Improved bioprocess for enhanced xylitol synthesis by newly isolate Meyerozyma caribbica (CP02)

doi:10.21203/rs.3.rs-3345022/v1

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Research Article

Improved bioprocess for enhanced xylitol synthesis by newly isolate Meyerozyma caribbica (CP02)

https://doi.org/10.21203/rs.3.rs-3345022/v1

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published 24 Feb, 2024

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The present work models the fermentation process parameters of the newly isolated, Meyerozyma caribbica CP02 for enhanced xylitol production and its fermentability study on rice straw hydrolysate. The impact of process variables was initially studied one at a time each followed by statistical validation. Temperature 32°C, pH 3.5, 200 rpm, 1.5% (v/v) inoculum, 80 gL^− 1 initial xylose was optimized and a sequential two-stage agitation in fermentation process was adopted. At optimized conditions, xylitol yield of 0.77 gg^− 1 and 0.64 gg^− 1 was actualized using media containing commercial and rice straw derived xylose, respectively. For scale up, in 3L batch bioreactor, the highest xylitol yield (0.63 gg^− 1) was attained at 72 h with media containing rice straw derived xylose (59.22 gL^− 1) along with inhibitors (1.82 gL^− 1 aliphatic acids, 0.141 gL^− 1 furans, 0.95 gL^− 1 total phenols). This implies, M. caribbica CP02 demonstrated good hydrolysate fermentability even at high initial xylose concentration. Therefore, isolate CP02 has potential commercial feasibility in bio-refineries for high yield xylitol production with minimal hydrolysate processing.

Rice straw

Lignocellulose

Xylitol

Bioreactor

Fermentation

Pretreatment

Rice is a staple food crop for most of the world's population, with a global production of 730 MT/yr and a massive generation of rice straw (RS) as a subsequent waste by-product. There is currently no sustainable method for managing the large amount of RS produced, 741–1111 MT/yr (290 kg RS/ton of milled rice). Most of it is either left on the ground or burned on site, which has several ecological, health, and environmental consequences (Louis et al., 2022; Toan et al., 2022). Moreover, straw burning culminates in the accumulation of atmospheric contaminants, such as carbon monoxide, carbon dioxide, volatile organic compounds, oxides of sulphur and, polycyclic aromatic hydrocarbons which deteriorates the air quality and crop yield (Li et al., 2023). Hence, there is an urgent need to develop large-scale conversion technologies to valorize RS for the development of commercially relevant value-added products such as xylitol. The biotransformation of xylose into xylitol from hemicellulose of lignocellulosic biomass such as rice straw is also of interest to modern biorefineries, as it can be processed into a variety of bio-based chemical products after the cellulosic fraction has been used for ethanol production. According to the US Department of Energy, xylitol is one of the top twelve carbohydrate-derived chemicals with the potential to be a co-product of plant biomass-led bio-refinery (Vallejos and Area, 2017) and, also with lactic acid, which is one of the most advanced productions from lignocellulosic biomass (Baptista and Domingues, 2022). Xylitol has been approved for use in food products in over 50 countries for dietary purposes, particularly for food, pharmaceutical and odontological applications. Additionally, xylitol also possesses good stabilizing, moisturizing and cryoprotectant properties and has applicability in cosmetics and polymer industries too (Umai et al., 2022). Currently, the annual market sale of xylitol is $823.6 million, with a projected increase to $1.37 billion by 2025(Hernández-Pérez et al., 2019). Commercially, xylitol production entails catalytic hydrogenation of D-xylose under extremely high temperature and pressure conditions, making the entire process very expensive and energy intensive. The biotechnological route allows for cheaper xylitol production while also allowing for the sustainable use of excess RS. Factors influencing microbial xylitol production include initial xylose concentration, the presence of other monosaccharides (primarily glucose and arabinose), process parameters (pH, temperature, oxygen supply, etc.), and the presence of microbial inhibitors (organic acids, furans, and phenolics), among others. Oxygen is required for yeasts to metabolize xylose, as it is linked to ATP formation, co-enzyme regeneration, and sugar transport throughout the oxidative phosphorylation pathway (Ahuja et al., 2020; Tizazu et al., 2018). On one hand, high shaking speed promotes cell biomass growth, while on the other it is detrimental to xylitol concentration as the latter is only accumulated under low oxygen conditions (Ding and Xia, 2006). This issue may be solved by achieving a balance between biomass growth and xylitol accumulation during the elongated stationary phase under microaerobic conditions. Furthermore, RS must be pretreated before it can be used for yeast-mediated xylitol production, and the composition of RS hydrolysate (RSH) directly influences the microbe's fermentation performance, cell biomass formation, and, ultimately, xylitol production (Singh, Arya and Krishania, 2021; Kaur et al., 2023). Levulinic and formic acids are produced during pretreatment as a result of furan decomposition, whereas acetic acid is produced by the release of acetyl groups from hemicelluloses. HMF and furfural are formed as a result of hexose and pentose degradation respectively, in addition to other phenolic compounds produced by lignin degradation. All these compounds act cumulatively to either deter or decrease xylitol production by microbes (Silva et al., 2013; Brás et al., 2014). Additionally, due to the inherently low xylose amount present in RS, 15.10–19.0% (w/w) as compared to other lignocellulosic biomasses such as corn cob, 28.0- 31.1% (w/w), and sugarcane bagasse, 21.80–27.0% (w/w), it needs to be extensively treated and concentrated to achieve the desirable xylose level, which is accompanied by a rise in the total inhibitor amount as well (Silva et al., 2010, Silva et al., 2015, Parades et al., 2015; Pippo et al., 2011). To facilitate low-cost fermentation operations, it is desirable to isolate novel yeast strains with good xylose to xylitol conversion ability and inhibitor tolerance (Silveira et al., 2020; Lopez et al., 2021). Candida sp. is the main xylitol producer; however, some of its species are also considered opportunistic pathogens, limiting its overall applicability, especially in food-led industries (Abdelgalil et al., 2021). In present bioconversion study, the two-step agitation and aeration pattern for xylitol synthesis improvement was investigated, beginning with a shake flask, and progressing to a 3L batch bioreactor, for aggrandized xylitol synthesis by considering all operational parameters. This work also studied the effects of compositional influence of RS hydrolysate at different concentrations on the behaviour of M. caribbica. The potential of wild isolate M. caribbica CP02 has also been validated, which produced remarkable xylitol yield from minimally processed RS acidic hydrolysate at high initial xylose and total inhibitor concentration, paving the way for an efficient bioprocess for commercialization.

