Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 1 5 9 3 e1 1 6 0 1

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Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus Jiali Feng, Qing Li, Jun Hu, Honghui Yang, Wen Cao, Liejin Guo* State Key Laboratory of Multiphase Flow in Power Engineering (MPFL), School of Energy and Power Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China

article info

abstract

Article history:

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H2

Received 5 January 2019

production after pretreatment. However, the pretreatment methods have different influ-

Received in revised form

ence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/

11 March 2019

cellusase. The hydrolysis rates increased from 45.51% by ddH2O-treatment to 60.79% by

Accepted 18 March 2019

diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar

Available online 10 April 2019

yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar

Keywords:

yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H2

Cornstalk hydrolysis

production vary among different hydrolysates. Photo-fermentative H2 production of strain

Biohydrogen

R. capsulatus JL1 and mutant JL1601 (cheR2-) with hydrolysates were investigated. The

Photo-fermentation

maximum H2 yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme

Rhodobacter capsulatus

pretreated cornstalk, while the H2 yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH2O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H2 production for mutant JL1601 (cheR2-) but not for strain JL1. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Transforming waste such as food, agriculture, and industry wastewaters to renewable energy eases the energy crisis while preventing environmental pollution. As an agricultural country, China is rich in crop straw resources, which is carbohydrate-rich material. However, due to the agricultural operation organization pattern and management, it is difficult

to realize the utilization of straw energy in large-scale. So far, burning, straw returning, and raising livestock are still the mainly utilization ways of stalks in China, energy utilization is less than 10% [1e6]. Converting crop straws to H2 via biological-fermentative routes attracts more and more attentions as a potential process for renewable and sustainable energy generation [7e9]. Cornstalk, as a kind of lignocellulosic raw material, is mainly composed of cellulose, hemicellulose and lignin, who

* Corresponding author. E-mail address: [email protected] (L. Guo). https://doi.org/10.1016/j.ijhydene.2019.03.143 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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are inter-wovened and clustered into bundles. Therefore, although it can provide as a potential substrate for biohydrogen production, pretreatment must be carried out before being fed to photosynthetic bacteria (PSB). Actually, there are several ways to break down the cornstalk into simple organic matters, among which, chemical pretreatments involving in the use of acid or alkali are considerable for its ease of implementation and inexpensive [9e13]. Acid or alkali pretreatment could be able to improve the solubilizations in cornstalk to increase the available substrates for PSB [9,14]. However, there are some byproducts generated along during chemical pretreatment, for example furan and phenol [15e17], which can work as inhibitors in fermentative H2 production. To improve the substrate-to-H2 conversion efficiency of cellulose, optimizing dilute acid or alkali hydrolysis conditions were carried out [9,11]. Enzymatic hydrolysis is

more environment-friendly, but the compact structure of cornstalk hindered the effective activity between enzyme and cellulose. Nevertheless, dilute acid or alkali pretreatment increased the internal surface area, combined dilute acid or alkali with enzyme hydrolysis was another more effective straw treatment [9]. Dark- or photo-fermentative H2 production from wastes is more and more popular (Fig. 1). Dark-fermentation is characterized by high H2 production rate but low yield, its theoretical H2 yield is 4 mol-H2/mol-glucose. To improve H2 yield, integrated dark-photo fermentative H2 production process dealing with various pretreated agricultural residue has been studied to improve the substrate-to-H2 conversion efficiency as an alternative to single dark-fermentation [18e21], where acetate and butyrate produced by dark fermentation are available for PSB. However, some drawbacks of this system

Fig. 1 e Schematic illustration of the biohydrogen production from cornstalk.

