 (5)	+1 (5)	+1 (80)	8.943 ± 0.63 *
9	0 (3)	0 (3)	0 (50)	7.610 ± 0.46 *
10	0 (3)	0 (3)	0 (50)	7.683 ± 0.40 *
11	0 (3)	0 (3)	0 (50)	7.393 ± 0.73 *
SPA	5	-	-	6.770 ± 0.65

x1 = peptone (g/L), x2 = yeast extract (g/L), and x3 = RPW (%). Asterisks (*) on the samples point to significant differences as compared to the standard (p < 0.05), according to the t-test.

). Exception made to treatment 5, all the remaining treatments resulted in significantly higher yields than those of the SPA standard medium. From a comparison among cell growth results, it can be seen that in this case, the cell concentration in the examined range was not a determining factor for yield. This suggests that although the SPA medium results in higher bacterial growth (Table 3), other factors related to medium composition, such as Fe, Zn, and Mn micromineral concentrations, could play an important role in yield increase.

The nitrogen source plays an essential role in the growth and production of xanthans by bacteria of the Xanthomonas gender [27]. According to the Pareto plot for xanthan yield (Figure 2), peptone was the most influential independent variable in the observed response, followed by yeast extract and RPW, in addition to the isolated RPW effect and the peptone–RPW combination. These results point out that peptone and yeast extract, when used by themselves, are able to provide good yields. However, RPW supplementation can render the cultivation medium still more efficient.

Previous studies already demonstrated the importance of organic nitrogen sources in xanthan production. Kurbanolgu and Kurbanolgu (2007) and Caegnatto et al. (2011) [28,29] highlighted that peptone and yeast extract provide soluble amino acids and minerals essential to the cultivation medium, creating favorable conditions for biopolymer synthesis. Ozdal and Kurbanolgu (2019) [9] reported a significant increase in xanthan production by using peptones extracted from hens’ feathers, reaching yields in excess of 24 g/L in a shaker at 200 rpm.

In spite of the positive impact of yeast extract, yields observed (4.02 to 4.18 g/L) were lower than those obtained in this study, suggesting that yeast extract per se may be not as efficient as when combined with other components, such as peptone and RPW. Da Silva et al. (2018) [30] also utilized agro-industrial wastes such as coconut and cocoa peel, reaching values of 3.89 to 4.48 g/L. In the present study, the use of RPW as an alternative source provided yields higher than those reported in these papers, indicating that this agro-industrial waste can be a viable alternative for optimizing xanthan production.

Demirci et al. (2019) [10] obtained a superior yield (14.3 g/L) by utilizing disposed bread waste; however, the hypothesis of an inflated yield cannot be discarded in view of the presence of starch. Trivunović et al. (2024) [12] reached yields between 4 and 10 g/L by utilizing rosé wine wastewater. Yield variations can be explained by differences in substrate composition and adopted fermentation conditions. Similar yields obtained from treatments tested in this study reinforce the viability of partial replacement of conventional medium components for RPW, peptone, and yeast extract, without compromising xanthan productivity. This finding is relevant for process scalability since it enables using agro-industrial wastes as sustainable, economically viable alternatives while maintaining a competitive performance as compared with conventional substrates.

3.3. Residual Nitrogen

Nitrogen contents measured in initial time (0 h) varied between 4.52 and 12.21 mg/dL, suggesting that culture medium composition was directly influenced by peptone, yield extract, and RPW concentrations (

Table 5. Residual nitrogen in media containing different peptone, yield extract, and RPW concentrations at 0 and 24 h X. arboricola pv. pruni strain 101 cell growth.

Treat.	x1	x2	x3	Residual Nitrogen 0 h (mg/L)	Residual Nitrogen 24 h (mg/L)
1	−1 (1)	−1 (1)	−1(20)	45.2 ± 1.3 *	102.8 ± 8.6 *
2	+1 (5)	−1 (1)	−1 (20)	78.2 ± 1.9 *	280.4 ± 4.5 *
3	−1 (1)	+1 (5)	−1 (20)	81.3 ± 3.2	236.9 ± 6.1 *
4	+1 (5)	+1 (5)	−1 (20)	94.4 ± 2.8 *	449.9 ± 4.6 *
5	−1 (1)	−1 (1)	+1 (80)	48.1 ± 2.5 *	108.2 ± 8.5 *
6	+1 (5)	−1 (1)	+1 (80)	85.7 ± 1.9 *	374.0 ± 6.1
7	−1 (1)	+1 (5)	+1 (80)	82.1 ± 2.5 *	286.4 ± 7.5 *
8	+1 (5)	+1 (5)	+1 (80)	122.1 ± 9.0 *	433.9 ± 8.6 *
9	0 (3)	0 (3)	0 (50)	82.9 ± 1.6	349.4 ± 3.2 *
10	0 (3)	0 (3)	0 (50)	90.5 ± 1.9 *	336.3 ± 5.5 *
11	0 (3)	0 (3)	0 (50)	89.7 ± 0.9	359.9 ± 9.3 *
SPA	5	-	-	91.8 ± 0.6	376.4 ± 1.8

x1 = peptone (g/L), x2 = yield extract (g/L), and x3 = RPW (%). Asterisks (*) on the samples point to significant differences as compared to the standard (p < 0.05), according to the t-test.

