Isolation, screening, and characterization of the newly isolated osmotolerant yeast Wickerhamomyces anomalus BKK11-4 for the coproduction of glycerol and arabitol.

This study explored the isolation and screening of an osmotolerant yeast, Wickerhamomyces anomalus BKK11-4, which is proficient in utilizing renewable feedstocks for sugar alcohol production. In batch fermentation with high initial glucose concentrations, W. anomalus BKK11-4 exhibited notable production of glycerol and arabitol. The results of the medium optimization experiments revealed that trace elements, such as H3BO3, CuSO4, FeCl3, MnSO4, KI, H4MoNa2O4, and ZnSO4, did not increase glucose consumption or sugar alcohol production but substantially increased cell biomass. Osmotic stress, which was manipulated by varying initial glucose concentrations, influenced metabolic outcomes. Elevated glucose levels promoted glycerol and arabitol production while decreasing citric acid production. Agitation rates significantly impacted the kinetics, enhancing glucose utilization and metabolite production rates, particularly for glycerol, arabitol, and citric acid. The operational pH dictated the distribution of the end metabolites, with glycerol production slightly reduced at pH 6, while arabitol production remained unaffected. Citric acid production was observed at pH 6 and 7, and acetic acid production was observed at pH 7. Metabolomic analysis using GC/MS identified 29 metabolites, emphasizing the abundance of sugar/sugar alcohols. Heatmaps were generated to depict the variations in metabolite levels under different osmotic stress conditions, highlighting the intricate metabolic dynamics occurring post-glucose uptake, affecting pathways such as the pentose phosphate pathway and glycerolipid metabolism. These insights contribute to the optimization of W. anomalus BKK11-4 as a whole-cell factory for desirable products, demonstrating its potential applicability in sustainable sugar alcohol production from renewable feedstocks.

d-Arabitol production by a high arabitol-producing yeast, Zygosaccharomyces sp. Gz-5 isolated from miso.

d-Arabitol, an alternative sweetener to sugar, has low calorie content, high sweetness, low glycemic index, and insulin resistance-improving ability. In this study, d-arabitol-producing yeast strains were isolated from various commercial types of miso, and strain Gz-5 was selected among these strains. Phylogenetic tree analysis of the internal transcribed spacer sequence revealed that strain Gz-5 was distinct from Zygosaccharomyces rouxii, a major fermenting yeast of miso. The strain, identified as Zygosaccharomyces sp. Gz-5, grew better than other Z. rouxii in 150 g/L NaCl and produced 114 g/L d-arabitol from 295 g/L glucose in a batch culture for 8 days (0.386 g/g-consumed glucose). In a fed-batch culture, the yeast produced 133 g/L d-arabitol for 14 days, and the total d-arabitol amount increased by 1.75-fold. These results indicated that Zygosaccharomyces sp. Gz-5, a non-genetically modified strain, has excellent potential for the industrial production of d-arabitol.

Production of arabitol from glycerol by immobilized cells of Wickerhamomyces anomalus WC 1501.

Polyalcohols such as arabitol are among the main targets of biorefineries aiming to upcycle wastes and cheap substrates. In previous works Wickerhamomyces anomalus WC 1501 emerged as an excellent arabitol producer utilizing glycerol. Arabitol production by this strain is not growth associated, therefore, in this study, pre-grown cells were entrapped in calcium alginate beads (AB) and utilized for glycerol transformation to arabitol. Flasks experiments aimed to assess the medium composition (i.e., the concentration of inorganic and organic nitrogen sources and phosphates) and to establish the appropriate carrier-to-medium proportion. In flasks, under the best conditions of ammonium limitation and the carrier:medium ratio of 1:3 (w/v), 82.7 g/L glycerol were consumed in 168 h, yielding 31.2 g/L arabitol, with a conversion of 38% and volumetric productivity of 186 mg/mL/h. The process with immobilized cells was transferred to laboratory scale bioreactors with different configurations: stirred tank (STR), packed bed (PBR), fluidized bed (FBR), and airlift (ALR) bioreactors. The STR experienced oxygen limitation due to the need to maintain low stirring to preserve AB integrity and performed worse than flasks. Limitations in diffusion and mass transfer of oxygen and/or nutrients characterized also the PBR and the FBR and were partially relieved only in ALR, where 89.4 g/L glycerol were consumed in 168 h, yielding 38.1 g/L arabitol, with a conversion of 42% and volumetric productivity of 227 mg/mL/h. When the ALR was supplied with successive pulses of concentrated glycerol to replenish the glycerol as it was being consumed, 117 g/L arabitol were generated in 500 h, consuming a total of 285 g/L glycerol, with a 41% and 234 mg/L/h. The study strongly supports the potential of W. anomalus WC 1501 for efficient glycerol-to-arabitol conversion using immobilized cells. While the yeast shows promise by remaining viable and active for extended periods, further optimization is required, especially regarding mixing and oxygenation. Improving the stability of the immobilization process is also crucial for reusing pre-grown cells in multiple cycles, reducing dead times, biomass production costs, and enhancing the economic feasibility of the process.

