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.

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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

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

b 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

A R T I C L E I N F O

Keywords:

Aureobasidium

Consolidated biosaccharification

Co-utilization

Lignocellulosic hydrolysate

Pigment-free pullulan

A B S T R A C T 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 pro-duction, 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 biosacchari fica-tion was used as the pullulan producfica-tion 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 lig-nocellulose bioconversion to pullulan

1 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

cross-linked by α-(1→6) glycosidic bonds, and the glucose units of

mal-totriose are attached byα-(1→4) linkages (Sugumaran & Ponnusami,

2017) The unique linkage pattern endows pullulan distinctive physical

traits, adhesive properties, and capability to formfibers, 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,

in-cluding targeted drug delivery, DNA carrier, tissue engineering,

vacci-nation, 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 com-mercial 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, var-ious 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

https://doi.org/10.1016/j.carbpol.2020.116400

Received 25 January 2020; Received in revised form 26 April 2020; Accepted 28 April 2020

⁎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)

1The authors contribute equally to this work

Available online 03 May 2020

0144-8617/ © 2020 Elsevier Ltd All rights reserved

T

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

strate-gies 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

en-zyme 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

bioconver-sion 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

lig-nocellulose bioconversion by separating fermentation from the

in-tegrated CBP process (Liu, Li, Feng, & Cui, 2020) CBS employs

cellu-losome-producing strains as the whole-cell biocatalyst for lignocellulose

deconstruction and determines fermentable sugars as the target

pro-ducts 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

con-centration 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

pull-ulan, 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-fer-ment glucose and xylose to achieve high yield Robust pullulan

pro-duction under various sugar ratios is also critical for industrial

appli-cations 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

ro-bust production of pullulan from lignocellulosic biomass

2 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

ther-mocellum strainΔpyrF::KBm (Liu et al., 2019) was cultivated

anaero-bically 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 5 g/L

Avicel (PH-101, Sigma-Aldrich LLC.) as the carbon source

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

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 in-ternal 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 phylo-genetic tree was constructed and visualized using Mega 7 software (Kumar, Stecher, & Tamura, 2016)

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 5 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 min with 20% (w/w, based on dry substrates) dosage of ammonium sulfite and a liquid to solid ratio of 6 Afterwards, the pretreated wheat straw samples were washed with tap water before the saccharification process The anae-robic bottles were horizontally shaken in a 55 °C incubator at 200 rpm for 8 days 1.5-mL cultures were sampled every 2 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 3 hours The mixtures were concentrated at 10,000 g for 10 min 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 concentra-tions 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-mLflasks containing 30.0 ml of pullulan production medium with xylose, glu-cose, 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 re-lative 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 begin-ning of fermentation The electrical strength of the cultures was mea-sured 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 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 wasfirst heated in a boiling water bath for 15 min, cooled to room temperature, and centrifuged at 14,000 g and 4 °C for 10 min to remove cells Two volumes of ice-cold ethanol were added

in the supernatant to precipitate polysaccharides at 4 °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

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

con-centration of 10 mg/mL and were hydrolyzed by a commercial

pull-ulanase (400 U/ml, Sigma-Aldrich LLC.) at 60 °C for 15 min The TLC

analysis was carried out with a solvent system of

N-butanol-pyridine-water (6:4:3) and a detection reagent comprising 20.0 g/L

diphenyla-mine 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, 5

μm) and a RID detector at 35 °C The mobile phase was ultra-pure water

at aflow rate of 0.5 mL/min The purity was calculated with the

fol-lowing equation (Eq1):

=

×

Purity(%)

The amount of the releasedmaltotriosefromthe purifiedpullulan

The amount of the releasedmaltotriosefrom thepullulanstandard 100%

(1) For NMR analysis, 20 mg of the purified sample was dissolved in 0.5

mL of deuterated water One-dimensional 13C NMR and1H NMR

ex-periments 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 CBS105.22Twere 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

modifica-tions (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 min and suspended in

