Robust production of pigment-free pullulan from lignocellulosic hydrolysate by a new fungus co-utilizing glucose and xylose

Cost-efficient production of pullulan is of great importance but remains challenging due largely to the high-cost carbon sources. Lignocellulosic biomass is considered an alternative carbon source for industrial pullulan production, while new fungus producers that co-utilizing lignocellulose-derived glucose and xylose are required.

Carbohydrate Polymers 241 (2020) 116400 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Robust production of pigment-free pullulan from lignocellulosic hydrolysate by a new fungus co-utilizing glucose and xylose T Guanglei Liua,c,1, Xiaoxue Zhaoa,1, Chao Chenb,d,e, Zhe Chia,c, Yuedong Zhangb,d,e, Qiu Cuib,d,e, Zhenming Chia,c, Ya-Jun Liub,d,e,* a College of Marine Life Science, Ocean University of China, China CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, China c Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, China d Dalian National Laboratory for Clean Energy, China e University of Chinese Academy of Sciences, Chinese Academy of Sciences, China b A R T I C LE I N FO A B S T R A C T Keywords: Aureobasidium Consolidated biosaccharification Co-utilization Lignocellulosic hydrolysate Pigment-free pullulan Cost-efficient production of pullulan is of great importance but remains challenging due largely to the high-cost carbon sources Lignocellulosic biomass is considered an alternative carbon source for industrial pullulan production, while new fungus producers that co-utilizing lignocellulose-derived glucose and xylose are required In this study, a new fungus Aureobasidium melanogenum TN2-1-2 showed simultaneously assimilation of glucose and xylose and could produce pigment-free pullulan due to its deficiency in melanin synthesis The ability of TN2-1-2 producing pullulan was remarkably robust in the presence of varying glucose to xylose ratios and ionic salt concentrations Furthermore, condensed lignocellulosic hydrolysates obtained by consolidated biosaccharification was used as the pullulan production medium without supplying any nutrients, and pigment-free pullulan was produced by TN2-1-2 with the titer and yield of 55.1 g/L and 0.5 gPullulan/gCBS hydrolysate, respectively Hence, this work provides a potential industrial pullulan producer TN2-1-2 and new insight into the lignocellulose bioconversion to pullulan Introduction Pullulan is an imperative natural polymer produced extracellularly by yeast-like fungus Aureobasidium spp (Li et al., 2015) Structurally, pullulan is primarily composed of maltotriose repeating units crosslinked by α-(1→6) glycosidic bonds, and the glucose units of maltotriose are attached by α-(1→4) linkages (Sugumaran & Ponnusami, 2017) The unique linkage pattern endows pullulan distinctive physical traits, adhesive properties, and capability to form fibers, compression moldings, and strong films that are impervious to oxygen Besides, pullulan can be derivatized by substituting its hydroxyl groups with desired chemical moieties to extend its biomedical applications, including targeted drug delivery, DNA carrier, tissue engineering, vaccination, molecular chaperons, and medical imaging (Singh, Kaur, & Kennedy, 2015; Singh, Kaur, Rana, & Kennedy, 2017) Owing to the important properties of pullulan, the bioprocess for pullulan production has been widely studied to enhance the production and yield So far, sucrose is used as the main substrate for the commercial production of pullulan (Jiang et al., 2018; Sugumaran & Ponnusami, 2017) However, the relative shortage of sucrose resource and its high cost limit the industrial production of pullulan Moreover, the by-product accumulation of fructooligosaccharides caused by the pullulan production from sucrose should also be concerned (Liu et al., 2017) To reduce the carbon source cost for pullulan production, various substrates, including glucose, molasses, hydrolyzed potato starch waste, and inulin, have been used to substitute sucrose (Goksungur, Uzunogullari, & Dagbagli, 2011; Jiang et al., 2018; Ma, Liu, Chi, Liu, & Chi, 2015; Srikanth et al., 2014), but it remains challenging to develop processes for cost-efficient pullulan production Lignocellulosic biomass is the most abundant sustainable carbon source on earth, thus has the potential to be used as an alternative carbon source for industrial fermentation Because of the complex and ⁎ Corresponding author at: CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, China E-mail address: liuyj@qibebt.ac.cn (Y.-J Liu) The authors contribute equally to this work https://doi.org/10.1016/j.carbpol.2020.116400 Received 25 January 2020; Received in revised form 26 April 2020; Accepted 28 April 2020 Available online 03 May 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 241 (2020) 116400 G Liu, et al previously described (Jiang et al., 2018) Fungal fermentation and carbon source assimilation analyses were performed according to a published standard method (Kurtzman, Fell, & Boekhout, 2011) The genomic DNA was extracted using a TIANamp Yeast DNA Kit (TIANGEN, Beijing, China) Amplification and sequencing of the internal transcribed spacer region (ITS) of the rRNA gene cluster were performed using the common primers ITS1 and ITS4 according to the methods described by (Ma et al., 2014) The ITS1 sequence obtained was aligned using BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast cgi) The sequence which shared over 98% similarity with the currently available sequence was considered to be the same species The phylogenetic tree was constructed and visualized using Mega software (Kumar, Stecher, & Tamura, 2016) recalcitrant structure of lignocellulose, one of the premises to produce pullulan from lignocellulosic substrates is the efficient bioconversion of the insoluble substrate into fermentable sugars So far, various strategies have been developed for lignocellulose bioconversion, including separate enzymatic hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), consolidated bioprocessing (CBP), and consolidated bio-saccharification (CBS) (Liu, Liu, Feng, Li, & Cui, 