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require attention, including additional energy consumption for complicated reactors and dark-fermentation effluent pretreatment such as sterilization and pH adjustment. For the practical applicability, simplified single-step photo-fermentation comes up with more potential [8,22e24], it is characterized by high yield at the presence of light with theoretical 12 mol-H2/mol-glucose, Abo-Hashesh et al. even enhanced the H2 yield to 9.0 ± 1.2 mol-H2/mol-glucose with pure R. capsulatus JP91 [25], which was much higher than the maximum H2 yield by two-step process. Nevertheless, the cornstalk hydrolysate obtained by chemical pretreatment is dark in color and complicated in composition. To improve waste utilization, some researchers employed mixed anaerobic bacteria to handle different substrates [8,26,27], but the microbial community structure is easily changed to affect the photo-fermentation stability. On the other hand, using genetic engineering to construct pure mutant strains to improve the photo-fermentation under stressful conditions was promising [24,28,29], for the metabolic routes in pure strain being detected easily. For the cornstalk and other cellulose waste as photofermentation substrates, in addition to optimizing the parameters of a certain hydrolysis method, comprehensive consideration of cornstalk hydrolysis method and PSB also makes sense. In this study, cornstalk was pretreated with HCl/ NaOH/cellusase to obtain the fermentable solution, then photo-fermentative H2 production with two Rhodobacter capsulatus strains JL1 and mutant JL1601 (cheR2-) were conducted using these hydrolysates as carbon source, in order to explore the effect of hydrolysis methods on photo-fermentation by PSB and provide theoretical reference for bioconversion efficiency improvement of cornstalk-to-H2.

Materials and methods Inoculum culture The isolated Rhodobacter capsulatus strain JL1 from Xingqing Park lake water in Xi'an, China and a derived mutant JL1601 (cheR2-) with cheR2 gene partly deleted were applied as photofermentative H2 production strains. The construction and characteristic of R. capsulatus JL1601 were described in detail [30]. Suitable pH range for H2 production with the cheR2mutant migrated from faintly acid to weak alkaline condition comparing to strain JL1, and mutant R. capsulatus JL1601

(cheR2-) showed higher NH3eN tolerance. The mutant JL1601 (cheR2-) performed better H2 production performance than the wildtype JL1 under the same illumination condition. Both photosynthetic bacteria were cultured in 50 mL screwed tubes fed with 10 mL MPYE medium at 35
C for about 48 h, shaking at 150 rpm in aerobic dark incubator.

Pretreatment of cornstalk Dried cornstalk was ground and passed through 60 meshes to obtain the powder sample, then 25.0 g of sample in 1 L conical flask was used for each set of experiment (solid-toliquid ratio ¼ 1:10, w/v). The experimental parameters in hydrolysis of cornstalk were listed in Table 1. Cornstalk powder was mixed with 1.5% HCl or 0.75% NaOH for 0.5 h at 108
C, then hydrolysate AcH and AlH were obtained after filtration, respectively. The control group was pretreated with ddH2O for 0.5 h at 108
C to obtain the hydrolysate WH. After chemical hydrolysis with HCl/NaOH/ddH2O as above, the pH values were adjusted to 4.80 with concentrated NaOH/HCl, and 2.25 g cellulase was fed into the mixture to keep at 50
C for 10 h. The corresponding hydrolysates were abbreviated to AcEH, AlEH and WEH. Pretreatments such as pH adjustment and sterilization were implemented before the hydrolysates being used as substrate for photo fermentation.

Photo-fermentative H2 production process Before H2 production process, the photosynthetic bacteria were pre-cultured under dark-aerobic condition at 35
C for 48 h as described previously [31], then harvested and diluted to OD660 ¼ 1. For photo-fermentation process, the pH value of modified MedA culture was adjusted to 7.0 with 1 M NaOH. The compositions of modified MedA are: Solution C 20 mL/L, L-sodium glutamate 1.0 g/L, cornstalk 10 g/L, L-glutamic acid 0.1 g/L, L-aspartic 0.04 g/L, NaCl 1.0 g/L, phosphate buffer 20 mM, 1000*Vitamin 1.0 mL/L. Solution C contains: Nitrilotriacetic acid 10 g/L, MgSO4$7H2O 29.5 g/L, CaCl2$2H2O 3.335 g/ L, FeSO4$7H2O 0.349 g/L, (NH4)6Mo7O24$4H2O 9.3 mg/L, ZnSO4$7H2O 0.5475 g/L, EDTA 0.125 g/L, H3BO3 5.7 mg/L, MnSO4$H2O 77 mg/L, CuSO4$5H2O 19.6 mg/L, Co(NO3)2$6H2O 12.4 mg/L 1000*Vitamin solution contains: Thiamine hydrochloride 0.5 g/L, Nicotinic acid 1 g/L, Biotin 10 mg/L. The photo-reactors were 30 mL disposable sterilized syringes (Fig. 2), in each syringe, 10 mL reaction culture was