1 mol/L NaOH, followed by autoclaving at 120 °C for 20 min The

supernatant of the autoclaved solution was further acidified to pH 2.0

with 1 N HCl to precipitate the pigment The precipitate was recovered

by centrifugation at 5,000 g for 10 min 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

ab-sorbance from 500 to 600 nm using a Multiskan Sky Microplate

Spectrophotometer (Thermo Fisher Scientific, USA) according to

pre-viously 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

de-scribed (Zhang, Liu, Cui, & Cui, 2015) Furfural and 5-hydroxymethyl

furfural (HMF) was detected using a UV detector (284 nm) and a

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 aflow rate of 1 mL/min Soluble lignin was estimated by UV spectro-photometry 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-dini-trosalicylic acid (DNS) method The carbon to nitrogen (C/N) ratio was determined by analyzing the carbon and nitrogen elements on an ele-mental 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 ex-periments 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 mul-tiple comparison test A probability value of p < 0.05 was considered significant

3 Results and discussion

3.1 The honey-derived strain TN2-1-2 converted both glucose and xylose to pullulan efficiently

Efficient co-utilization of glucose and xylose is essential for the economically feasible production of pullulan from lignocellulosic hy-drolysates because lignocellulosic hyhy-drolysates 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 pull-ulan production from xylose is relatively less investigated So far, only

A pullulans AY82 and A pullulans ATCC 42023 were reported to pro-duce pullulan from xylose with titers of 17.63 and 11.2 g/L, respec-tively, 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 pullulan-producing 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 inFig 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

Fig 1 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.ABCDData in the xylose group with different superscripts differ (p < 0.05).abcdData in the glucose group with different superscripts differ (p < 0.05)

strain P16 (8.7 and 45.1 g/L) (Ma et al., 2014) and another

honey-derived 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 6 days’

cultivation, and was barely observed after 8 days’ cultivation (Fig 2A)

In contrast, the type strain A melanogenum CBS105.22T produced a

large amount of melanin within 6 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 5 days (Sugumaran & Ponnusami, 2017) and

such long-term cultivation usually causes severe accumulation of

mel-anin 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

A melanogenum NG swollen cells producing melanin-free pullulan be-cause of its pH-based regulation of cell morphogenesis and melanin biosynthesis (Li et al., 2009) The melanin biosynthesis after the pull-ulan production process was also analyzed As shown in Fig S1, after cultivation in the pullulan production medium for 5 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 inFig 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, al-though A pullulans strains are generally regarded as the important pullulan producer (Sugumaran & Ponnusami, 2017), strains of A mel-anogenum also have great potential in the commercial production of pullulan

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 glu-cose and xylose on pullulan production and cell growth were vestigated The results indicated that as the glucose concentration in-creased 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/gGlucosewas obtained with the pullulan titer of 58.3 g/L, which were higher than previously reported A pull-ulans strains For example, A pullpull-ulans CCTCCM2012259 produced 39.8 g/L from glucose under nitrogen-limiting conditions in a 5 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

Fig 2 Phenotypic and molecular ana-lyses of the fungal strain TN2-1-2 A, The colonies of the strain TN2-1-2 and the type strain A melanogenum CBS105.22Ton the PDA plate after 3, 6, and 8-day cultivation B, The cell mor-phology of the strain TN2-1-2 C, The phylogenetic tree of TN2-1-2 with other yeast relatives based on neighbor-joining analysis of D1/D2 26S rDNA sequences Bootstrap values at the notes are from 1000 replicates

yield of 0.51 gPullulan/gGlucoseby fermenting inflasks (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

con-ditions (Chen et al., 2014) This suggested the excellent capability of the

strain TN2-1-2 to produce pullulan from not only glucose but also

xy-lose In addition, when the xylose concentration increased, the pullulan

production by TN2-1-2 maintained at a similar level under our

condi-tion but the xylose utilizacondi-tion decreased significantly especially when

the xylose concentration increased from 120 g/L to 140 g/L This

in-dicated 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

lig-nocellulosic hydrolysates

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

ra-tios To address this, the effects of glucose to xylose ratios on the cell

growth, substrate utilization, and pullulan production of the strain

TN2-1-2 were tested As shown inFig 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