).

After 24 h, a significant increase in nitrogen concentrations was observed, reaching values between 102.8 and 449.9 mg/L. This behavior was consistent for all treatments, with the highest increases being observed for those containing higher peptone and yeast extract concentrations. For example, for treatment 4 (+1 peptone and +1 yeast extract), nitrogen concentration increased from 9.44 to 44.99 mg/dL, while for treatment 8 (having the same peptone and yeast extract, but higher RPW percentage), the increase was from 12.21 to 43.99 mg/dL. Such an increase after 24 h cultivation was also observed by Macagnan et al. (2021) [31] when studying the influence of different yeast extracts on X. arboricola pv. pruni strain 101 cell growth.

The cause of nitrogen increase was not completely revealed, but it is suggested that the X. arboricola bacterium can be involved in a process of atmospheric nitrogen fixation [32]. Alternatively, gradual nitrogen release from the degradation of compounds present in peptone and in yeast extract may have contributed to the observed increase. Further studies are required to confirm possible nitrogen fixation and identify the mechanisms at the origin of this phenomenon.

As regards the effects of independent variables, peptone exhibited a positive relationship with initial nitrogen content (Figure 3). At higher concentration (+1, corresponding to 5 g/L), nitrogen contents were significantly higher, as could be observed from treatments 4 (9.44 mg/dL) and 8 (12.21 mg/dL), nitrogen values at 0 h were consistently higher, as in treatments 3 (8.13 mg/dL) and 7 (8.21 mg/dL).

3.4. Viscosity

The rheological properties of xanthan were described by the Ostwald–de Waele model using K and ɳ parameters, with a determination coefficient (R²) of 0.99 (

Table 6. X. arboricola pv. pruni strain 101 rheological parameters obtained from different RPW, peptone, and yeast extract concentrations.

Treat.	x1	x2	x3	Consistency (K)	Flow behavior (ɳ)
1	−1 (1)	−1 (1)	−1(20)	4.874 ± 0.051 *	0.181 ± 0.004 *
2	+1 (5)	−1 (1)	−1 (20)	1.549 ± 0.018 *	0.308 ± 0.006
3	−1 (1)	+1 (5)	−1 (20)	1.702 ± 0.011 *	0.361 ± 0.000 *
4	+1 (5)	+1 (5)	−1 (20)	0.205 ± 0.002 *	0.605 ± 0.000 *
5	−1 (1)	−1 (1)	+1 (80)	2.102 ± 0.099 *	0.291 ± 0.013
6	+1 (5)	−1 (1)	+1 (80)	0.798 ± 0.024 *	0.436 ± 0.004 *
7	−1 (1)	+1 (5)	+1 (80)	0.737 ± 0.004 *	0.461 ± 0.005 *
8	+1 (5)	+1 (5)	+1 (80)	0.290 ± 0.002 *	0.590 ± 0.019 *
9	0 (3)	0 (3)	0 (50)	0.853 ± 0.018 *	0.417 ± 0.000 *
10	0 (3)	0 (3)	0 (50)	1.816 ± 0.027 *	0.337 ± 0.009 *
11	0 (3)	0 (3)	0 (50)	0.989 ± 0.071 *	0.402 ± 0.015 *
SPA	5	-	-	2.399 ± 0.066	0.298 ± 0.007

x1 = peptone (g/L), x2 = yeast extract (g/L), and x3 = RPW (%). Asterisks (*) on the samples point to significant differences as compared to the standard (p < 0.05), according to the t-test.

). The consistency index (K), which translates the resistance to fluid flow, varied from 4.874 to 0.205 for treatments 1 and 4, respectively. The flow behavior index (ɳ) varied from 0.605 and 0.181, indicating the relationship between shear rate and viscosity. A value of (ɳ) below 1 (ɳ < 1) confirms the pseudoplastic behavior [1].