Table 1 e Experimental parameters in hydrolysis of cornstalk. Hydrolysatea AcH AlH WH AcEH AlEH WEH a

b

Pretreatment

Enzymatic

1.5% HCl at 108
C for 30 min 0.75% NaOH at 108
C for 30 min ddH2O at 108
C for 30 min 1.5% HCl at 108
C for 30 min 0.75% NaOH at 108
C for 30 min ddH2O at 108
C for 30 min

e e e 2.25 g cellulaseb at 50
C for 10 h 2.25 g cellulase at 50
C for 10 h 2.25 g cellulase at 50
C for 10 h

AcH: acid hydrolysis; AlH: alkali hydrolysis; WH: ddH2O hydrolysis; AcEH: acid-enzymatic combined hydrolysis; AlEH: alkali-enzymatic combined hydrolysis; WEH: ddH2O-enzymatic combined hydrolysis. Commercial cellulose from ChengDe TianFeng Co., 100 U/g (CMC), U: mmol glucose/min/g.

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Reducing sugar conversion efficiency ðRSY; g=gÞ ¼ reducing sugar yield=degraded cornstalk

(2)

Substrate to H2 conversion efficiency ðSCE; %Þ ¼

actual H2 yield ðmolÞ theoretical H2 yield ðmolÞ

(3)

In Eq. (3), the theoretical H2 production is calculated according to Eq. (4)- Eq. (6): CH3 COOH þ 2H2 O/2CO2 þ 4H2

(4)

CH3 CH2 CH2 COOH þ 6H2 O/4CO2 þ 10H2

(5)

C6 H12 O6 þ 6H2 O/6CO2 þ 12H2

(6)

The light energy conversion efficiency (LCE) was defined according to Eq. (7): LCE ¼

H2 energy contant  H2 yield  100% absorded light energy

(7)

In Eq. (7), the H2 energy constant is 286 kJ/mol-H2, the absorbed light energy is instead by the incident light energy.

Results and discussions Comparison of pretreatment method Fig. 2 e The photo-reactor in this experiment.

inhaled, and the redundant volume of the air was drained away. The opening of the syringe was sealed by the silicone tube, and the gas generated during the reaction pushed the plug upward and was collected. The gas output can be easily obtained according to the scale of the syringe. The light source of illumination incubator was provided by 10 W filament lamps (OSRAM, 90 lm) to keep the light intensity at 4000 ± 100 Lux (equal to 5.9 ± 0.1 W/m2). Temperature was kept at 30 ± 0.5
C. Each test was carried out with three independent duplicates.

Analytical methods The reducing sugar concentration in hydrolysate, biomass concentration, pH value and light intensity were determined as described previously [30]. The biogas composition was measured by a gas chromatograph (GC, Agilent, 7820A) equipped with thermo conduct detector (TCD) and a 4 m  3 mm stainless column filled with Hayesep padding. The acetate and butyrate concentration in the liquid samples of photo fermentation were determined by the same GC equipped with a flame ionization detector (FID) and a 30 m  0.32 mm glass column. The cornstalk hydrolysis rate ¼

mI  mR  100% mI

(1)

In Eq. (1), mI is the weight of initial dried cornstalk, mR is the weight of dried cornstalk residue.