Development of an Integrated Bioprocess System for Bioethanol and Arabitol Production from Sugar Beet Cossettes.

Research background: An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. Experimental approach: The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). Results and conclusions: The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L.h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. Novelty and scientific contribution: An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable 'zero waste' production system.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Non-canonical D-xylose and L-arabinose metabolism via D-arabitol in the oleaginous yeast Rhodosporidium toruloides.

R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical L-arabinose and D-xylose metabolism proceeding through D-arabitol and D-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that D-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative D-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of D-xylose to D-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative D-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative L-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

Isolation of Zygosaccharomyces siamensis kiy1 as a novel arabitol-producing yeast and its arabitol production.

Arabitol is gaining attention in the food industry as an alternative sweetener owing to its low-caloric and non-cariogenic characteristics. The yeast strain kiy1 was newly isolated from unpasteurized honey for arabitol production. Based on internal transcribed spacer sequence analysis, the isolated strain was identified as Zygosaccharomyces siamensis. In this study, the effects of different substrates and sugar concentrations on arabitol production were investigated. When three types of carbon sources (glycerol, fructose, and glucose) were used, glucose was the most suitable substrate for arabitol production (68.7 g/L). Maximum arabitol production (101.4 g/L) was observed at a glucose concentration of 30%, and the highest arabitol production yield was 0.34 g/g of initial glucose. In the time-course production of sugar alcohols by strain kiy1, glucose was completely consumed for 8 days. The concentration of arabitol exceeded that of glycerol after 3 days, and the final arabitol concentration reached 83.6 g/L after 10 days. The maximum production rate was 16.7 g/L/day. The yeast produced glycerol as an intracellular sugar alcohol in the early stage of culture and switched its metabolism to arabitol production after the middle stage. Z. siamensis kiy1 possessed an NADP+-dependent arabitol dehydrogenase, which indicated that it probably produces arabitol via ribulose from glucose. These results suggest that the novel yeast strain, Z. siamensis kiy1, is promising for arabitol production. The proposed arabitol production approach can contribute toward its production at the industrial scale.

Structural properties of optically clear bacterial cellulose produced by Komagataeibacter hansenii using arabitol.

Bacterial cellulose (BC) exhibits beneficial properties for use in biomedical applications but is limited by its lack of tunable transparency capabilities. To overcome this deficiency, a novel method to synthesize transparent BC materials using an alternative carbon source, namely arabitol, was developed. Characterization of the BC pellicles was performed for yield, transparency, surface morphology, and molecular assembly. Transparent BC was produced using mixtures of glucose and arabitol. Zero percent arabitol pellicles exhibited 25% light transmittance, which increased with increasing arabitol concentration through to 75% light transmittance. While transparency increased, overall BC yield was maintained indicating that the altered transparency may be induced on a micro-scale rather than a macro-scale. Significant differences in fiber diameter and the presence of aromatic signatures were observed. Overall, this research outlines methods for producing BC with tunable optical transparency, while also bringing new insight to insoluble components of exopolymers produced by Komagataeibacter hansenii.

Identification of an l-Arabitol Transporter from Aspergillus niger.

l-arabitol is an intermediate of the pentose catabolic pathway in fungi but can also be used as a carbon source by many fungi, suggesting the presence of transporters for this polyol. In this study, an l-arabitol transporter, LatA, was identified in Aspergillus niger. Growth and expression profiles as well as sugar consumption analysis indicated that LatA only imports l-arabitol and is regulated by the arabinanolytic transcriptional activator AraR. Moreover, l-arabitol production from wheat bran was increased in a metabolically engineered A. niger mutant by the deletion of latA, indicating its potential for improving l-arabitol-producing cell factories. Phylogenetic analysis showed that homologs of LatA are widely conserved in fungi.