2019; Parisutham, Kim, & Lee, 2014) SHF and SSF are off-site saccharification strategies depending on fungal cellulases, and the enzyme cost severely limits their commercial applications (Lynd et al., 2017; Taha et al., 2016) CBP integrates enzyme production, cellulose hydrolysis, and fermentation in one step for lignocellulose bioconversion to greatly reduce the enzyme cost (Lynd, van Zyl, McBride, & Laser, 2005) and is mainly used for lignocellulosic biofuel production (Xu, Singh, & Himmel, 2009) CBS is a newly proposed strategy for lignocellulose bioconversion by separating fermentation from the integrated CBP process (Liu, Li, Feng, & Cui, 2020) CBS employs cellulosome-producing strains as the whole-cell biocatalyst for lignocellulose deconstruction and determines fermentable sugars as the target products The produced sugar-rich CBS hydrolysates can be potentially used as the carbon sources for various downstream fermentations (Liu et al., 2019, 2020), including pullulan production It should be noted that CBS hydrolysates derived from complex lignocellulosic biomass are usually with low sugar purity and concentration For example, the CBS end-products from pretreated wheat straw contained 22.9 g/L glucose and 7.0 g/L xylose (Liu et al., 2019) Thus, to construct a complete bioprocess from lignocellulose to pullulan, the pullulan production process should be compatible with the CBS process, and the Aureobasidium strains should co-ferment C6/C5 sugars under various sugar ratios and tolerate high salt conditions To be specific, the pullulan producers should be able to efficiently co-ferment glucose and xylose to achieve high yield Robust pullulan production under various sugar ratios is also critical for industrial applications Additionally, because CBS hydrolysate will be concentrated to improve the reducing sugar concentrations, the high osmotic pressure caused by increased salinity in the CBS system should be tolerated by the pullulan producers as well Therefore, in the present study, a new pullulan producing yeast-like fungal was screened and characterized, and a CBS-compatible fermentation process was developed for the robust production of pullulan from lignocellulosic biomass 2.3 Preparation of CBS hydrolysates The CBS process was performed as previously described (Liu et al., 2019) In brief, the strain C thermocellum ΔpyrF::KBm was initially cultivated with g/L Avicel as the sole carbon source to the exponential stage 5% (v/v) of the cells were then inoculated into the GS-2 medium to initiate the saccharification process with 40 g/L sulfite pretreated wheat straw (SPS) as a cellulosic substrate The sulfite pretreatment was performed in a cooking reactor (VRD-42SD-A China Pulp and Paper Research Institute, Beijing, China) at 160 °C for 60 with 20% (w/w, based on dry substrates) dosage of ammonium sulfite and a liquid to solid ratio of Afterwards, the pretreated wheat straw samples were washed with tap water before the saccharification process The anaerobic bottles were horizontally shaken in a 55 °C incubator at 200 rpm for days 1.5-mL cultures were sampled every days to determine sugar production The obtained lignocellulosic hydrolysates were then treated with 3% (w/v) activated carbon in a water bath at 80 °C shaking for hours The mixtures were concentrated at 10,000 g for 10 to remove carbon powder The supernatants were placed in a 50 °C drying container for moisture evaporation until the reducing sugars were concentrated to the determined concentration The protein concentrations of the hydrolysates were determined as previously described (Bradford, 1976) 2.4 Fungal fermentation for pullulan production The fungal strains were aerobically grown in YPD medium at 28 °C for 24 h, and then 5-ml cultures were inoculated into the 250-mL flasks containing 30.0 ml of pullulan production medium with xylose, glucose, or a mixture of xylose and glucose as the carbon source The concentration of supplemented sugar varied from 100.0 to 140.0 g/L When a mixture of xylose and glucose was used as the carbon source, the total sugar concentration was 110.0 g/L with different glucose to xylose ratios (100%:0, 75%:25%, 50%:50%, 25%:75%, 0:100%) After treatment with activated carbon and concentration, CBS hydrolysates were used as the medium for pullulan production without adding relative nutrients If required, different concentrations of NaCl (0.0, 10.0, 20.0, 30.0, 40.0, 50.0, and 60.0 g/L) was supplemented at the beginning of fermentation The electrical strength of the cultures was measured using an AquaPro Water Quality Tester (HM Digital, US) at 20 °C All cultivations were performed aerobically at 28 °C, 180 rpm for 120 h Materials and methods 2.1 Bacterial and fungal strains and cultivation The yeast-like fungal strains TN12-1, TN12-2, TN5-3, TN1-2, TN3-3 and TN2-1-2 used in this study were isolated from natural honeycomb (Jiang et al., 2018) A melanogenum strain P16 was isolated from a mangrove ecosystem (Ma, Fu, Liu, Wang, & Chi, 2014) The yeast-like fungal strains were maintained in yeast-polypeptone-dextrose (YPD) medium and on potato dextrose agar (PDA) at 28 °C The pullulan production medium was composed of 110.0 g/L carbon source (xylose and glucose), 2.0 g/L yeast extract, 0.2 g/L (NH4)2SO4, 5.0 g/L Na2HPO4·12H2O, and 0.15 g/L MgSO4·7H2O, pH 6.5 Clostridium thermocellum strain ΔpyrF::KBm (Liu et al., 2019) was cultivated anaerobically at 55 °C in GS-2 medium (1.5 g/L KH2PO4, 3.8 g/L K2HPO4·3H2O, 2.1 g/L Urea, 1.0 g/L MgCl2·6H2O, 150 mg/L CaCl2·2H2O, 1.25 mg/L FeSO4·6H2O, 1.0 g/L cysteine-HCl, 10 g/L MOPS-Na, 6.0 g/L yeast extract, 3.0 g/L trisodium citrate·2H2O, 0.1 mg/L resazurin, pH 7.4) (Johnson, Madia, & Demain, 1981) with g/L Avicel (PH-101, Sigma-Aldrich LLC.) as the carbon source 2.5 Pullulan purification and quantification The pullulan purification and quantitative determination were performed according to a previously reported method (Ma et al., 2014) The fermentation broth was first heated in a boiling water bath for 15 min, cooled to room temperature, and centrifuged at 14,000 g and °C for 10 to remove cells Two volumes of ice-cold ethanol were added in the supernatant to