All the components used for media formulation were of analytical grade procured from Hi-media, India, and standards from Sigma Aldrich.

2.1 Screening of xylose-utilizing microorganisms for xylitol production

1g of soil samples collected from RS dumping yard, garden, sugarcane field, cow dung and decaying wood, were inoculated each in 500 ml Erlenmeyer flasks containing 100 ml sterile YPX media (% w/v): 0.25 yeast extract, 0.50 peptone, 3 xylose (pH 5.5) and incubated at 32°C for 96 h in a rotary shaker (Climo-Shaker ISF1-X, Basel, Switzerland) at 150 rpm. 100µl of broth from each of the inoculated flasks was serially diluted and spread on nutrient agar plates containing 1% (w/v) xylose as the principal carbon source and incubated overnight at 32°C. A loopful of the resulting distinct microbial colonies was taken from each plate and inoculated in 100 ml flasks with 40 ml of YPX media and incubated at 150 rpm for 96 h. The isolate CP02 was chosen for further research based on its xylitol production efficiency, and identification was performed at IMTECH, Chandigarh.

2.2 Morphological analysis and identification of CP02

Inverted microscopy was used to determine the colony and cell morphology of the selected isolate. Phylogenetic analysis was performed using the sequences that were downloaded based on Blast search similarity of ITS and 26S rRNA gene (LSU) regions and recently published data

(Yurkov et al., 2017, Zhai et al., 2019). The multiple sequence alignment for individual gene regions were carried out online at the MAFFT server (http://mafft.cbrc.jp/alignment/server/) (Katoh et al., 2019), and alignments were manually corrected using BioEdit (Hall 1999). Maximum likelihood trees under the GTR + GAMMAI (GTR substitution model with gamma- distributed rate heterogeneity) model with 1000 bootstrap replicates was constructed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2014; Stamatakis et al., 2008) at the CIPRES Science Gateway platform (Miller et al., 2010).

2.3 Determination of factors affecting xylitol production – OVAT

For preliminary analysis of the operational factors determining fermentation, one variable at a time (OVAT) approach was used. In a 100 mL flask, 40 mL production medium containing (gL^− 1) 50.0 xylose, 10 yeast extract, 10 peptone, 1 MgSO₄.2H₂O, 5 CaCl₂.7H₂O, 5 KH₂PO₄, pH 5.5 was inoculated with 1.0% (v/v) of a 18 h old seed culture and incubated at 32°C for up to 120 h. The effect of incubation temperature on xylitol yield was studied by carrying out the fermentation from 28 to 36 $^\circ$C. The influence of media pH was studied at several pH values ranging from 3.0 to 7.0 at above mentioned conditions. The effect of rate of agitation was analyzed by varying the rpm from 100 to 300. Likewise, the influence of inoculum size was studied by varying the inoculum concentration from 0.5 to 9% (v/v). The effect of initial substrate concentration was analyzed in the range of 50 to 100 gL^− 1 xylose.

2.5 Modelling and optimization of the xylitol production process by RSM design

Based on the optimal operational values of process variables attained, the Box Behnken Design of Response surface methodology (RSM) was used to statistically optimize the important fermentation process parameters further. Also, the interaction of these variables on xylitol production was studied. Based on this, a more specific range of variables in RSM was considered and coded as A (coded: 50–110 gL^− 1 xylose), B (coded: 0.5–2.5% (v/v) inoculum, C (coded: 2.5–4.5 pH), and D (coded: 100–300 rpm agitation rate). All experiments were performed at 32°C as determined previously by OVAT. Regression analysis was performed using the statistical software Design Expert 11.1.2.0 (Stat Ease, Minneapolis, USA) to determine the cumulative influence of independent variables on the response. A set of 30 runs with 6 central points was performed discretely and in randomized manner (Table 1T). The significance of every model value was established with ANOVA.