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 in-dustrial strain for pullulan production from various lignocellulosic hy-drolysates

Carbon catabolite repression is known as a wide-spread cellular regulation that cells utilize one of two or more carbon sources pre-ferentially 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 subse-quently 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 simulta-neously 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

TN2-1-2 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)

3.5 Halotolerance of the strain TN2-1-2 in pullulan production

According to a previous study, 40 g/L SPS was considered the op-timal 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 con-centration of the CBS hydrolysates would be required Since the GS-2 medium used for CBS contained phosphate and various metal ion ele-ments, the residual medium components in the CBS hydrolysate might

be concentrated along with the reducing sugar, resulting in increased

Fig 3 Effects of glucose (A) and xylose (B) concentrations on pullulan produc-tion 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).abcData

in the pullulan production group with different superscripts differ (p < 0.05)

Fig 4 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 produc-tion, yield, cell dry weight, and sugar utilization Values were means of three independent determinations ABC Data

in the yield group with different su-perscripts differ (p < 0.05).abcData in the pullulan production group with

different superscripts differ (p < 0.05)

B, The time course of residual glucose during the fermentation with 50% glu-cose and 50% xylose as the carbon source

salinity To confirm whether the pullulan producer TN2-1-2 could

tol-erate high salt concentration, different amount of NaCl was supplied

into the pullulan production medium, and the total salinity was

esti-mated by monitoring the corresponding electrical conductivities As

shown inFig 5, the cell growth was not significantly influenced when

the NaCl concentration increased from 0 to 50 g/L (electrical

ductivity increased from 0.46 to 6.37 S/m) When the NaCl

con-centration 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

ductivity increased to 3.86 S/m) It is known that the electrical

con-ductivity of standard seawater with a salinity of 3.5% is about 3 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 strainΔpyrF::KBm was

previously constructed as a whole-cell biocatalyst to produce

lig-nocellulose-derived sugars via CBS process (Liu et al., 2019) After

pre-cultivation with Avicel as the sole carbon source, theΔpyrF::KBm cells

were inoculated into the saccharification system with 40 g/L SPS as the

substrate following the previously reported CBS process (Liu et al.,

2019) As determined by the DNS and HPLC methods, 32.84 g/L re-ducing sugar including 25.1 g/L glucose and 7.0 g/L xylose con-centration 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 or-ganisms (Kont, Kurasin, Teugjas, & Valjamae, 2013) But for fermen-tation 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 produc-tion, activated carbon was used to discolorize the lignocellulosic hy-drolysates before using as the carbon source As shown inFig 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 con-centration 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 con-centrations 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 thefinal electrical conductivity

of the condensed hydrolysates was 3.35 S/m, which was in the elec-trical 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 hydro-lysates would have little effect on the cell growth and pullulan

Fig 5 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

de-terminations.ABCData in the cell dry weight with different superscripts differ

(p < 0.05).abcData in the pullulan production group with different superscripts

differ (p < 0.05)

Fig 6 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 thermo-cellum strainΔpyrF::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 de-colorized 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

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

nu-trients, 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

pro-duction, and sugar consumption were all similar, indicating that no

further supplementation of nutrients was required using the CBS

hy-drolysate 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, 5 ml of cells grown in the YPD medium were

in-oculated 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

pro-duction 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

de-termined 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

re-pression 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

ni-trogenous metabolites such as protein-derived amino acids,

lipid-de-rived 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

fer-mentation, 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

sup-plementation 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

pullulan production from glucose is usually with relatively low effi-ciency (Sugumaran & Ponnusami, 2017) Recently, Chen et al devel-oped 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 de-veloping countries, the use of grains for non-food production should be avoided

Agro-wastes are promising non-food substrates for pullulan pro-duction (Mishra, Zamare, & Manikanta, 2018) For example, protein-rich 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) Al-though 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 supple-mented (Chen et al., 2014;Prakash, Varma, Prabhune, Shouche, & Rao,