precipitate polysaccharides at °C for 12 h The precipitate was then dissolved in deionized water at 80 °C and the ethanol precipitation step was repeated The obtained precipitate was 2.2 Phenotypic, biochemical and molecular analyses of the fungal strain The colonies formed on the PDA plates were photographed, and the phenotypic analysis of the cells in the cultures was performed as Carbohydrate Polymers 241 (2020) 116400 G Liu, et al SunFire C18 (4.6 mm × 250 mm) chromatographic column at 35 °C with the mobile phase of ethanol/water (volume ratio of 1:4) and a flow rate of mL/min Soluble lignin was estimated by UV spectrophotometry at 280 nm (Zhang et al., 2014) and calculated according to a previous method (Mussatto & Roberto, 2006) The concentration of reducing sugar in CBS hydrolysate was determined by the 3,5-dinitrosalicylic acid (DNS) method The carbon to nitrogen (C/N) ratio was determined by analyzing the carbon and nitrogen elements on an elemental analyzer (Vario EL cube, Elementar Co., Germany) through burning with oxygen at the temperature of 1200 °C for 70 s The statistical analysis was performed based on three separate experiments using the GraphPad Prism 6.01 (GraphPad Software Inc., USA) Mean values were compared and analyzed using either t-test or one-way analysis of variance (ANOVA) with Tukey HSD post hoc multiple comparison test A probability value of p < 0.05 was considered significant lyophilized and weighed 2.6 Pullulan characterization For thin-layer chromatography (TLC), the purified pullulan and the pullulan standard were dissolved in deionized water to reach a concentration of 10 mg/mL and were hydrolyzed by a commercial pullulanase (400 U/ml, Sigma-Aldrich LLC.) at 60 °C for 15 The TLC analysis was carried out with a solvent system of N-butanol-pyridinewater (6:4:3) and a detection reagent comprising 20.0 g/L diphenylamine in acetone, 20.0 g/L aniline in acetone, and 850.0 g/L phosphoric acid (5:5:1, v/v/v) (Silica gel 60, MERCK, Germany) (Jiang et al., 2018) For determining the pullulan purity, 10 mg/mL of either the purified pullulan or the pullulan standard was completely hydrolyzed by the commercial pullulanase at 37 °C The released maltotriose was determined by high-performance liquid chromatography (HPLC) using Agilent 1260 equipped with a Venusil HILIC column (4.6 × 250 mm, μm) and a RID detector at 35 °C The mobile phase was ultra-pure water at a flow rate of 0.5 mL/min The purity was calculated with the following equation (Eq1): Results and discussion 3.1 The honey-derived strain TN2-1-2 converted both glucose and xylose to pullulan efficiently Purity(%) = The amount of the releasedmaltotriosefromthe purifiedpullulan ×100% The amount of the releasedmaltotriosefrom thepullulanstandard Efficient co-utilization of glucose and xylose is essential for the economically feasible production of pullulan from lignocellulosic hydrolysates because lignocellulosic hydrolysates usually contain both C6 and C5 sugars However, most microorganisms prefer glucose over other monomeric sugars (Gancedo, 1992) For pullulan production, numerous reported Aureobasidium strains can efficiently synthesize pullulan from glucose (Sugumaran & Ponnusami, 2017) but the pullulan production from xylose is relatively less investigated So far, only A pullulans AY82 and A pullulans ATCC 42023 were reported to produce pullulan from xylose with titers of 17.63 and 11.2 g/L, respectively, under their optimized conditions (Chen et al., 2014; Kennedy & West, 2018) To increase pullulan titer, high sugar concentrations are generally required but may cause high osmotic stress to pullulan producers (Choudhury, Saluja, & Prasad, 2011) Thus, the robust osmotolerant ability is regarded as a desirable criterion for commercial pullulanproducing strains Honey is a saturated or supersaturated solution of sugars with extremely low water activity and is considered a natural environment for the isolation of osmophilic yeasts (Cadez, Fulop, Dlauchy, & Peter, 2015) In this study, six yeast-like fungal strains isolated from natural honey samples in our previous study (Jiang et al., 2018) were used for pullulan production with a high concentration of xylose as the carbon source As shown in Fig 1, the strain TN2-1-2 showed the highest pullulan yield of 49.5 and 58.3 g/L from 110 g/L xylose and glucose, respectively (Fig 1), which were significantly higher than those of the other tested strains, including a mangrove (1) For NMR analysis, 20 mg of the purified sample was dissolved in 0.5 mL of deuterated water One-dimensional 13C NMR and 1H NMR experiments were performed on a Bruker Avance III 600 MHz NMR spectrometer equipped with a z-gradient triple resonance cryoprobe using the internal DSS as previously described (Lazaridou, Roukas, Biliaderis, & Vaikousi, 2002) The molecular weight of pullulan was determined using a Waters™ 1515 Gel Permeation Chromatography (GPC) system with a Multi-angle laser light scattering detector (MALLS) as described by (Jiang et al., 2018) 2.7 Melanin extraction and determination The strain TN2-1-2 and the type strain CBS105.22T were cultured in the pullulan production medium for 120 h to determine the production and accumulation of melanin after the fungal fermentation for pullulan production according to previously described methods with modifications (Kumar, Mongolla, Pombala, Kamle, & Joseph, 2011) In detail, the fungal cells grown in the pullulan production medium for 120 h were separated by centrifugation at 8000 g for 10 and suspended in mol/L NaOH, followed by autoclaving at 120 °C for 20 The supernatant of the autoclaved solution was further acidified to pH 2.0 with N HCl to precipitate the pigment The precipitate was recovered by centrifugation at 5,000 g for 10 and washed with deionized water for three times The purified melanin was lyophilized and weighted to determine melanin production Melanin in the purified pullulan was determined based on the absorbance from 500 to 600 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, USA) according to previously reported methods (Li et al., 2009) 2.8 Analysis methods The cell biomass was determined by monitoring