2.6 Study of two stage agitation pattern for improved xylitol production

Next a sequential variation in agitation was investigated as an approach to further increase xylitol yield by the isolate CP02. The experiment was performed with a higher agitation in the first phase which enables a better oxygenation followed by lowering the agitation in second phase of fermentation process for xylitol accumulation under oxygen-deficient condition. Fermentation was carried out at optimized conditions in 500 ml shake flask containing 100 ml production media (80 gL^− 1, initial xylose) at 32°C for 96 h by varying the rpm after time interval of 34 h, as optimized previously (data not included) in various combinations such as 250/200, 250/100, 250/150, 200/150 and 200/100 (Zhang et al., 2012). The flask shaking constantly at 200 rpm, throughout the entire incubation period was take as control.

2.7 Xylose rich RS hydrolysate preparation

RS was procured from local farms in Mohali region of Punjab, India. The RS was subjected to dilute acid catalyzed steam pretreatment using 1.5% v/v H₂SO₄ for 30 min according to the method proposed by Singh et al. (2021) and modified accordingly. After bringing the acidic RSH (rice straw hydrolysate) to pH 3.5 with Ba(OH)₂, it was centrifuged for 25 min to remove the insoluble salt, Ba(SO₄)₂. The supernatant was mixed with 2% w/v activated charcoal powder at 60°C for 1 h before being removed by vacuum filtration through a 0.45µm cellulose acetate membrane filter. The detoxified hemicellulosic hydrolysate liquor was subdivided into three parts and concentrated under vacuum using a rotary evaporator (Rotavapor RV.10, IKA Staufen, Germany) to achieve final xylose levels of 60, 80, and 100 gL^− 1. The concentration of monosaccharides, aliphatic acids, and furans in the detoxified and concentrated hydrolysate was estimated using the collected aliquots.

2.8 Shake-flask study of detoxified and concentrated RSH for xylitol production

500 mL flasks containing 100 mL RSH set to different xylose levels were prepared according to standardized media conditions and supplemented with gL^− 1: 7.5 yeast extract, 1 MgSO₄.7H₂O, 0.5 CaCl₂, 0.5 KH₂PO₄, pH 3.5 and sterilized at 110°C for 15 min (see supplementary material 1, Fig. 1SF). The Erlenmeyer flasks were inoculated with 1.5% (v/v), 18 h old inoculum culture and incubated at 32°C and 200 rpm for 34 h before switching to 150 rpm for the remaining period.

2.9 Xylitol production using from RSH in a 3L batch bioreactor

Batch process for xylitol production was performed in a 3L bench top Bioreactor (Bio-Spin series, Bio-Age Equipment and services, Mohali, India) with 2.5 L working volume, equipped with pH probe (Broadley James, USA), dissolved oxygen sensing probe (Broadley James, USA) and a temperature sensor. As previously described, detoxified RSH medium with xylose level adjusted to approximately 60 gL^− 1 was used as fermentation medium upon nutrient supplementation and steam sterilization. Prior to sterilization, the pH of the media was adjusted to 3.5 with 0.1N HCl. The inoculum size was 1.5% v/v and the temperature was held constant at 32°C. As an anti-foaming agent, 1% (v/v) polypropylene glycol was used. For the first 24 h, the airflow was kept at 1.2 Lmin^− 1 and 200 rpm agitation, then reduced to 0.6 Lmin^− 1 and 150 rpm respectively, for the remaining time. Samples were taken on a regular basis for metabolite and cell biomass quantification.

2.10 Analytical methods

Microbial cell biomass was evaluated by computing absorbance at 600 nm in spectrophotometer (UV-1900i Shimadzu Corporation Kyoto, Japan). Morphological analysis of the microbial cell and colony characteristics was performed by Inverted microscope (Nikon H600L, Nikon, Japan) equipped with Nikon’s Digital Sight TS2-S-SM and Scanning electron microscope (Jeol, JCM-6000 benchtop SEM, Tokyo, Japan). Extracellular metabolite (acetic acid, formic acid, levulinic acid, glucose, xylose, arabinose, xylitol, arabitol, HMF and furfural) quantification was conducted by HPLC (Agilent, HiPlex, Santa Clara, California, USA) equipped with a Hi-Plex H column (300 × 7.7 mm, Agilent) coupled to a Refractive index detector. Five mM H₂SO₄ was used as the mobile phase at a flow rate of 0.6 mL/min. Column temperature was maintained at 60°C and that of detector at 55°C. The concentration of total phenols was estimated by Folins Method (Lamuela-Raventos, 2017).