2011) The fungus A pullulans ATCC 42023 was used to produce pull-ulan from prairie cordgrass hydrolysate obtained by cellulase hydro-lysis 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 lig-nocellulosic 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 break-throughs 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 pur-ification process should be carried out, if the sugar yield of CBS could increase to the optimal sugar concentration required for pullulan pro-duction (110 g/L), the cost of both cell cultivation and sugar puri fica-tion would be greatly reduced Addifica-tionally, pH control and optimi-zation would also enhance pullulan production performance and reduce the cost (Xia, Wu, & Pan, 2011)

3.8 Characterization of pullulan produced from lignocellulosic hydrolysate

Pullulan is connected byα-1,6-D-glucosidic and α-1,4-D-glucosidic linkages (Fig 6), and pullulanase could selectively hydrolyze α-1,6-D-glucosidic 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 hydro-lysates using HPLC (Fig S4) By comparing the amounts of released

maltotriose, the purity of the produced pullulan was calculated to be

93.7%

Furthermore, the produced pullulan was verified by1H-NMR and

13

C-NMR structural analyses (Fig 8) As indicated in the

unidimen-sional 1H-NMR optical spectrum (Fig 8A), the proton peak

displace-ments of both the pullulan standard and purified pullulan were

dis-tributed between W3.3and W5.4 Moreover, the anomeric proton at the

site ofα-(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

(pro-duced 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

che-mical shifts corresponding to theα-(1→6) linkages (100.5473 ppm for

the pullulan standard and 100.5434 ppm for the produced pullulan)

andα-(1→4) linkages (102.8521 and 102.3890 ppm for the pullulan

standard, and 102 8753 and 102.4281 ppm for the produced pullulan) All these results suggested that the pullulan produced from CBS hy-drolysates 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 × 105and 1.377 × 105g/mol, respectively, which were higher than the values of the pullulan produced from pure glucose (1.537 × 105and 0.77 × 105 g/mol for Mw and Mn, respectively, suggesting the feasibility of uti-lizing CBS hydrolysate for pullulan production by TN2-1-2 The pro-duced pullulan was pure white observed by eyes (Fig 6) and the mel-anin 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

4 Conclusion

To adapt the conditions of the lignocellulosic hydrolysate con-taining mixed C5/C6 sugars and high salt concentration, the pullulan production by the strain TN2-1-2 was investigated with various con-centrations and ratios of glucose and xylose and different salinities The results suggested robust pullulan production by TN2-1-2 utilizing glu-cose and xylose simultaneously As shown inFig 6, based on the robust and melanin-free pullulan producer TN2-1-2 and the previously de-veloped 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

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,

Fig 7 TLC analysis of the pullulan hydrolyzed by commercial pullulanase

Lane 1, glucose; Lane 2, maltotriose; Lane 3 and 4, TN2-1-2 produced pullulan

hydrolyzed by activated and inactivated pullulanase, respectively; Lane 5,

pullulan produced by TN2-1-2 without treatment

Fig 8.1H-NMR (A) and13C-NMR (B) spectra of pullulan standard and the pullulan produced by TN2-1-2

Validation.Qiu Cui: Writing - review & editing, Resources Zhenming

Chi: Writing - review & editing, Resources Ya-Jun Liu:

Conceptualization, Visualization, Supervision, Writing - review &

editing, Funding acquisition

Acknowledgments

This research was supported by the National Natural Science

Foundation of China [grant number 31970069], QIBEBT and Dalian

National Laboratory For Clean Energy (DNL), CAS (Grant number

QIBEBT I201905), Key Laboratory of Biofuels, Qingdao Institute of

Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

(Grant number CASKLB201803), the“Transformational Technologies

for Clean Energy and Demonstration”, Strategic Priority Research

Program of the Chinese Academy of Sciences (Grant Number XDA

21060201), and the Major Program of Shandong Provincial Natural

Science Foundation (Grant Number ZR2018ZB0208)

Appendix A Supplementary data

Supplementary material related to this article can be found, in the

online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116400

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