the cell dry weight according to the methods described by (Chen et al., 2019) The CBS hydrolysates were analyzed by HPLC Glucose, xylose, arabinose and cellobiose concentrations were determined using a refractive index detector equipped with a Bio-Rad HPX-87H column as previously described (Zhang, Liu, Cui, & Cui, 2015) Furfural and 5-hydroxymethyl furfural (HMF) was detected using a UV detector (284 nm) and a Fig Pullulan producing ability of different yeast-like fungal strains isolated from natural honey 110 g/L of glucose or xylose were used as the carbon source Values were means of three independent determinations ABCD Data in the xylose group with different superscripts differ (p < 0.05) abcd Data in the glucose group with different superscripts differ (p < 0.05) Carbohydrate Polymers 241 (2020) 116400 G Liu, et al Fig Phenotypic and molecular analyses of the fungal strain TN2-1-2 A, The colonies of the strain TN2-1-2 and the type strain A melanogenum CBS105.22T on the PDA plate after 3, 6, and 8-day cultivation B, The cell morphology of the strain TN2-1-2 C, The phylogenetic tree of TN2-1-2 with other yeast relatives based on neighborjoining analysis of D1/D2 26S rDNA sequences Bootstrap values at the notes are from 1000 replicates A melanogenum NG swollen cells producing melanin-free pullulan because of its pH-based regulation of cell morphogenesis and melanin biosynthesis (Li et al., 2009) The melanin biosynthesis after the pullulan production process was also analyzed As shown in Fig S1, after cultivation in the pullulan production medium for days, the type strain CBS105.22T produced 0.026 ± 0.003 g/gcell dry weight melanin while almost no melanin was detected in the culture of TN2-1-2 Thus, the strain TN2-1-2 showed natural deficiency in melanin biosynthesis thereby is considered a promising candidate for the production of pigment-free pullulan For molecular identification, the ITS sequence of the strain TN2-1-2 (Accession number MN752213) exhibited 99 % similarity to that of the type strain A melanogenum CBS105.22T Thus, as the topology of the phylograms in Fig 2D confirmed, the strain TN2-1-2 belonged to the species A melanogenum So far, three A melanogenum strains, including TN2-1-2, P16, and TN1-2 have been proved to produce high titer of pullulan (Jiang et al., 2018; Ma et al., 2014) This suggested that, although A pullulans strains are generally regarded as the important pullulan producer (Sugumaran & Ponnusami, 2017), strains of A melanogenum also have great potential in the commercial production of pullulan strain P16 (8.7 and 45.1 g/L) (Ma et al., 2014) and another honeyderived strain TN1-2 (15.2 and 48.7 g/L) (Jiang et al., 2018) It was also noteworthy that the strain TN2-1-2 had similar pullulan productivity with either xylose or glucose as the sole carbon source compared to most of the other tested strains (Fig 1), suggesting that TN2-1-2 could efficiently assimilate both glucose and xylose to produce pullulan 3.2 The strain A melanogenum TN2-1-2 was naturally deficient in melanin biosynthesis The TN2-1-2 colonies grown on the PDA plate showed weak pink and sticky and were surrounded by extracellular polysaccharides (Fig 2A) All the yeast-like fungal cells were ellipsoidal and oval and were budding to generate the secondary conidia without the formation of chlamydospores and arthroconidia (Fig 2C), which were similar to reported strains of Aureobasidium spp (Li et al., 2015) Carbon source assimilation experiments were performed and the results also showed that TN2-1-2 had characteristics closely related to the type strain A melanogenum CBS105.22 T 584.75 (Table S1) Interestingly, unlike most of known Aureobasidium spp strains that usually synthesize melanin thereby being named as “black yeast” (Li et al., 2015), TN2-1-2 showed significantly reduced ability to synthesize melanin because the blackish color was undetectable in the colonies on the PDA plate after days’ cultivation, and was barely observed after days’ cultivation (Fig 2A) In contrast, the type strain A melanogenum CBS105.22T produced a large amount of melanin within days under the same condition (Fig 2B) The melanin production is well-known as an obstacle to pullulan industrial production by increasing the cost of pullulan purification (Singh, Saini, & Kennedy, 2009) The pullulan fermentation process commonly goes on for days (Sugumaran & Ponnusami, 2017) and such long-term cultivation usually causes severe accumulation of melanin pigment Many studies have been carried out to obtain strains deficient in pigment formation by mutagenesis and genetic engineering (Chen et al., 2019; Yu, Wang, Wei, & Dong, 2012) For instance, Chen et al construct a mutant strain of A melanogenum producing no melanin by inactivating two copies of the PKS1 and PKS2 genes involved in the DHN-melanin biosynthesis (Chen et al., 2019) Additionally, pH control during fermentation was considered effective and convenient to harvest 3.3 Effect of glucose and xylose concentrations on pullulan production It has been well documented that a high initial carbon/nitrogen ratio (nitrogen starvation) is required to boost pullulan biosynthesis (Li et al., 2015) Therefore, the effects of different concentrations of glucose and xylose on pullulan production and cell growth were investigated The results indicated that as the glucose concentration increased from 100 to 140 g/L, the pullulan titer was gradually improved The glucose utilization maintained at a level of above 99% and the production of cell biomass also showed no significant change (Fig 3A) When 110.0 g/L of glucose was used as the carbon source, the highest pullulan yield of 0.53 gPullulan/gGlucose was obtained with the pullulan titer of 58.3 g/L, which were higher than previously reported A pullulans strains For example, A pullulans CCTCCM2012259 produced 39.8 g/L from glucose under nitrogen-limiting conditions in a L fermenter (Wang, Chen, Wei, Jiang, & Dong, 2015) Yu et al reported an A pullulans SZU 1001 mutant which produced 25.65 g/L pullulan with a Carbohydrate Polymers 241 (2020) 116400 G Liu, et al Fig Effects of glucose (A) and xylose (B) concentrations