2.11 Estimation of fermentation kinetics parameters

The yield of xylitol (Y_xylitol/S; g/g) was computed by angular coefficient from a linear regression of plot between xylitol concentration, P (gL^− 1) and xylose concentration, S (gL^− 1). Xylitol productivity, Q_p, (gL^− 1h^− 1) was calculated by the highest xylitol production (gL^− 1) and fermentation time (h), using formula: Q_p= $\frac{\text{g}/\text{L}}{\text{h}}$. The efficiency of xylitol conversion (η_xylitol) was calculated by formula: η_xylitol=$\frac{{\mathbf{Y}}_{\frac{\mathbf{P}}{\mathbf{S} \left(\mathbf{x}\mathbf{y}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{o}\mathbf{l}\right)}}}{{\mathbf{Y}}_{\frac{\mathbf{P}}{\mathbf{S} \left(\mathbf{t}\mathbf{h}\mathbf{e}\mathbf{o}\mathbf{r}\mathbf{e}\mathbf{t}\mathbf{i}\mathbf{c}\mathbf{a}\mathbf{l} \mathbf{y}\mathbf{i}\mathbf{e}\mathbf{l}\mathbf{d}\right)}}}$

Where, the estimated theoretical xylitol yield coefficient is 0.91 g/g of xylose (Silveira et al., 2019), based on the following reaction: xylose + NADH + H⁺→ xylitol + NAD⁺ .

Further, also modeled by software LAPFIT, using the modified Gompertz Model:

$$\varvec{Y}= \varvec{A}\varvec{*}\mathbf{exp}\left\{\varvec{B} \varvec{e}\varvec{x}\varvec{p}\left(\varvec{C}\varvec{*}\varvec{X}\right)\right\}$$

Where Y = Specific Xylitol Production (gL^− 1)

A = Xylitol Production Potential (gL^− 1)

B = Lag Phase Duration (h)

C = Maximum Conversion Rate (gL^− 1h^− 1)

3.1 Isolation and identification of xylitol producing yeast strain

The isolate CP02 showed a desirable xylitol yield and therefore, it was selected for further study and identified using phylogenetic analysis. Based on Maximum Likelihood (ML) analysis of a combined dataset of the ITS and D1/D2 domain of 26S rDNA regions showed that our taxon clustered in a monophyletic clade with Meyerozyma carribica strains, with 99.68% sequence similarity. Hence, the isolate CP02 was named by its scientific nomination plus the lab code, i.e., Meyerozyma caribbica CP02 and submitted under the GenBank accession number ON077350 (CP02). M. caribbica CP02 was characterized by an ovoid to elongate, 1.4 × 4–6 µm, which either occurred singly, or in short chains. The colony appeared smooth, butyrous, glistening, and off-white in colour. Furthermore, M. caribbica has been shown to be a non-pathogenic, non-conventional, safe, and rapidly growing yeast even under microaerophilic conditions (Prabhu et al., 2020). Additionally, M. caribbica has also been reported to possess antagonistic properties and maximizes the shelf life of food and beverages by inhibiting the growth of bacteria and fungi, therefore, making it an attractive candidate for a broad range of applications (Qiu et al., 2022).

3.2 Optimization of operational parameters affecting fermentation by M. caribbica CP02

3.2.1 Effect of temperature

A relatively stable xylitol titer of 27.06 to 29.40 gL^− 1 and productivity of 0.28 to 0.30 gL^− 1h^− 1 was obtained in the temperature range of 30–34°C (Fig. 2a). Though, the highest xylitol yield of 29.40 gL^− 1 and productivity (Q_p) of 0.30 gL^− 1h^− 1 was observed at 32°C. Temperature impacts microbial growth rate and xylitol production which might be the reason for almost 24% decrease in xylitol titer (23.4 gL^− 1) at 36°C as compared to xylitol titer at 32°C. A subsequent reduction in xylose consumption by 27% was also observed as temperature also affects the regulation of transport proteins involved in xylose sequestration and enzyme activity (Wang et al., 2011).

3.2.2 Effect of pH

The experimental findings of a set of four flasks for batch xylitol bioconversion were studied to determine the effect of pH ranging from 3.5 to 7.5. Maximum xylitol production (30.5 gL^− 1), yield (0.61 gg^− 1 xylose consumed), and productivity (0.32 gL^− 1h^− 1) were observed at pH 4.0 after 96 h (Fig. 2b). Apart from being acid tolerant, the xylitol yield obtained from M. caribbica CP02 ranged from 0.51gg^− 1 to 0.61gg^− 1 xylose consumed, indicating its activity over a wide pH range. In a similar study using corn cob hydrolysate, Nagarajan et al. (2021) reported the optimum working pH of M. caribbica to be 3.69. Candida tropicalis produced 26.12 gL^− 1 xylitol in the optimum pH range of 3.0 to 4.0 at 200 rpm in a fermentation study on oil palm empty fruit bunch hydrolysate (Mohamad and Kamal, 2009). Moreover, resistance to acidic pH, is not just beneficial for improving the fermentation capacity during second generation fermentation where pH generally falls < 5, which heavily impacts the cell viability, but also for lowering the instance of bacterial contamination under process conditions operational at pH below 5.0 (Vanmarcke et al., 2021).