Treatments obtained from different RPW, peptone, and yeast extract concentrations enabled us to obtain xanthan products having different properties. For treatment 1, with the lower peptone concentrations, yeast extract, and RPW, the consistency index (K) was the highest, 4.874, pointing to high viscosity, while the flow behavior index (ɳ) was the lowest, 0.181, featuring a more accentuated pseudoplastic behavior. High viscosity provides better water retention capacity, which contributes to higher stability and improvement in food texture for the food industry. On the other hand, for treatment 4, with high peptone (5 g/L) and yeast extract (5 g/L) concentrations, the consistency index (K) was the lowest, at only 0.205, and the flow behavior index (ɳ) was the highest, 0.605.

Comparatively, Cancella et al. (2024) [1] investigated xanthan production from milk substrates, such as milk permeate and deproteinized whey in a shaker. The consistency index (K) of the obtained xanthan products was 1.829 and 0.874, respectively, which were lower than those obtained from rice parboiling water. Crugeira et al. (2023) [33] also assessed xanthan biosynthesis using wet olives bagasse, with 15% and 20% concentrations, resulting in consistency indices (K) of 4.353 and 4.216. Besides, flow behavior (ɳ) indices were 0.2939 for 15% concentration and 0.2534 for 20%. Trivunović et al. (2024) [12] made use of vineyard wastewater and obtained viscosity values between 40 and 60 mPa.

These values are lower than those found in the present study signaling that RPW can be a highly efficient substrate for obtaining high-viscosity xanthan products. It is important to consider that cultivation medium composition, Xanthomonas strain employed, and production conditions, such as temperature, time, pH, agitation, and aeration, can significantly influence polymer structure and consequently the rheological properties of the produced gum [34]. Based on the results of Figure 4, it can be observed that the response value is mainly influenced by peptone and yeast extract concentration, with negative effects of 4.49 and 4.35, respectively, at a 5% significance level. This means that the higher the contents of these components, the lower the gum viscosity at the tested concentration range. This is desirable since it involves input savings.

In Figure 5, it is possible to observe the independent variables’ effect on the flow behavior index (ɳ). Yeast extract was the highest positive impact factor on this parameter, with a standardized effect of 6.66, followed by peptone with an effect of 5.36. This means that both components, at higher concentrations, in the range examined, significantly increase the value of (ɳ), which means lower pseudoplasticity.

3.5. Minerals

The xanthan gum mineral composition varied among the treatments, influenced by the medium components. As expected, high RPW, peptone, and yeast extract concentration treatments favored the incorporation of minerals into the biopolymer. P was the element that most differed from the value ascertained for xanthan pruni synthesized in the standard medium—SPA. P, Si, Ca, and Mn exhibited higher concentration in high-RPW treatments. K was more influenced by peptone concentration and yeast extract. Na, Zn, Mg, and Mn were kept relatively stable.

Other elements, besides those listed in the

Table 7. Mineral salts of xanthan gum obtained from Xanthomonas arboricola pv. pruni strain 101 from different concentrations of rice parboiling water, peptone, and yeast extract.