Development of an enzymatic cascade to systematically utilize lignocellulosic monosaccharide.

BACKGROUND: The fermentation valorization of two main lignocellulosic monosaccharides, glucose and xylose, is extensively developed; however, it is restricted by limited yield and process complexity. An in vitro enzymatic cascade reaction can be an alternative approach. RESULTS: In this study, a three-stage, five-enzyme cascade was developed to convert pretreated biomass to valuable chemicals. First, a ribose-5-phosphate isomerase B mutant isomerized xylose to d-xylulose with high substrate specificity, and a d-arabinose dehydrogenase continued to reduce d-xylulose to d-arabitol. Simultaneously, glucose was utilized for the coenzyme regeneration catalyzed by a glucose dehydrogenase, generating useful gluconic acid and achieving 73% of total conversion rate after 36 h. Then, six kinds of pretreated biomass lignocellulose were hydrolyzed by cellulase and hemicellulase, and corn cob was identified as the initial substrate for providing the highest monosaccharide content. A 65% conversion rate of the lignocellulosic xylose was obtained after 24 h. CONCLUSIONS: This study presents a proof of concept to convert main lignocellulosic monosaccharides systematically by an enzymatic cascade at stoichiometric ratio. © 2022 Society of Chemical Industry.

High-level production of d-arabitol by Zygosaccharomyces rouxii from glucose: Metabolic engineering and process optimization.

d-Arabitol is a top value-added compound with wide applications in the food, pharmaceutical and biochemical industries. Nevertheless, sustainable biosynthesis of d-arabitol is limited by lack of efficient strains and suitable fermentation process. Herein, metabolic engineering and process optimization were performed in Zygosaccharomyces rouxii to overcoming these limitations. Adopting systems metabolic engineering include enhancement of innate biosynthetic pathway, supply of precursor substrate d-ribulose-5P and cofactors regeneration, a novel recombinant strain ZR-5A with good performance was obtained, which boosted d-arabitol production up to 29.01 g/L, 59.31 % higher than the parent strain. Further with the optimum medium composition and fed-batch fermentation, the strain ZR-5A finally produced 149.10 g/L d-arabitol with the productivity of 1.04 g/L/h, which was the highest titer ever reported by Z.rouxii system. This is the first report on the use of metabolic engineering to construct Z. rouxii chassis for the sustainable production of d-arabitol.

d-Arabitol Ameliorates Obesity and Metabolic Disorders via the Gut Microbiota-SCFAs-WAT Browning Axis.

d-Arabitol, which is typically found in mushrooms, lichens, and higher fungi, might play an effective role in alleviating visceral fat accumulation and insulin resistance particularly for its low calorie and glycemic index. However, the regulatory mechanisms of d-arabitol for alleviating obesity and associated metabolic disorders remain poorly understood. This study aimed to investigate and analyze the underlying relationship between d-arabitol-mediated gut microbiota and obesity. The results showed that d-arabitol dramatically ameliorated body weight gain, fat accumulation, and insulin resistance in HFD-fed rats. Likewise, d-arabitol remarkably increased the relative abundance of the genera Blautia, Anaerostipes, and Phascolarctobacterium and decreased the genera Romboutsia and Clostridium_sensu_stricto_1. Furthermore, these alterations in gut microflora increased SCFAs, which in turn indirectly promoted AMPK-PGC-1α-related white adipose tissue (WAT) browning. Therefore, d-arabitol would have the potential to alleviate obesity through the gut microbiota-SCFAs-WAT browning axis. It could be considered as a sugar substitute for the obese population and diabetic patients.

Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of D-arabitol from pure glycerol.