3.2.3 Effect of agitation rate

The amount of residual xylose in the fermented broth was 0.15 gL^− 1 at 200 rpm, compared to 29.69 gL^− 1 and 32.70 gL^− 1 xylitol in flasks agitated at 250 and 150 rpm, respectively. However, xylose was completely consumed at 250 rpm, whereas 2.01 gL^− 1 xylose remained unconsumed at 150 rpm during the same incubation period (96 h), as shown in Fig. 2c. The xylitol titer obtained at 100 rpm agitation was much lower (30.71 gL^− 1) with 9.35 gL^− 1 residual xylose. The lowest amount of xylitol was obtained at 300 rpm (26.08 gL^− 1) with complete xylose consumption. This is consistent with the study of Prabhu et al. (2020), which showed that the xylose utilization time decreased as agitation increased, with the maximum time taken at 120 rpm (96 h) and the shortest time taken at 300 rpm (72 h) for an initial xylose concentration of 60 gL^− 1 using mutated Pichia fermentans. The xylitol titer also increased linearly up to 250 rpm, after which the yield dropped. The highest xylitol titer and yield obtained by them was 36.6 gL^− 1 and 0.61 gg^− 1 respectively, at 250 rpm.

3.2.4 Effect of inoculum size

The effect of inoculum size on xylitol synthesis by M. caribbica CP02 was investigated at concentrations ranging from 0.5 to 9% (v/v). The highest xylitol production (30.3 gL^− 1) was found at 1% (v/v) inoculum size, with approximately 98% xylose utilization after 96 h of incubation, as shown in Fig. 2d. Furthermore, when the size of the inoculum was increased from 1% (v/v) to 3 and 5% (v/v), the xylitol titer showed a gradual and insignificant decrease to 29.08 and 29.2 gL^− 1, with 98.2 and 98.4% consumption of the initial xylose, respectively. At 7% (v/v) and 9% (v/v) inoculum sizes, the minimum xylitol titer was 27.17 gL^− 1 and 28 gL^− 1, respectively, indicating that using a larger amount of seed culture resulted in the use of xylose for cell growth rather than xylitol production. Furthermore, using the aforementioned inoculum concentrations, only 90.58% and 88.12% of the xylose was utilized after 96 h. The decrease in xylose consumption could be since oxygen is essential for xylose uptake rate by pentose assimilating yeasts, and higher inoculum concentration reduces the oxygen level in the medium (Raj et al., 2013).

3.2.5 Effect of initial substrate

Flasks containing production media were formulated with 50–100 gL^− 1 xylose (Fig. 2e). The highest xylitol titer (50.3 gL^− 1) with a specific xylitol yield of 0.64 gg^− 1, 0.52 gL^− 1h^− 1 productivity, 1.87 gL^− 1 residual xylose was recorded with the xylose amount of 80 gL^− 1. An increase in the xylitol titer to, 56 gL^− 1 and 61.5 gL^− 1 with xylose consumption of 92.13% and 86.02% was seen with 90 gL^− 1 and 100 gL^− 1 initial xylose concentration, respectively after 96 h. The xylitol titer further increased to 57 gL^− 1 and 67 gL^− 1 after 120 h of incubation with yield of 0.67 gg^− 1 and 0.75 gg^− 1 xylose consumed respectively. This implies that increasing the concentration of initial substrate, increases xylitol production in a batch process, but at the expense of a longer incubation period. However, after a certain limit, substrate inhibition occurs and productivity drops as the microorganisms are unable to tolerate the elevated osmotic pressure in a batch process (Zhang and Geng, 2012). Also, from the standpoint of economic feasibility, it may be desirable to shorten the fermentation time.

3.3 Statistical validation

Statistical analysis is an effective way to identify and validate the significant factors affecting the yield of product. Analysis of variance (ANOVA) was employed to examine the studied experimental factors and their statistical significance in the performance of the recommended method.

The F-value (Fischer variance ratio) of the computed model was 73.80 and the p-value of < 0.0001, which implies p < 0.05 for regression model equation confirmed that the quadratic model fit well to the experimental results. The lack of fit value of 2.96 implied that the model’s variables significantly correlated with process response which was xylitol production (Vázquez et al., 2017). Also, the difference between adjusted R² (0.9733) and predicted R² (0.9297) in the current study was less than 0.20, indicating that the model was reliable (Table 2T).

The regression equation for xylitol production after a complete fermentation cycle as a function of the quadratic terms and associated interactions can be presented as:

Xylitol = 57. 13 + 19.34A + 0.7600B -0.7342C + 2.84D + 1.39AB -1.72AC -1.16AD -0.4000 BC + 0.5225 BD + 0.0475 CD -9.23A² -4.67B² -2.06C² -7.65D²