The cornstalk used in this study was kindly provided by China agricultural university, which contains 24.3% cellulose, 22.5% hemicellulose, 17.5% lignin and a certain amount of low molecular carbohydrate, protein and inorganic salt. As shown in Table 2, the cornstalk hydrolysis rate and RSY by hydrothermal pretreatment (WH) were 45.51% and 0.13 g/g, respectively. The reducing sugar concentration (RSC) was only 9.13 g/L. Hydrothermal pretreatment increased the dissolution of low molecular sugars in cornstalk, and kept at 108
C to hinder reducing sugar degradation, while the hemicellulose was remain different to dissolution [32]. By way of acid hydrolysis (AcH) and alkali hydrolysis (AlH), the hydrolysis rates were increased to 60.79% and 51.6%, respectively. The crystallinity degree of cornstalk is relatively high, cellulose and lignin are intertwined to form its dense and stable structure, which is almost non-degradable at 108
C. However, b-1,4-glycosidic bond in cellulose molecule is sensitive to acid, appropriate Hþ concentration makes it rupture to decrease the polymerization degree of cellulose to form small molecules of sugars, glycosides and organic acids. Although hemicellulose is water-insoluble, it is almost degraded completely in dilute acid under optimized conditions [33]. Lignin includes acidinsoluble lignin and acid-soluble lignin, so the dilute acid solution can also dissolve a small amount of lignin. Alkali pretreatment leads to a decrease in the polymerization degree and crystallinity of the cornstalk, the chemical bond between lignin and carbohydrate was broken to form easily-digestible hydroxyl lignin, which increased the soluble substances, alkaline pretreatment also partially dissolves the hemicellulose fraction [34], but the hydrolysis rate of alkali pretreatment

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Table 2 e Effect of cornstalk hydrolysis by different methods. Hydrolysate AcH AlH WH AcEH AlEH WEH a b

Hydrolysis rate (%)

Cornstalk concentration (g/L)

RSCa (g/L)

RSYb (g/g)

pH

60.79 51.60 45.51 67.60 64.10 50.81

87.24 78.47 69.04 78.24 80.57 69.79

36.92 0.95 9.13 48.45 20.57 15.06

0.42 0.01 0.13 0.62 0.26 0.22

1.1 7.6 5.2 4.6 5.4 3.9

Reducing sugar concentration. Reducing sugar conversion efficiency.

is still lower than that of acid pretreatment. On the other hand, the RSY of acid-treated cornstalk was 0.42 g/g, and the RSC was 36.92 g/L, which increased by 2.26-fold and 3.04-fold comparing with hydrolysate of hydrothermal treatment, respectively. Dilute acid increased the solubility of cornstalk and converted the dissolved components into reducing sugar. On the contrary, the RSY of alkali hydrolysate cornstalk was only 0.01 g/g, with a RSC of 0.95 g/L, because alkali mainly destroyed the cornstalk compactness, with un-conspicuous effect on the further degradation of cellulose into reducing sugar. Cellulase can degrade cellulose materials efficiently and specifically. As shown in Table 2, the hydrolysis rate of WEH was increased slightly comparing to that of WH, nonetheless the RSY was increased by 69%, which was consistent with the changing trend of RSC. Although water-heat pretreatment has a slight effect on the crystallinity of cornstalk, shredding makes a spot of cellulase action site to be exposed, for the cellulose degradation by enzyme. The hydrolysis rate of AcEH was 67.6%, the RSY and RSC were 0.62 g/g and 48.45 g/L, which were increased by 11.2%, 47.6% and 31.2% respectively in contrast with those of AcH without enzyme treatment. For hydrolysate pretreated with dilute alkali, further enzymatic hydrolysis increased the RSY from 0.01 g/g to 0.26 g/g, while the hydrolysis rate was increased by 24.2%. Both dilute acid and alkali have destructive effects on straw crystal structure. However, the main role of dilute alkali treatment is to tear the complex formed by cellulose and lignin leading to the cellulose absorb water and expand, the straw cell wall becomes loose so that it is easy to be broken down by digestive fluids such as cellulase. pH is one of the important factors affecting photofermentative H2 production. Table 2 shows the final pH value of the hydrolysate. The pH differences among hydrolysates were significant, none but that of alkaline hydrolysate (AlH) was close to the neutral condition. Although the pH of the reaction system was adjusted to 4.8 before enzymatic hydrolysis, the pH values of the three enzymatic hydrolysates were diverse with the changes in composition and concentration of the hydrolysates. The light shielding effect also plays an important role in photo fermentation [35]. As demonstrated by Fig. 3, the colors of hydrolysates were significantly variation. The final hydrolysates obtained were featured by fine transmittance, AcH and WH were orange, while AlH was dark black, and the effect of enzymatic treatment on color was weak. McIntosh, S. et al. also observed that pretreatment of wheat straw with dilute

Fig. 3 e The color of the cornstalk hydrolysate.1:AcH; 2:AlH; 3:WH; 4:AcEH; 5:AlEH; 6:WEH; 7:MedA medium.