on pullulan production and yield, cell dry weight, and sugar utilization Values were means of three independent determinations ABC Data in the yield group with different superscripts differ (p < 0.05) abc Data in the pullulan production group with different superscripts differ (p < 0.05) yield of 0.51 gPullulan/gGlucose by fermenting in flasks (Yu et al., 2012) As shown in Fig 3B, the xylose utilization ratio of TN2-1-2 was above 95% except for the setup containing 140.0 g/L xylose When the xylose concentration in the pullulan production medium ranged from 100 to 120 g/L, the pullulan yield and titer were all at the highest level of 50.2 g/L and 0.46 gPullulan/gGlucose, respectively According to a previous report, A pullulans AY82 could produce pullulan from xylose with a maximal pullulan titer of 17.63 g/L under the optimized conditions (Chen et al., 2014) This suggested the excellent capability of the strain TN2-1-2 to produce pullulan from not only glucose but also xylose In addition, when the xylose concentration increased, the pullulan production by TN2-1-2 maintained at a similar level under our condition but the xylose utilization decreased significantly especially when the xylose concentration increased from 120 g/L to 140 g/L This indicated the effect of xylose concentration on the pullulan production by TN2-1-2 was more pronounced than that of glucose, and 110.0 g/L was determined as the optimum concentration for both glucose and xylose in terms of the pullulan production and yield For further experiments, the concentration of supplemented reducing sugars was adjusted to 110.0 g/L for fermentations based on either pure sugar or lignocellulosic hydrolysates was used as the sole carbon source (100% xylose) This result indicated that the strain TN2-1-2 was capable of robust production of pullulan with different glucose to xylose ratios ranging from 100%:0 to 50%:50% Thus, TN2-1-2 could be potentially used as a robust industrial strain for pullulan production from various lignocellulosic hydrolysates Carbon catabolite repression is known as a wide-spread cellular regulation that cells utilize one of two or more carbon sources preferentially when multiple carbon sources are provided (Deutscher, 2008) The presence of glucose may inhibit the utilization of xylose and thus cause decreased sugar utilization and product yield (Kwak & Jin, 2017) Thus, besides the ability to utilize mixed glucose and xylose to produce pullulan, whether the supplemented xylose and glucose were utilized simultaneously by the strain should be concerned as well The co-assimilation ability of glucose and xylose by TN2-1-2 was subsequently determined by fermentation using a mixed sugar with an equal amount of glucose and xylose as the carbon source (Fig 4B) The result showed that the amount of glucose and xylose decreased simultaneously along with the cultivation, indicating the C6/C5 co-fermenting capability of TN2-1-2 It took 72 hours and 120 hours to exhaust 55 g/L of glucose and xylose under this condition, respectively, indicating a higher assimilation rate of glucose than that of xylose Although TN2-12 could assimilate glucose and xylose simultaneously, it still showed preference to glucose as the carbon source as other reported yeast strains (Agbogbo, Coward-Kelly, Torry-Smith, & Wenger, 2006) 3.4 Robust pullulan production by the strain TN2-1-2 with mixed sugars The C6 and C5 sugar compositions in lignocellulosic hydrolysates vary depending on the type of substrate, pretreatment method, and cellulolytic enzymes (Singh, Shukla, Tiwari, & Srivastava, 2014) Thus, the industrial pullulan producers should have the robustness in the efficient utilization of mixed sugars with various glucose to xylose ratios To address this, the effects of glucose to xylose ratios on the cell growth, substrate utilization, and pullulan production of the strain TN21-2 were tested As shown in Fig 4A, the glucose to xylose ratio varied from 100%:0 to 0:100% and the total sugar concentration was 110 g/L The utilization rates of the total sugar maintained at a level of above 99% with various sugar ratios, and high pullulan titers and yields were detected without significant difference (p > 0.05) except when xylose 3.5 Halotolerance of the strain TN2-1-2 in pullulan production According to a previous study, 40 g/L SPS was considered the optimal substrate load for the current CBS process (Liu et al., 2019), and approximately 30 g/L of reducing sugar would be produced Because 110 g/L sugar was required for pullulan production, further concentration of the CBS hydrolysates would be required Since the GS-2 medium used for CBS contained phosphate and various metal ion elements, the residual medium components in the CBS hydrolysate might be concentrated along with the reducing sugar, resulting in increased Fig The co-utilization of glucose and xylose by the strain TN2-1-2 to produce pullulan A, Effects of glucose (G) to xylose (X) ratios on pullulan production, yield, cell dry weight, and sugar utilization Values were means of three independent determinations ABC Data in the yield group with different superscripts differ (p < 0.05) abc Data in the pullulan production group with different superscripts differ (p < 0.05) B, The time course of residual glucose during the fermentation with 50% glucose and 50% xylose as the carbon source Carbohydrate Polymers 241 (2020) 116400 G Liu, et al substrate following the previously reported CBS process (Liu et al., 2019) As determined by the DNS and HPLC methods, 32.84 g/L reducing sugar including 25.1 g/L glucose and 7.0 g/L xylose concentration was produced, and a trace amount of cellobiose was also detected (Fig S2) Lignocellulosic hydrolysates may contain various lignin-derived compounds that have an inhibitory or toxic effect on fermenting organisms (Kont, Kurasin, Teugjas, & Valjamae, 2013) But for fermentation with CBS hydrolysate as the carbon source, this may not be a big issue as CBS itself is basically a biological process involving alive cells and the pretreatment is generally performed under the mild conditions and produce a low amount of toxins (Liu et al., 2020) According