Under microaerobic conditions, the maximum xylitol production, was 57.13 gL^− 1. A, B and D., were the factors that had a positive influence on the production of xylitol. Likewise, interaction of AB, BD and CD also affected the xylitol production positively. Variables with higher F values and lower p values are more significant (Khataee et al., 2010). The independent model variables (p < 0.05) having a significant effect on response, were determined from ANOVA table provided in supplementary material 2, Table 3T. From the results, it was inferred that A and D, were the factors that had a positive influence on the production of xylitol as compared to other variables under study, similar to the observations made in OVAT. Likewise, interaction of AB, BD and CD also affected the xylitol production positively. 3D interaction plots between the two experimental variables have been presented in Fig. 3. Figure 3a shows the response surface plot showing the interactive effect of (AB) of initial xylose concentration (A) and inoculum size (B) on xylitol titer (gL^− 1) which validates that the xylitol yield (gL^− 1) rises with increase in xylose titer from 50 to 110 gL^− 1 for inoculum size between 0.5 to 2.5% (v/v). The influence of AC (xylose and pH) on the response revealed that the xylitol titer (gL^− 1) increased as the initial xylose concentration of the fermentation media approached from 50 to 110 gL^− 1 irrespective of the changes in initial pH from 2.5 to 4.5 (Fig. 3b). Furthermore, the interactive effect of BD (inoculum size and agitation rate) demonstrated that the xylitol yield enhanced as the inoculum size increased from 0.5 to 2.5% (v/v) for the studied rate of agitation from 100 to 300 rpm (Fig. 3c). In a similar study, Yewale et al. (2017), conducted central composite design (CCD) and found that xylose concentration was one of the major factors for xylitol production.

3.4 Influence of variation of agitation speed on xylitol production

A batch fermentation with sequential two stage agitation, which involved altering the agitation speed at previously optimized time duration of 34 h, was efficient for enhancing the xylitol yield.

The highest agitation level, 250/200 rpm (250 rpm in the first stage for 34 h and 200 rpm in the second stage), resulted in the highest cell biomass (22.83 gL^− 1) with the lowest xylitol yield (0.64 gg^− 1). The lowest shaking speed level of 150/100 rpm resulted in the lowest cell biomass concentration (11.20 gL^− 1), xylitol titer (53.93 gL^− 1) and a moderately high xylitol yield (0.74 gg^− 1) with a maximum residual xylose concentration of 7.06 gL^− 1 as shown in Table 4T (Supplementary material 2).

However, agitation at 200/150 rpm showed the highest xylitol production (61.10 gL^− 1), xylitol yield (0.77 gg^− 1), xylose to xylitol bioconversion efficiency (84.61%), the best xylitol productivity (0.64 gL^− 1h^− 1) and complete consumption of xylose at 96 h (refer Fig. 4a, 4b) as compared to control which yielded 57.10 gL^− 1 xylitol, 0.72 xylitol gg^− 1 xylose consumed and 0.59 gL^− 1h^− 1 productivity. This could be due to increment in shaking speed which reduces xylitol titer by promoting biomass production via Tricarboxylic acid cycle. On the contrary, oxygen deficient conditions entirely abolish xylitol formation because of absence of enough cofactor for reductase activity (Branco et al., 2007). Zhang and Geng, (2012) used C. athensensis SB18 and reported that the agitation at 200 rpm for first 24 h followed by 100 rpm for rest of the incubation period exhibited the maximum xylitol titer (115.62 gL^− 1) from 150 gL^− 1 initial xylose and xylitol yield (0.77 gg^− 1) at 30°C for 108 h. Another study using corn cob hydrolysate found that increasing the aeration rate from 1.5 vvm to 3 vvm after 24 h resulted in higher xylitol yield (0.76 gg^− 1) (Ding and Xia, 2005). In shake flask study, maintaining micro aerobic conditions by lowering the agitation speed alone during the second phase improves the buildup of xylitol in broth by encouraging NADPH generation and limiting substrate utilization for cell maintenance (Branco et al., 2007).

3.5 Composition of RS hemicellulosic hydrolysate

Pretreated and detoxified RSH was vacuum concentrated to obtain 100 ml of RSH fractions, RSH1, RSH2 and RSH3 with initial xylose concentrations of 59.12, 78.52 and 98.63 gL^− 1 respectively. The major components of 1.5% v/v H₂SO₄ catalyzed untreated hydrolysate were (gL^− 1): sugars (1.0 glucose, 14.91 xylose, and 3.60 arabinose), aliphatic acids (1.5 acetic acid, 0.28 formic acid, and 0.04 levulinic acid), furans (0.029 HMF and 0.112 furfural) and total phenols (0.95). As previously reported, there was a significant decrease in the amount of these inhibitors after detoxification and evaporation due to vacuum concentration (Singh et al., 2021). However, inhibitors began to accumulate again as RSH concentration increased, with RSH1 having the least amount of total organic acids (1.54 gL^− 1), furans (0.049 gL^− 1), total phenols (0.64 gL^− 1) and RSH3 having the most, i.e., 2.43 gL^− 1, 0.078 gL^− 1 and 2.47 gL^− 1, respectively. Similarly, as shown in Table 1, the increase in initial xylose concentration was accompanied by an increase in the amount of glucose, arabinose and inhibitors as well.