BACKGROUND: D-Arabitol, a five-carbon sugar alcohol, represents a main target of microbial biorefineries aiming to valorize cheap substrates. The yeast Wickerhamomyces anomalus WC 1501 is known to produce arabitol in a glycerol-based nitrogen-limited medium and preliminary fed-batch processes with this yeast were reported to yield 18.0 g/L arabitol. RESULTS: Fed-batch fermentations with W. anomalus WC 1501 were optimized using central composite design (CCD). Dissolved oxygen had not a significant effect, while optimum values were found for glycerol concentration (114.5 g/L), pH (5.9), and temperature (32.5 °C), yielding 29 g/L D-arabitol in 160 h, a conversion yield of 0.25 g of arabitol per g of consumed glycerol, and a volumetric productivity of 0.18 g/L/h. CCD optimal conditions were the basis for further improvement, consisting in increasing the cellular density (3✕), applying a constant feeding of glycerol, and increasing temperature during production. The best performing fed-batch fermentations achieved 265 g/L D-arabitol after 325 h, a conversion yield of 0.74 g/g, and a volumetric productivity of 0.82 g/L/h. CONCLUSION: W. anomalus WC 1501 confirmed as an excellent producer of D-arabitol, exhibiting a remarkable capability of transforming pure glycerol. The study reports among the highest values ever reported for microbial transformation of glycerol into D-arabitol, in terms of arabitol titer, conversion yield, and productivity.

Biocatalytic conversion of a lactose-rich dairy waste into D-tagatose, D-arabitol and galactitol using sequential whole cell and fermentation technologies.

Dairy industry waste has been explored as a cheap and attractive raw material to produce various commercially important rare sugars. In this study, a lactose-rich dairy byproduct, namely cheese whey powder (CWP), was microbially converted into three low caloric sweeteners using whole-cell and fermentation technologies. Firstly, the simultaneous lactose hydrolysis and isomerization of lactose-derived D-galactose into D-tagatose was performed by an engineered Escherichia coli strain co-expressing ß-galactosidase and L-arabinose isomerase, which eventually produced 68.35 g/L D-tagatose during sequential feeding of CWP. Subsequently, the mixed syrup containing lactose-derived D-glucose and residual D-galactose was subjected to fermentation by Metschnikowia pulcherrima E1, which produced 60.12 g/L D-arabitol and 28.26 g/L galactitol. The net titer of the three rare sugars was 156.73 g/L from 300 g/L lactose (equivalent to 428.57 g/L CWP), which was equivalent to 1.12 mol product/mol lactose and 52.24% conversion efficiency in terms of lactose.

Enhanced Biosynthesis of D-Arabitol by Metschnikowia reukaufii Through Optimizing Medium Composition and Fermentation Conditions.

D-Arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31,858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 6 days, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2, 0.5, and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O, and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7 g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Enhanced culturing techniques for the mycobiont isolated from the lichen Xanthoria parietina.

Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well as methods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, on the growth of the axenically cultured mycobiont isolated from the lichen Xanthoria parietina were tested. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1% D-glucose used in the originally described LBM by either 1%, 2% or 3% of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11557-021-01707-7.

High D-arabitol production with osmotic pressure control fed-batch fermentation by Yarrowia lipolytica and proteomic analysis under nitrogen source perturbation.

D-arabitol, a five-carbon sugar alcohol, is widely used in food and pharmacy industry as a lower calorie sweetener or intermediate. Appropriate osmotic pressure was confirmed to facilitate polyol production by an osmophilic yeast strain of Yarrowia lipolytica with glycerol. In this study, an osmotic pressure control fed-batch fermentation strategy was used for high D-arabitol producing by Y. lipolytica ARA9 with crude glycerol. Glycerol was added to the broth quantitatively not only as a substrate but also as an osmotic agent. Meanwhile, NH3·H2O was fed as a nitrogen source and pH regulator. The maximum D-arabitol production reached 118.5 g/L at 108 h with the yield of 0.49 g/g and productivity of 1.10 g/L/h, respectively. Furthermore, a comparative proteomic analysis was used to study the cellular responses under excess and deficient nitrogen sources. Thirty-one differentially expressed protein spots belonging to seven different biological processes were identified. Excess nitrogen source enhanced gluconeogenesis and pentose phosphate pathways, both of which were involved in arabitol synthesis. In addition, cell growth was facilitated by increased expression of nucleotide and structural proteins. Enhanced energy and NADPH biosynthesis were employed to create a reductive environment and quell reactive oxygen species, improving D-arabitol production. Nitrogen deficiency resulted in cell rescue and stress response mechanisms such as reactive oxygen species elimination and heat shock protein response. The identified differentially expressed proteins provide information to reveal the mechanisms of the cellular responses under nitrogen source perturbation, and also provide guidance to improve D-arabitol production in metabolic engineering or process optimization methodologies.