to the HPLC analysis, furfural and 5-hydroxymethyl furfural (HMF) was not detectable in the CBS hydrolysate, indicating the slight inhibitory effect on downstream fungal fermentation and pullulan production Although lignin can be partially removed by pretreatment, there is still lignin left in pretreated substrates which can be gradually released during the saccharification process (Li, Liu, Yu, Zhang, & Mu, 2017; Tian, Zhao, & Chen, 2018) Both soluble lignin and extracts contain chromophores and auxophores, resulting in the deep color of the hydrolysates (Korntner et al., 2015; Sixta, 2006) Furthermore, the hydrolysates should be concentrated to reach the optimal sugar concentration of 110 g/L for pullulan production by TN2-1-2, and the color would become even deeper as the sugar solution is concentrated Because the dark color of the cultivation medium must be avoided for pullulan production, activated carbon was used to discolorize the lignocellulosic hydrolysates before using as the carbon source As shown in Fig 6, the hydrolysate color changed from dark red to light yellow The protein, sugar, and soluble lignin concentrations were determined before and after discolorization The results showed that the reducing sugar concentration maintained at a similar level, only slightly reduced by 3.3% (from 32.84 g/L to 31.74 g/L) but the protein and soluble lignin concentrations decreased significantly from 0.16 g/L and 1.05 g/L to 0.01 g/L and 0.20 g/L, respectively The C/N ratio of the decolorized CBS hydrolysate was determined as 13.07 ± 0.63 based on the element analysis The decolorized hydrolysates were then concentrated until the reducing sugar concentration reached 110 g/L and were used for pullulan production It was notable that the final electrical conductivity of the condensed hydrolysates was 3.35 S/m, which was in the electrical conductivity tolerance range of the strain TN2-1-2 (0-3.86 S/m) (Fig 5) This suggested that the salinity of the prepared CBS hydrolysates would have little effect on the cell growth and pullulan Fig Effects of NaCl concentrations on pullulan production, yield, cell dry weight, and sugar utilization The corresponding electrical conductivity values were also given for comparison Values were means of three independent determinations ABC Data in the cell dry weight with different superscripts differ (p < 0.05) abc Data in the pullulan production group with different superscripts differ (p < 0.05) salinity To confirm whether the pullulan producer TN2-1-2 could tolerate high salt concentration, different amount of NaCl was supplied into the pullulan production medium, and the total salinity was estimated by monitoring the corresponding electrical conductivities As shown in Fig 5, the cell growth was not significantly influenced when the NaCl concentration increased from to 50 g/L (electrical conductivity increased from 0.46 to 6.37 S/m) When the NaCl concentration increased to 60 g/L and resulted in electrical conductivity of 7.60 S/m, the cell dry weight only declined by 13.9%, indicating high halotolerance of the strain TN2-1-2 In terms of pullulan production, the strain TN2-1-2 maintained a relatively high pullulan production level when the NaCl concentration increased to 30 g/L (electrical conductivity increased to 3.86 S/m) It is known that the electrical conductivity of standard seawater with a salinity of 3.5% is about S/m (20 °C), implying that the strain TN2-1-2 was able to tolerate a high level of salinity comparable to seawater for pullulan production 3.6 Preparation of CBS hydrolysates for pullulan fermentation The genetically engineered C thermocellum strain ΔpyrF::KBm was previously constructed as a whole-cell biocatalyst to produce lignocellulose-derived sugars via CBS process (Liu et al., 2019) After precultivation with Avicel as the sole carbon source, the ΔpyrF::KBm cells were inoculated into the saccharification system with 40 g/L SPS as the Fig Schematic representation of the whole process for pullulan production from lignocellulosic biomass The whole process contains two main steps, consolidated biosaccharification (CBS) and fungal fermentation In the CBS processs, the engineered C thermocellum strain ΔpyrF::KBm was used as the whole-cell biocatalyst to solubilize sulfite pretreated wheat straw (SPS) to sugar-rich CBS hydrolysates Cellulose and hemicellulose of the lignocellulosic biomass were converted to glucose and pentose (mainly xylose), respectively The obtained CBS hydrolysate was decolorized using activated carbon and concentrated to reach a reducing sugar concentration of 110 g/L Afterwards, the CBS hydrolysate was directly used for fungal fermentation to produce pullulan using a newly isolated fungus strain A melanogenum TN2-1-2 Because TN2-1-2 is deficient in melanin biosynthesis, the produced pullulan is pigment-free without blackish color Carbohydrate Polymers 241 (2020) 116400 G Liu, et al pullulan production from glucose is usually with relatively low efficiency (Sugumaran & Ponnusami, 2017) Recently, Chen et al developed an engineered strain TN3-1 that can produce 103.5 g pullulan from 140 g glucose under the experimental conditions (Chen et al., 2019) Nevertheless, glucose is mainly produced from corn starch Due to the big concern of the worldwide food shortage, especially in developing countries, the use of grains for non-food production should be avoided Agro-wastes are promising non-food substrates for pullulan production (Mishra, Zamare, & Manikanta, 2018) For example, proteinrich corn steep liquor and de-oiled seed cake were used to grow A pullulans strains, and over 70 g/L pullulan were produced (Choudhury, Sharma, & Prasad, 2012; Sharma, Prasad, & Choudhury, 2013) Although high pullulan yields were obtained, these agro-wastes were usually supplemented as a nitrogen nutrient and starch-derived glucose was still considered the main carbon source Among all the carbon sources of agro-wastes, lignocellulose is known as the most abundant alternative carbon source for industrial fermentation but difficult to utilize due to its recalcitrant structure Chen et al reported that the maximal production of pullulan