Table 1

Chemical composition of pretreated RSH before and after detoxification, followed by concentration
Pretreat-ment	Sugars (gL^− 1)			Aliphatic acids (gL^− 1)			Furans (gL^− 1)		Total phenols (gL^− 1)
Pretreat-ment	Glucose	Xylose	Arabinose	Acetic acid	Formic acid	Levulinic acid	5-HMF	Furfural	Phenols
UTRSH^#	1.0	14.91	3.60	1.5	0.28	0.04	0.029	0.112	0.95
RSH*1	3.70	59.22	12.04	1.43	0.09	0.02	0.015	0.034	0.64
RSH2	5.53	78.62	20.30	2.06	0.11	0.03	0.018	0.044	1.80
RSH3	7.82	98.73	27.45	2.17	0.22	0.04	0.021	0.057	2.47
Note: # UTRSH; Rice straw hydrolysate obtained after steam pretreatment * RSH; Detoxified and vacuum concentrated rice straw hydrolysate consisting of initial xylose concentration adjusted at, 1) 59.12, 2) 78.52 and, 3) 98.63 gL^-1

3.6 Study of xylitol production by M. caribbica CP02 with RSH

Two stage agitation during fermentation was carried out in 500 mL shake flasks in batch mode using RSH derived media (RSHM) at optimized conditions to assess the RSH fermentability of M. caribbica CP02 under high initial xylose concentrations as compiled in Table 2. As can be seen, the xylitol titer decreased as the level RSH concentration increased. RSHM1 had the highest xylose titer (36.10 gL^− 1), xylitol yield (0.62 gg^− 1), and productivity (0.38 gL^− 1h^− 1) (Fig. 5a). In comparison to the 500 mL shake flask model fermentation with a similar amount of commercial xylose (80 gL^− 1) taken initially under the same conditions, a reduction of nearly 75% of xylitol titer (15.01 gL^− 1) and 48% of yield (0.44 gg^− 1) was observed after 96 h incubation (Section 3.5). A similar study using C. tropicalis GS18 yielded 0.60 gg^− 1 and a xylitol titer of 34.21 gL^− 1 from detoxified RS hydrolysate concentrated to obtain an initial xylose level of 58.78 gL^− 1 (Kaur et al., 2022). Similarly, the accumulated biomass concentration decreased from 18.30 gL^− 1 (RSHM1) to 13.71 gL^− 1 (RSHM2) and 10.23 gL^− 1 (RSHM3). This decrease in fermentation parameters could be attributed to the combined stress exerted by elevated levels of total solids and inhibitors at highly concentrated RSH. Furans and their corresponding alcohols have been shown to reduce cell biomass yield, whereas aliphatic acids inhibit cell growth by facilitating the influx of dissociated form, thereby increasing H⁺ ions into the cytoplasm (Palmqvist and Hägerdal, 2000). Moreover, lag phase of M. caribbica CP02 stretched from about 12 h in RSHM1 to 48 h and 72 h in RSHM2 (Fig. 5b) and RSHM3 (Fig. 5c) respectively. Although, a sudden increase in xylitol production (38.22 gL^− 1) was observed at the beginning of stationary phase i.e., 120 h on RSHM2, indicating the good adaptability, tolerance, and fermentation efficiency of M. caribbica CP02 even with high initial xylose concentration (78.52 gL^− 1) and inhibitors such as acetic acid (1.08 gL^− 1), formic acid (0.10 gL^− 1), levulinic acid (0.03), 5-HMF (0.018 gL^− 1) and furfural (0.044 gL^− 1). This implies that M. caribbica CP02 could produce good xylitol yields in RS-derived media with initial xylose concentrations as high as 80 gL^− 1, but at the expense of a longer process time. A reduction in sugar consumption and partial inhibition was observed when the initial xylose concentration in RS hydrolysate was increased to 98.63 gL^− 1. The minimum xylitol titer obtained was 4.24 gL^− 1 after 96 h, while the xylitol concentration rose to 25.15 gL^− 1 after 120 h. The decrease in xylose to xylitol bioconversion efficiency of M. caribbica CP02 could also be attributed to an increase in RSHM viscosity due to an increase in the amount of dry matter with volume reduction of RSH to achieve the desired xylose concentration. High viscosity may influence yeast metabolic activity and fermentation kinetics (Wang et al., 2020). RSHM1 was chosen for further research after considering all the factors influencing the feasibility of the fermentation process, such as incubation time, nutrient consumption, and total inhibitor accumulation.

Table 2

Shake flask study summarizing fermentation variables of xylitol production by *M. caribbica* CP02 using RSHM
	Residual xylose (gL^− 1)	Xylitol^a (gL^− 1)	X (gL^− 1)	Y_P/S (gg^− 1)	Y_P/X (gg^− 1)	Qp (gL^− 1h^− 1)	ⴄ (%)
RSHM* 1	1.28	36.10	18.30	0.62	1.97	0.38	68.13
RSHM 2	42.17	15.01	13.71	0.40	1.09	0.16	43.95
RSHM 3	75.46	4.24	10.23	0.18	0.41	0.04	19.78
Note: *, Rice straw hydrolysate derived media; ^a, Xylitol titer after 96 h of fermentation
X: biomass, Y_P/S: yield (g xylitol) (g xylose consumed)^-1, Y_P/S: yield (g xylitol) (g biomass produced)^-1, Qp: xylitol productivity, ⴄ: xylose to xylitol bioconversion efficiency

