Carbide slag pretreatment enhances volatile fatty acid production in anaerobic fermentation of four grass biomasses

Energy Conversion and Management 199 (2019) 112009

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Carbide slag pretreatment enhances volatile fatty acid production in anaerobic fermentation of four grass biomasses ⁎

T

⁎

Xue Taoa,b, Panyue Zhanga, , Guangming Zhangc, , Mohammad Nabia, Siqi Wanga, Junpei Yea, Shuai Baoa, Qian Zhanga, Na Chena a

College of Environmental Science & Engineering, Beijing Forestry University, Beijing 100083, China Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL 60208, United States c School of Environment & Natural Resources, Renmin University of China, Beijing 100872, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Grass biomasses Carbide slag pretreatment Volatile fatty acid VS removal Microbial community

Anaerobic fermentation of biomasses for volatile fatty acid (VFA) production has attracted widespread attention. Grass biomasses have the advantages of high yield, rapid and perennial growth, low lignin content and low input, etc. In order to improve VFA production from grass biomasses, carbide slag was applied to pretreat four grass biomasses (panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning) before anaerobic fermentation. Results showed that carbide slag pretreatment changed the structure of grass biomasses and enhanced their crystallinity index (CrI). The reducing sugar yield and VFA production from four pretreated grass biomasses were significantly improved. Pretreated turfgrass pruning was the most promising grass compared with other grass biomasses, with the highest VFA production of 8803.87 mg/L and volatile solid removal of 56.76%. Kinetic results indicated that the first order kinetic model, cone model and modified Gompertz model all fitted well with VFA production (R2 > 0.96), and pretreated turfgrass pruning achieved the highest VFA production potential of 11270 mg/L. Turfgrass pruning was the most suitable grass biomass for enzymatic hydrolysis and anaerobic fermentation due to a lower lignin content and CrI. High VS removal revealed that carbide slag pretreatment promoted substrate hydrolysis and further VFA production. There existed a positive correlation between enzymatic hydrolysis and anaerobic fermentation of grass biomasses (R2 = 0.9064). Results of microbial community structure indicated that the high VFA production from turfgrass pruning after carbide slag pretreatment was mainly contributed to enrichment of phylum Firmicutes and genus Clostridium.

1. Introduction Grass biomasses are one of the most abundant and renewable feedstocks around the world, especially more than 40 million acres of turfgrass were estimated in the U.S, which covered almost one-fifth of the country’s land area [1]. They contain a large amount of organic matters, such as carbohydrate and protein, which indicates that grass biomasses are a potential substrate for bioenergy production through anaerobic fermentation technology [2,3]. Furthermore, comparing to other lignocellulosic biomasses, grass biomasses contain low lignin and have relatively soft structure, which makes them more easily to be degraded. Anaerobic fermentation converting these bio-wastes to valuable products (e.g., CH4, volatile fatty acids (VFAs) and ethanol) in an environment-friendly and sustainable way has the potential to replace fossil fuels and reduce environmental pollution [4,5].

VFAs are short-chain fatty acids that consist of six or fewer carbon atoms, which have been employed to a wide range of applications, such as, as feedstock for bioenergy production [4] and bioplastics production [6] or as external carbon sources for nutrient removal in wastewater treatment plants [7]. At present, commercial VFAs are mostly produced by chemical process, which consumes large amounts of fossil carbon resources and causes serious environmental problems. Therefore, the researchers have focused on developing biological routes for VFA production from potential organic wastes, for example, waste activated sludge [8], food waste [9], algal [10], swine manure [11] and rice straw [12], etc. However, there has been a few researches on VFA production from grass biomasses [13,14]. Compared with common feedstocks, anaerobic fermentation of grass biomasses for VFA production shows some significant advantages of high organics, low lignin content, soft structure, renewability and so on [13]. Therefore, the

⁎ Corresponding authors at: College of Environmental Science and Engineering, Beijing Forestry University, Qinghua East Road 35, Haidian District, Beijing 100083, China. E-mail addresses: [email protected] (P. Zhang), [email protected] (G. Zhang).

https://doi.org/10.1016/j.enconman.2019.112009 Received 23 June 2019; Received in revised form 27 August 2019; Accepted 28 August 2019 0196-8904/ © 2019 Elsevier Ltd. All rights reserved.