Treat.	P (mg/g)	Zn (mg/g)	Fe (mg/g)	Si (mg/g)	Ca (mg/g)	K (mg/g)	Mg (mg/g)	Mn (mg/g)	Na (mg/g)
1	13.72 ± 0.53 *	0.03 ± 0.01	0.03 ± 0.004	0.05 ± 0.003	1.84 ± 0.02	21.53 ± 0.39 *	3.51 ± 0.12	0.05 ± 0.001 *	2.85 ± 0.04
2	13.54 ± 0.29 *	0.03 ± 0.01	0.03 ± 0.004	0.05 ± 0.01	1.98 ± 0.05 *	25.33 ± 0.96	3.52 ± 0.28	0.05 ± 0.005 *	2.53 ± 0.01 *
3	15.42 ± 0.32 *	0.02 ± 0.004	0.06 ± 0.005 *	0.04 ± 0.005	1.67 ± 0.16	22.63 ± 0.35 *	3.75 ± 0.09 *	0.09 ± 0.14 *	2.67 ± 0.08 *
4	16.08 ± 0.19 *	0.05 ± 0.02	0.07 ± 0.006 *	0.10 ± 0.01 *	1.87 ± 0.01	26.36 ± 1.35	4.06 ± 0.10 *	0.05 ± 0.001 *	2.92 ± 0.08
5	31.49 ± 1.73 *	0.04 ± 0.005	0.05 ± 0.006 *	0.23 ± 0.01 *	2.41 ± 0.08 *	22.28 ± 0.15 *	4.12 ± 0.07 *	0.18 ± 0.01	2.95 ± 0.09
6	25.66 ± 2.03 *	0.06 ± 0.01	0.08 ± 0.01 *	0.16 ± 0.001 *	2.76 ± 0.30 *	26.06 ± 0.78	4.33 ± 0.25 *	0.19 ± 0.02	2.41 ± 0.13 *
7	29.21 ± 1.37 *	0.02 ± 0.008	0.05 ± 0.003 *	0.14 ± 0.01 *	2.33 ± 0.11 *	24.95 ± 0.10	4.34 ± 0.28 *	0.15 ± 0.004 *	2.40 ± 0.11 *
8	27.10 ± 0.05 *	0.02 ± 0.001	0.05 ± 0.008 *	0.15 ± 0.02 *	3.73 ± 0.07 *	26.11 ± 0.03	4.61 ± 0.40 *	0.11 ± 0.005 *	2.59 ± 0.08 *
9	16.18 ± 0.33 *	0.06 ± 0.001	0.05 ± 0.003 *	0.06 ± 0.01	2.21 ± 0.04 *	25.66 ± 0.16	3.87 ± 0.07 *	0.11 ± 0.004 *	2.59 ± 0.05 *
10	17.10 ± 0.05 *	0.06 ± 0.01	0.06 ± 0.01 *	0.08 ± 0.01 *	1.86 ± 0.003	22.28 ± 0.15 *	4.24 ± 0.10 *	0.09 ± 0.005 *	2.48 ± 0.11 *
11	18.33 ± 0.50 *	0.02 ± 0.001	0.07 ± 0.004 *	0.12 ± 0.01 *	2.24 ± 0.04	24.14 ± 0.85	3.57 ± 0.02 *	0.13 ± 0.006 *	3.12 ± 0.08 *
SPA	12.81 ± 0.03	0.03 ± 0.001	0.03 ± 0.004	0.05 ± 0.001	1.59 ± 0.13	25.80 ± 1.09	3.43 ± 0.05	0.19 ± 0.11	2.93 ± 0.04

Asterisks (*) on the samples point to significant differences as compared to the standard (p < 0.05), according to the t-test.

, were also analyzed. Ti, B, and Al were found only as trace elements. Analyzes for Cd, Ba, Cu, Ni, Pb, Cr, and As were performed, but these elements were not detected in the obtained xanthan. According to the monography provided by the FAO/WHO Expert Committee on Food Additives [35], xanthan should not contain more than 2 mg/Kg lead in its general form, with the limit reduced to 0.5 mg·kg⁻¹ for infant formulae. Besides, the Food Chemicals Codex [36] monography sets a limit of 3 mg/Kg for As in xanthan gum. However, the results of the present research pointed out that xanthan pruni did not exhibit any detectable lead or arsenic concentration, thus it meets the safety standards required for human consumption.

Xanthan gums exhibited variable concentrations of monovalent salts, between 2.44% and 2.93%. Torres et al. (1993) [37] observed 4.97% monovalent salts in xanthan gums produced by X. Campestris. Similarly, Borges (2007) [38] reported 5% monovalent salts for X. arboricola pv. pruni 115 strain, while commercial xanthan gums showed monovalent salts content variation from 0.67% to 3.2%. It should be mentioned that the total cation concentration is directly related to the negative hydroxyl, pyruvyl, and acetyl groups/ions [39].

The relationship among ions is crucial for understanding xanthan’s functional properties. Among monovalent salts, Na’s contribution to viscosity increase is higher than that of K. Moreover, the replacement of K with Na through ionic exchange [40] constitutes a strategy to increase xanthan viscosity. Treatment 1, with lower K content, had the highest viscosity (K 4.874) as compared with both the remaining treatments and the standard. In spite of the fact that monovalent salts contribute to viscosity increase, generally, these are not responsible for forming strong gels due to their weaker interactions as compared with divalent and trivalent salts [41].

4. Conclusions

In the face of growing environmental challenges caused by increasing agro-industrial wastes, the use of RPW for producing biopolymers like xanthan is a sustainable, economically viable alternative. The partial (80%) replacement of peptone for rice parboiling water and yeast extract increased xanthan pruni yield and improved its rheological properties, increasing viscosity and pseudoplasticity. Besides, RPW fostered higher incorporation of minerals such as phosphorus, silicon, calcium, and magnesium. Potassium directly depended on P and YE concentration. Toxic elements such as arsenic and lead were not detected in the obtained xanthan. These findings evidence the potential of agro-industrial wastes as efficient alternatives for sustainable biopolymer production. Besides, obtained data provides important subsidies for bioprocess optimization, focusing on yield increment and end product quality. Aiming at scalability, complementary studies are being carried out in agitated flasks and bioreactors to assess the effect of RPW addition to the xanthan pruni production medium, which will contribute to future industrial production in line with sustainable development.