NaOH resulted in a dark colored slurry [36], and that the color intensity increased with pretreatment severity. Our research proved that the contribution of alkali is greater than acid to color formation, the second step of further enzymatic hydrolysis played little part in decoloration.

Photo fermentation from cornstalk hydrolysate 10 g-cornstalk/L was used as substrate to explore the H2 production performance of the wildtype strain R. capsulatus JL1 and mutant R. capsulatus JL1601 (cheR2-). The hydrolysates without decolorization were adjusted to pH 7.0 using 1 M NaOH/HCl. Based on the cornstalk concentration in the hydrolysate (Table 2), we calculated the volume of hydrolysate that needed to reach the concentration of 10 g/L cornstalk in the medium. The reaction volume was then supplemented with culture medium to reach 10 mL. As shown in Table 3, the hydrogen yield (HY) of the wildtype strain R. capsulatus JL1 from hydrothermal-treated cornstalk (WH) was 96.7 ± 7.7 mL/g, it was decreased to 82.7 ± 11.8 mL/g with WEH. Even the RSC of AcH was considerably higher than that of WH, the HY from AcH was reduced slightly to 93.8 ± 4.9 mL/g, and it kept going down to 82.8 ± 13.6 mL/g from corresponding further enzyme-treated hydrolysate AcEH. Interestingly, its H2 yield from AlH was 34.9 ± 0.0 mL/g, which was increased to the highest 123.8 ± 14.2 mL/g with AlEH. On the other hand, the cumulative H2 yield by mutant JL1601 (cheR2-) was 10.53% lower than that of strain JL1 with AcH as carbon source, while that of mutant JL1601 (cheR2-) was about 2.7 times with AcEH

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Table 3 e Hydrogen yield (HY) from different hydrolysates (mL-H2/g-cornstalk). Inoculum

Strain JL1 Mutant JL1601 DHYa a

Hydrolysate AcH

AlH

WH

AcEH

AlEH

WEH

93.8 ± 4.9 83.9 ± 4.9 10.53%

34.9 ± 0.0 52. ±5.8 þ50%

96.7 ± 7.7 129.6 ± 10.1 þ34.09%

82.8 ± 13.6 224.9 ± 5.2 þ171.43%

123.8 ± 14.2 218.2 ± 0.0 þ76.25%

82.7 ± 11.8 167.3 ± 3.3 þ102.38%

The increased rate of mutant JL1601 comparing to that of wild strain JL1.

comparing to that of strain JL1. The H2 yield of mutant JL1601 (cheR2-) was 52.3 ± 5.8 mL/g with AlH, and it was significantly increased to 218.2 ± 0.0 mL/g when AlEH was used as substrate. The H2 yield of mutant JL1601 (cheR2-) 167.3 ± 3.3 mL/g from WEH was enhanced by 29.09% contrasting with that from WH. Fig. 4 demonstrated the cumulative H2 production of both strains using different hydrolysates for photo fermentation. It can be seen that the maximum H2 production of strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H2 production of mutant JL1601 (cheR2-) was obtained when the hydrolysates pretreated with acid-enzyme using as the substrates. It was worth noting that the hydrolysis rate of cornstalk in Table 2 was not always positively correlated to photo-fermentative H2 yield for both strain JL1 and mutant JL1601 (cheR2-), only part of the soluble substances in hydrolysate could be converted into H2. Furthermore, pretreatment increased turbidity of the straw mixture, and the color of the hydrolysate will be deepened, especially for alkali hydrolysis. The dark would lead to serious light shielding effect. According to our previous exploration, the light energy absorption of cheR2- mutant JL1601 was relatively stable, and the optimal conversion rate can be achieved without excessive light intensity. Meanwhile, H2 production inhibitors such as NHþ 4 and furfural were inevitable in the process of acid/alkali treatment [37], but mutant JL1601 (cheR2-) showed stronger ammonium tolerance, thus it obtained enhanced H2 yield comparing to the wildtype JL1 from five of six cornstalk hydrolysate. In previous researches on substrate hydrolysis [9,38e40], hydrolysis rate and reducing sugar concentration were