3.7 RSH based two-stage agitation and aeration fermentation in a 3L batch bioreactor

58.20 gL^− 1 of xylose was consumed to produce maximum xylitol titer of 37.13 gL^− 1 and a bioconversion efficiency of 70.32%. A xylitol yield of 0.63 gg^− 1 and, productivity of 0.52 gL^− 1h^− 1 was obtained in batch bioreactor as compared to 0.38 g L^− 1h^− 1 productivity obtained in the 500-mL shake flask, with a similar initial xylose concentration using RSHM. Whereas, the kinetic modeling revealed with the Gompertz Model that the xylose consumption and xylitol production were properly fitting with the experimental data (Fig. 6b, c). The optimized process indicates an improvement in xylitol productivity due to the improved mixing phenomena and higher mass transport efficiency achieved in the growth vessel. Furthermore, in a 500-mL shake flask study, even though the rate of biomass growth decreased during the xylitol accumulation stage, cell growth continued to reach its maximum at 60 h. In bioreactor fermentation, on the other hand, the biomass concentration peaked at 48 h and the final cell dry weight was 18.41 gL^− 1. The rapid biomass growth during the cell growth phase can be linked to a faster xylitol accumulation rate and, as a result, improved productivity. This is in correspondence with the findings of Silva et al. (2006), who found a similar increase in xylitol productivity when switching from aerated flasks to 1L batch bioreactors while keeping the xylitol yield in the same range. Using C. guilliermondii, the researchers reported a xylitol yield, volumetric productivity, and efficiency of 0.59 gg^− 1, 0.54 gl^− 1h^− 1, and 64.3%, respectively, from RSH with an initial xylose concentration of 81.4 gL^− 1. Similarly, a 1L bioreactor study on sugarcane bagasse hydrolysate using M. caribbica JA9 with 40 gL^− 1 xylose yielded 0.54 gg^− 1, which was found to be greater than the control strain M. guilliermondii (0.44 gg^− 1) under the same conditions (Trichez et al., 2019).

Bioprospecting lignocellulosic agricultural waste and selective enrichment on RS derived xylose-rich medium resulted in the isolation and purification of Meyerozyma caribbica CP02, a new xylitol producing yeast. M. caribbica CP02's tolerance to extreme acidic conditions reduces the risk of bacterial contamination during fermentation. In shake flask experiments, commercial xylose yielded 0.74 gg^-1 (ⴄ, 86.81%) and RSH-based media yielded 0.64 gg^-1 (ⴄ, 70.32%) xylitol, respectively. On RSH media, a 3-L bioreactor study of M. caribbica CP02 produced a high xylitol titer of 37.13 gL^-1 with a xylitol yield of 0.63 gg^-1, which is the highest in the literature from rice straw hydrolysate. Therefore, the current study establishes the competence of M. caribbica CP02 for further scale-up as well as its candidature as a potential yeast strain for commercial xylitol bioproduction and as an alternative by-product in biorefineries aiming for holistic biomass utilization.

Ethics approval and consent to participate: Not applicable

Consent for publication: Agreed by all authors

Availability of data and materials: Available on reasonable request

Competing interests: The authors declare no conflict of interest.

Funding: Not applicable

Authors' contributions: Saumya Singh: Methodology, Investigation, Formal analysis, validation, Writing – original draft preparation. Shailendra Kumara Arya: Conceptualization, Supervision, Formal analysis, validation, Writing – review & editing. Meena Krishania: Conceptualization, Supervision, Writing – review & editing Project administration

Acknowledgements: The authors are grateful towards Department of Biotechnology, Government of India for providing financial support in the form of flagship project (BT/CIAB-Flagship/2018), to CIAB, Mohali.

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Improved bioprocess for enhanced xylitol synthesis by newly isolate Meyerozyma caribbica (CP02)

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Abstract

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1. Introduction

2. Materials and Methods

2.1 Screening of xylose-utilizing microorganisms for xylitol production

2.2 Morphological analysis and identification of CP02

2.3 Determination of factors affecting xylitol production – OVAT

2.5 Modelling and optimization of the xylitol production process by RSM design

2.6 Study of two stage agitation pattern for improved xylitol production

2.7 Xylose rich RS hydrolysate preparation

2.8 Shake-flask study of detoxified and concentrated RSH for xylitol production

2.9 Xylitol production using from RSH in a 3L batch bioreactor

2.10 Analytical methods

2.11 Estimation of fermentation kinetics parameters

3. Results and discussions

3.1 Isolation and identification of xylitol producing yeast strain

3.2 Optimization of operational parameters affecting fermentation by M. caribbica CP02

3.2.1 Effect of temperature

3.2.2 Effect of pH

3.2.3 Effect of agitation rate

3.2.4 Effect of inoculum size

3.2.5 Effect of initial substrate

3.3 Statistical validation

3.4 Influence of variation of agitation speed on xylitol production

3.5 Composition of RS hemicellulosic hydrolysate

3.6 Study of xylitol production by M. caribbica CP02 with RSH

3.7 RSH based two-stage agitation and aeration fermentation in a 3L batch bioreactor

Conclusion

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