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determined according to the optimization of reducing sugar production in the preliminary of this study. For the carbide slag pretreatments, 10.0 g dried grass biomasses were immersed in 200 ml carbide slag solution of 1.75% (w/v) at 120 ℃ for 40 min in a sealed stainless-steel hydrothermal reaction kettle (Beijing Yanzheng Biotechnology co. LTD, China). After carbide slag pretreatment, the mixture was cooled to room temperature, and the solid was separated from the liquor by filtration and washing with tap water until a neutral pH. The solid fraction was then dried at 65 °C for 12 h as the substrate for subsequent enzymatic hydrolysis and anaerobic fermentation, which were marked as PV-CS, TL-CS, MS-CS and TP-CS, respectively. Raw grass biomasses were used as control and marked as PV-control, TL-control, MS-control and TPcontrol, respectively.

anaerobic fermentation of grass biomasses for VFA production has significant research value and applications. Despite the potential of grass fermentation, the low yield of VFAs (1.64 g/L) from raw grass biomasses is the main obstacle for developing a viable industrial process [13]. Grass biomasses have the rigid cellulose-hemicellulose-lignin structure, which prevents it to be utilized by enzymes or microbes, thereby resulting in low biodegradability and VFA production. Therefore, pretreatment of grass biomasses to disrupt their rigid structure is necessary prior to VFA fermentation [14] and digestion [15]. Various pretreatments have been applied, including physical treatment [16], chemical treatment [13] and combined treatment [13]. Among these pretreatment methods, alkaline treatment has attracted more and more attention in effective removal of lignin and hemicellulose from biomasses [13]. Compared with traditional alkalis, carbide slag, an alkaline industrial waste, is cheap, which will significantly reduce the chemical cost of pretreatment, meanwhile achieve “waste treating waste”. In our previous study, carbide slag was used to replace traditional alkalis to treat grass biomasses including turfgrass pruning and switchgrass [17,18] for enhancement of reducing sugar production, and the reducing sugar production increased by 79%. However, up till now, there is no study regarding to the VFA production from grass biomasses using carbide slag pretreatment. The present work was therefore to explore the feasibility of carbide slag pretreatment to enhance anaerobic fermentation for VFA production from four grass biomasses. Effects of carbide slag pretreatment on the compositions change, enzymatic hydrolysis and anaerobic fermentation of grass biomasses were investigated, with the aim to provide an effective technology for maximizing the fermentation efficiency for VFA production. In addition, the microbial community structures in fermentation systems of raw and pretreated turfgrass pruning were analyzed.

2.3. Enzymatic hydrolysis for reducing sugar production The enzymatic hydrolysis of grass biomasses was conducted at a solid loading of 2.5% (w/v), a temperature of 50 °C and a rotation speed of 150 rpm. The reaction system contained 1 g biomass sample and 40 ml citrate buffer (0.1 mol/L, pH = 4.8), and the enzyme loading was 30 FPU/g substrate. The reaction mixture was filtered, and the liquid was used to analyze the reducing sugar production. All enzymatic hydrolysis experiments were conducted in triplicates. Results were presented as mean values and standard deviations. 2.4. Anaerobic fermentation for VFA production The raw and pretreated grass biomasses were anaerobically fermented in serum bottles with a working volume of 250 ml. The ratio of substrate to inoculum was 2:1 based on VS, and distilled water was added into serum bottles to reach a constant working volume of 250 ml. The initial pH of all systems was adjusted to neutral using 1 mol/L NaOH or HCl. Sodium 2-bromoethanesulfonate was added to anaerobic fermentation system to selectively inhibit methanogenesis [19]. After flushing with high purity nitrogen (N2) for 10 min to remove oxygen, these serum bottles were sealed and placed in a gas-bath shaker with a mesophilic temperature of 35 ± 1 °C and a rotating speed of 100 rpm for anaerobic fermentation. Each experiment was performed for 14 days in triplicates.

2. Materials and methods 2.1. Materials and inoculum In this study, four grass biomasses, panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning, were chosen for VFA production. The medicago sativa and turfgrass pruning present relatively low lignin content and soft structure, while panicum virgatum and triarrhena lutarioriparia show relatively high lignin content and rigid structure. Panicum virgatum and triarrhena lutarioriparia were harvested at the Grass and Biofuels Center (Beijing, China). Medicago sativa was purchased from Shiyang River experimental station (Gansu, China). Turfgrass pruning was collected from Beijing Forestry University (Beijing, China). After air-drying, the grass biomasses were cut to less than 3 cm, dried in an oven at 85–105 ℃ for more than 24 h, milled by a laboratory mill (DF-25S, Dade, China), screened to obtain a fraction of 20–60 mesh, and then stored in plastic bags at room temperature for later use. The commercial cellulose enzyme with filter paper activity of 200 FPU/g generated by Trichoderma reesei was purchased from Hunan Chemical Ltd. (Hunan, China). Carbide slag was purchased from Hengyang Ruijie Trade Co., Ltd. (Hunan, China), and the main components of carbide slag included 70.06% CaO, 3.03% SiO2, 0.99% Al2O3, 0.40% Fe2O3 and 24.94% loss on ignition [17]. Other chemicals were of analytical grade and purchased from Beijing Chemical Industry Group Co., China (Beijing, China). Seed sludge from Hefei Bida Environment Protection Technology Co., Ltd (Anhui, China) was used as inoculum of anaerobic fermentation, which presented the following characteristics: pH of 7.17, VS (volatile solid)/TS (total solid) of 66.84%, soluble protein content of 543 mg/L, soluble carbohydrate content of 259 mg/L, water content of 90.13% and SCOD of 3160 mg/L.