Fig. 4 e The cumulative H2 yield with hydrolysates by strain JL1 and mutant JL1601.

employed to evaluate the pretreatment efficiency, few evidences were presented about the matching of pretreatment method and H2 production strain. However, our results suggested that the suitable pretreatment method would be different among PSB for H2 production, because the environmental tolerance and optimum H2 production conditions are various, especially for various mutant strains. It is necessary to determine the substrate pretreatment method according to the vaccinated bacteria in practice. Table 4 and Fig. 5 summarized the carbon sources compositions and SCEs from different cornstalk hydrolysates by the wildtype JL and mutant JL1601 (cheR2-). In addition to the reducing sugar, there are also different concentrations of acetate and butyrate, which can be used by photosynthetic bacteria. Due to the least inhibitors, both strains obtained the highest SCEs of 51.75% and 69.36% with WH, respectively. In spite of enzymatic pretreatment enhancing the reducing sugar concentration, the SCE of strain JL1 was severely reduced to 31.71% with WEH as substrate, while that of mutant JL1601 (cheR2-) was weakly decreased. Inhibitors appeared during the process of enzymatic hydrolysis, besides, the pH of WH was closer to neutral condition than that of WEH leading to less accumulated salt for pH adjustment (Table 2), and cheR2-deletion enhanced the salt tolerance of strain JL1 to keep a higher conversion rate. Acid/alkali pretreatment reduced the SCEs of both strain JL1and mutant JL1601 (cheR2-) sharply. Due to sugars degradation, the acetate and butyrate were increased. And the pH values of AcH, AcEH and AlEH were all deviation from neutrality, what would be harmful for photo-fermentative H2 production. Moreover, the SCE of mutant JL1601 (cheR2-) from acetate was inherently lowered [30]. The main effective components in alkali hydrolysate (AlH) was acetate accompanying by serious light shielding effect, therefore the SCE of mutant JL1601 (cheR2-) from AlH was only 20.81% with a LCE of 0.8%. Alkali-enzyme hydrolysis (AlEH) significantly improved the effective components in the hydrolysate substrate, the LCE by JL1601 (cheR2-) was increased to 3.2%, even the SCE was only enhanced to 26.18%. Under the same light shielding condition by AlH and AlEH, increased reducing sugar concentration promoted the H2 production rate of AlEH by mutant JL1601 (cheR2-) to enhance its LCE, however, the more acetate and butyrate would hinder the H2 production by mutant JL1601 (cheR2-), therefore the increase rates of SCE and LCE with AlH and AlEH as substrate by mutant JL1601 (cheR2-) do not agree with each other. The SCE of mutant JL1601 (cheR2-) was slightly lower than that of JL1 only when AcH was used as substrate, which was consistent with previous single-factor study that cheR2 gene deletion enhanced the alkali tolerance of photosynthetic bacteria, while the acid resistance was

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Table 4 e The conversion efficiency of strain JL1 and mutant JL1601 from cornstalk hydrolysates. Hydrolysate

AcH AlH WH AcEH AlEH WEH

Carbon source (mM)

SCE (%)

LCE of JL1601

Reducing sugar

Acetate

Butyrate

JL1

JL1601

14.29 0.35 3.34 23.25 9.09 6.09

34.67 27.00 10.83 34.67 36.50 10.83

9.20 0.00 0.00 6.02 11.70 0.00

10.41 13.89 51.75 7.73 14.85 31.71

9.31 20.81 69.36 21.01 26.18 64.16

1.2% 0.8% 1.9% 3.3% 3.2% 2.4%

Fig. 5 e (a)Components of carbon source in different cornstalk hydrolysates and (b) SCE with cornstalk hydrolysates by strain JL1 and mutant JL1601.

slightly damaged. However, the highest LCE of mutant JL1601 (cheR2-) was up to 3.3% with AcEH as substrate. It can be speculated that the effect of pH on photo-fermentative H2 production exceeds other inhibitors in the hydrolysate. To seek more economical substrates pretreatment method with strain-friendly is one of the important ways to improve the photo fermentation H2 production.