by A pullulans AY82 from sugarcane bagasse hydrolysate was 12.65 g/L with a yield of 0.25 g/g after 7-day cultivation (Chen et al., 2014) While the hemicellulose hydrolysate was obtained by stream explosion and sulfuric acid hydrolysis rather than biosaccharification and various medium nutrients were supplemented (Chen et al., 2014; Prakash, Varma, Prabhune, Shouche, & Rao, 2011) The fungus A pullulans ATCC 42023 was used to produce pullulan from prairie cordgrass hydrolysate obtained by cellulase hydrolysis A high yield of 0.79 g/g was obtained but the pullulan titer was only 11.2 g/L after cultivation for 168 h with supplementation of yeast extract (Kennedy & West, 2018) Wang et al isolated an adapted A pullulans mutant to produce pullulan from the hydrolysate of untreated rice hull and obtained a maximal yield of 22.2 g/L (Wang, Ju, Zhou, & Wei, 2014) Thus, although the pullulan production from lignocellulosic hydrolysates has been reported previously, we obtained higher pullulan yield and titer by coupling CBS and fermentation of the strain TN2-1-2 The competitiveness of CBS sugars compared to starch sugar would affect the feasibility of pullulan production from lignocellulose to a great extent As we have calculated previously, the cost of CBS sugar should be competitive to starch sugar (∼500 US$ per ton currently) (Liu et al., 2020) As a newly developed technology, CBS is considered promising because it enjoys a major advantage in reducing enzyme costs but further improvements, including process optimization and development of new biocatalysts, are still required to make breakthroughs in terms of practical cost-effectiveness (Liu et al., 2020) It is worth noting that the cost of sugar purification using activated carbon accounted for over 50% of the total cost when the sugar yield of CBS was 30 g/L in this study Although further optimization on the purification process should be carried out, if the sugar yield of CBS could increase to the optimal sugar concentration required for pullulan production (110 g/L), the cost of both cell cultivation and sugar purification would be greatly reduced Additionally, pH control and optimization would also enhance pullulan production performance and reduce the cost (Xia, Wu, & Pan, 2011) production by the strain TN2-1-2 3.7 Pullulan production by the strain TN2-1-2 from lignocellulosic hydrolysates Because the GS-2 medium used for the cultivation of wholt-cell biocatalyst contained all metal ions and macronutrients that pullulan production medium required, together with the remained YPD medium in the inoculum, the residual nutrients in the CBS hydrolysate might support the pullulan production by TN2-1-2 Besides, the pH value of the CBS hydrolysate which was detected as 6.2 was close to that of the pullulan production medium (pH 6.5) Thus, the concentrated CBS hydrolysates containing 110 g/L reducing sugar was directly used as the medium for pullulan production without supplementation of any nutrients, and the strain TN2-1-2 could produce 55.1 ± 2.1 g/L pullulan with a yield of 0.50 gPullulan/gCBS hydrolysate We also carried out the fermentation using CBS hydrolysates supplemented with nutrients of the pullulan production medium, and obtained the pullulan titer and yield of 55.7 ± 2.3 g/L and 0.50 gPullulan/gCBS hydrolysate, respectively As shown in Fig S3, the time courses of the cell growth, pullulan production, and sugar consumption were all similar, indicating that no further supplementation of nutrients was required using the CBS hydrolysate for pullulan production The pH value decreased to 3.6 after 120-h fermentation which was similar to the previous study (Chen et al., 2014) In this study, ml of cells grown in the YPD medium were inoculated in 30-ml fermentation systems for pullulan production and the remained yeast extract and peptone in the inoculum might contain sufficient nitrogen sources for pullulan production The C/N ratio of the pullulan production media containing 110 g/L glucose was calculated as 42.56 based on the content of nitrogen sources in the pullulan production medium and YPD medium, the inoculum size, and the nitrogen contents according to a previously reported method (Guerfali et al., 2019) The C/N ratio of the decolorized CBS hydrolysate was determined as 13.07 based on the element analysis Taking account with the nitrients derived from the inoculum, the C/N ratio was calculate to be 10.96 It is known that a high C/N ratio usually plays an important role in pullulan production (Li et al., 2015) because high concentration of ammonium and glutamine mediates severe nitrogen metabolite repression in fungi (Tudzynski, 2014) Interestingly, although the CBS hydrolysate had a lower C/N ratio than the pullulan production medium, similar pullulan titers of 58.3 g/L and 55.1 g/L were obtained (Fig 3A and S3A) This result might be explained by the various nitrogenous metabolites such as protein-derived amino acids, lipid-derived phosphocholine and colamine, and nucleotides produced by C thermocellum in the CBS hydrolysate that could be detected by element analysis but showed slight nitrogen repression effects on fungal fermentation, and also implied that the strain had the robustness on C/N ratios for pullulan production and the tolerance to the metabolites in reused media The glucose to xylose ratio in CBS hydrolysate was about 78%:22%, thus the pullulan production was compared to that with mixed sugars (glucose to xylose ratio of 75%:25%) as the carbon source, which was 58.9 g/L (Fig 4A) The result suggested the pullulan titer and yield of TN2-1-2 using the concentrated CBS hydrolysate were comparable to those with pure sugars as the carbon source even without further supplementation of medium components Because the fungal fermentation and pullulan purification processes are relatively mature in industry, the cost-effectiveness of the pullulan production process may greatly depend on the cost of carbon sources, i.e., sugars Sucrose is mainly used as the substrate for the industrial pullulan production and the pullulan titer and yield could reach 67.4 g/ L and 0.56 gPullulan/gsucrose, respectively (Ma et al., 