Comparison with other similar studies As shown in Table 5, Mirza et al. [41] investigated the effect of dilute H2SO4 and ammonia pretreatment on photo-

biohydrogen production from wheat straw with Rhodobacter capsulatus-PK. Wheat straw was pretreated using 30% ammonia and then hydrolyzed with cellulase and b-glucosidase to obtain 712 mL/L of H2 which was higher than those obtained by using dilute H2SO4 pretreatment. In contrast, the HYs of Rhodobacter capsulatus JL1 and Rhodobacter capsulatus JL1601 with acid-pretreatment cornstalk were higher than those corresponding alkali-treated hydrolysate, the maximum H2 yield was up to 224.9 ± 5.2 mL/g (equivalent to 2249 ± 52 mL/g). Zhang, Z. P. et al. [26] collected different types of crop stalks, such as corn, sorghum, rice, soybean and cotton, then dried, crushed and hydrolyzed with cellulase to

Table 5 e Comparison with similar papers. Substrate

Inoculum

H2 yield

Ref.

Corn Ear Corn stover Sorghum straw Rice straw Soybean straw Cotton stalks Corn cob wheat straw

Enzyme-treated

Pretreatment

Photosynthetic consortium

[26,42]

H2SO4-treated

wheat straw

Bagasse

4% H2SO4-treated 4%H2SO4eCa(OH)2 30%NH3e Enzyme 4% H2SO4

R. sphaeroides NRLL R. sphaeroides DSZM R. sphaeroides RV R. capsulatus PK

Cornstalk Cornstalk Rice straw

NaOH-cellulase Acid-cellulase HCl-enzymatic

230.1 mmol/L 145.8 mmol/L 150.4 mmol/L 140.3 mmol/L 130.7 mmol/L 119.3 mmol/L 589.2 mmol/L 115.3 mL H2 135.1 mL H2 178 mL H2 254 mL/L 372 mL/L 712 mL/L 0.55 L/L 0.005 L/L 339.5mL/g-cornstalk 3.56 ± 0.02 mol/mol 4.39 mL/g-rice straw

R. marinum NBRC 100434 R. marinum (Sanur) R.sphaeroides HY01 R.sphaeroides HY01 mixed culture

[22]

[41]

[43] [9] [24] [27]

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obtain the corresponding hydrolysate. In addition, the PSB flora was used as the hydrogen-producing strain from these hydrolysates, and the hydrogen conversion efficiency of different raw materials varied significantly. The corn stalk ingredients were sundry, the pretreatment method should be chosen according the substrate. For NaOH-cellulase treated cornstalk, R. sphaeroides HY01 produced 339.5 mL/g hydrogen, which was far more than 218.2 ± 0.0 mL/g by Rhodobacter capsulatus JL1601 in our study. For acid-cellulase treated cornstalk, R. sphaeroides HY01 got a SCE of 29.7%, which was slightly more than 21.01% by Rhodobacter capsulatus JL1601 in our study, but much lower than 64.16% by mutant JL1601 from WEH.

Conclusions In this study, cornstalk was pretreated by HCl/NaOH/cellulose alone or in combination to obtain six hydrolysates, acid and alkali were beneficial for hydrolysis rate, however, the RSCs and RSYs changed differently. Especially for the NaOH-treated hydrolysate, it contained less reducing sugar and dark color. A wildtype R. capsulatus JL1 and mutant R. capsulatus JL1601 (cheR2-) were used to generate H2 from these hydrolysates. The results showed that the maximum H2 production of strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the maximum H2 production of mutant JL1601 (cheR2-) was obtained from acid-enzyme substrates. The hydrolysis rates of cornstalk and RSCs were not always positively correlated to photo-fermentative H2 yield, it is necessary to explore the suitable substrate pretreatment method for different inoculums. Furthermore, more attention should be paid on the inhibitory factor removal without harming the strain.

[6]

[7]

[8]

[9]

[10]

[11]

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Acknowledgments This work is supported by Natural Science Foundation of China (No.51888103) and Science and Technology Planning Project of Sichuan Province (No.2016JZ0018).

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