2014) However, the high cost of carbon source (over 900 US$ per ton sucrose) still limits the industrial production of pullulan Glucose is a universal carbon source with lower price (∼500 US$ per ton) compared to sucrose, but the 3.8 Characterization of pullulan produced from lignocellulosic hydrolysate Pullulan is connected by α-1,6-D-glucosidic and α-1,4-D-glucosidic linkages (Fig 6), and pullulanase could selectively hydrolyze α-1,6-Dglucosidic linkages of pullulan to release maltotriose (Sugumaran & Ponnusami, 2017) Indeed, as indicated by the TLC analysis (Fig 7), the pullulan produced from CBS hydrolysate by the strain TN2-1-2 was hydrolyzed by a commercial pullulanase to maltotriose Additionally, we performed pullulanase hydrolysis of equal amount of the produced pullulan sample and the pullulan standard and analyzed the hydrolysates using HPLC (Fig S4) By comparing the amounts of released Carbohydrate Polymers 241 (2020) 116400 G Liu, et al standard, and 102 8753 and 102.4281 ppm for the produced pullulan) All these results suggested that the pullulan produced from CBS hydrolysates by the strain TN2-1-2 is with identical structure with the commercial pullulan standard The product quality of the CBS hydrolysate-derived pullulan was determined by GPC chromatogram analysis As showed in Fig S5, the weight-average (Mw) and number-average molecular weight (Mn) of the pullulan produced from CBS hydrolysates were 1.862 × 105 and 1.377 × 105 g/mol, respectively, which were higher than the values of the pullulan produced from pure glucose (1.537 × 105 and 0.77 × 105 g/mol for Mw and Mn, respectively, suggesting the feasibility of utilizing CBS hydrolysate for pullulan production by TN2-1-2 The produced pullulan was pure white observed by eyes (Fig 6) and the melanin in the purified pullulan was rarely detected (Fig S6), suggesting that the pullulan produced by TN2-1-2 was pigment-free Thus, the pullulan-producing strain that deficient in melanin biosynthesis would have a wide range of application prospects in the food and medicine industry Conclusion Fig TLC analysis of the pullulan hydrolyzed by commercial pullulanase Lane 1, glucose; Lane 2, maltotriose; Lane and 4, TN2-1-2 produced pullulan hydrolyzed by activated and inactivated pullulanase, respectively; Lane 5, pullulan produced by TN2-1-2 without treatment To adapt the conditions of the lignocellulosic hydrolysate containing mixed C5/C6 sugars and high salt concentration, the pullulan production by the strain TN2-1-2 was investigated with various concentrations and ratios of glucose and xylose and different salinities The results suggested robust pullulan production by TN2-1-2 utilizing glucose and xylose simultaneously As shown in Fig 6, based on the robust and melanin-free pullulan producer TN2-1-2 and the previously developed CBS biocatalyst, we constructed a complete bioprocess to produce pigment-free pullulan from lignocellulosic biomass effectively Thus, this study provided an insight into the cost-effective pullulan industrial production maltotriose, the purity of the produced pullulan was calculated to be 93.7% Furthermore, the produced pullulan was verified by 1H-NMR and 13 C-NMR structural analyses (Fig 8) As indicated in the unidimensional 1H-NMR optical spectrum (Fig 8A), the proton peak displacements of both the pullulan standard and purified pullulan were distributed between W3.3 and W5.4 Moreover, the anomeric proton at the site of α-(1→6) linkages was detected based on the chemical shifts at 4.9483 ppm for the pullulan standard and 4.9681 ppm for the produced pullulan, and the signal distribution at 5.3717 and 5.4090 ppm (produced pullulan) could be attributed to α-(1→4) linkages while the analogs of pullulan standard were 5.3562 and 5.3928, respectively Furthermore, as shown in the 13C-NMR results (Fig 8B), the signal distribution of anomeric carbon region of the pullulan standard and produced pullulan appeared to be consistent, especially for the chemical shifts corresponding to the α-(1→6) linkages (100.5473 ppm for the pullulan standard and 100.5434 ppm for the produced pullulan) and α-(1→4) linkages (102.8521 and 102.3890 ppm for the pullulan Fig 1H-NMR (A) and 13 Declaration of Competing Interest The authors declare that they have no competing interests CRediT authorship contribution statement Guanglei Liu: Conceptualization, Data curation, Writing - original draft, Funding acquisition Xiaoxue Zhao: Investigation, Data curation Chao Chen: Investigation, Data curation Zhe Chi: Visualization, Validation, Writing - review & editing Yuedong Zhang: Visualization, C-NMR (B) spectra of pullulan standard and the pullulan produced by TN2-1-2 Carbohydrate Polymers 241 (2020) 116400 G Liu, et al Validation Qiu Cui: Writing - review & editing, Resources Zhenming Chi: Writing - review & editing, Resources Ya-Jun Liu: Conceptualization, Visualization, Supervision, Writing - review & editing, Funding acquisition 1870–1874 Kurtzman, C., Fell, J W., & Boekhout, T (2011) The yeasts: a taxonomic study Elsevier Kwak, S., & Jin, Y S (2017) Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective Microbial Cell Factories, 16 Lazaridou, A., Roukas, T., Biliaderis, C G., & Vaikousi, H (2002) Characterization of pullulan produced from beet molasses by Aureobasidium pullulans in a stirred tank reactor under varying agitation Enzyme and Microbial Technology, 31(1-2), 122–132 Li, B., Liu, C., Yu, G., Zhang, Y., & Mu, X (2017) Recent progress on pretreatment and fractionation of 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Sharmila, G., Muthukumaran, C., Jaganathan, M K., et al (2014) Statistical optimization of molasses based exopolysaccharide and biomass production by Aureobasidium pullulans MTCC 2195 Biocatalysis... pullulan sample and the pullulan standard and analyzed the hydrolysates using HPLC (Fig S4) By comparing the amounts of released Carbohydrate Polymers 241 (2020) 116400 G Liu, et al standard, and 102... al reported that the maximal production of pullulan by A pullulans AY82 from sugarcane bagasse hydrolysate was 12.65 g/L with a yield of 0.25 g/g after 7-day cultivation (Chen et al., 2014) While