2.5. Analytical methods 2.5.1. Composition analysis of grass biomasses Cellulose, hemicellulose and lignin of grass biomasses were analyzed using a fiber analyzer (A200i, Ankom, USA). Protein was analyzed by a Kjeldahl Analyzer (2300, FOSS, Switzerland). 2.5.2. Analysis of reducing sugar production After 72 h enzymatic hydrolysis, samples were collected from the serum bottles, and then centrifuged at 10,000 rpm for 5 min. The supernatant was used to analyze the reducing sugar production. The reducing sugar concentration was detected using the 3,5-dinitrosalicyclic acid method [20]. 2.5.3. Analysis of VFA production The fermented samples were taken from serum bottles every two days for the determination of VFA production. The fermented samples were centrifuged at 10000 rpm and 4 °C for 10 min, and then filtrated through a 0.25 μm membrane. 1 ml filtered sample was acidified by addition of 0.1 ml 3% phosphoric acid. Then the VFA concentration and compositions were measured by Gas Chromatograph (GC-2018, Shimadzu Co., Japan) equipped with analytical column DB-FFAP (30 m × 0.25 mm × 0.50 mm). Nitrogen was used as the carrier gas with a flow rate of 2.5 ml/min, and both the temperatures of injector and FID were 250 ℃. The Gas Chromatograph oven was programmed to begin at 90 °C for 3 min, then increase to 180 ℃ at a rate of 15 °C/min,

2.2. Carbide slag pretreatment The carbide slag dosage, pretreatment temperature and time were 2

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and hold at 180 ℃ for 3 min. The sample injection volume was 1.0 μl. All the detections were carried out independently in triplicates. The total VFAs were the sum of acetate, propionate, n-butyrate, iso-butyrate, n-valerate and iso-valerate acid during fermentation.

within lignocellulosic structure, thus preventing the biomass biodegradation. Previous studies reported that an increase of CrI could improve the enzymatic hydrolysis after acid or alkaline pretreatment [25,26]. The CrI changes of grass biomasses in this study are illustrated in Fig. 1B. After carbide slag pretreatment, the CrI of panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning increased by 15.2%, 77.1%, 9.9% and 56.4%, respectively, compared with that from raw grasses. The possible reason for this increase was the solubilization of amorphous region (lignin and hemicellulose) from lignocellulosic biomass after carbide slag pretreatment (Fig. 1A). Chen et al. reported that the CrI of sugarcane bagasse increased by 13.93% after NaOH pretreatment, and the subsequent enzymatic hydrolysis increased by more than two folds [27]. Based on the change of biomass compositions and structure, carbide slag pretreatment might be able to enhance the anaerobic fermentation of grass biomasses.

2.5.4. Other analysis TS and VS were analyzed following the APHA standard methods [21]. Mixture samples of anaerobic fermentation were taken from fermentation bottles every two days to analyze the SCOD, soluble protein and soluble carbohydrate. The mixture samples taken were centrifuged at 10000 rpm and 4 ℃ for 10 min. And the supernatant was filtered through a 0.45 μm membrane for further analysis of soluble fractions. SCOD of filtrate was analyzed by a Water Quality analyzer (Lianhua Tech Co. Ltd.), soluble protein was measured with Lowry’s method using serum protein as a standard solution [22], and soluble carbohydrate was measured with phenol-sulfuric acid method using glucose as a standard solution [23]. The CrI of grass biomasses was analyzed through X-ray diffractometer (XRD) (D8 Advance, Bruker, Germany), and the specific method and process was recorded in our previous report [18]. High-throughput sequencing technology was used to analyze the microbial community biodiversity, richness and compositions during anaerobic fermentation of raw and pretreated turfgrass pruning, and the details of the methods and procedure was reported in our previous study [13].

3.2. Effects of carbide slag pretreatment on reducing sugar production from grass biomasses Reducing sugar is generated from the process of enzymatic hydrolysis, and enzymatic hydrolysis is equivalent to the hydrolysis process of anaerobic fermentation. Therefore, it is necessary to investigate the reducing sugar production from four grass biomasses, and the results are shown in Fig. 2. Obviously, the pretreated grass biomasses showed much higher reducing sugar production than the raw ones. The maximum reducing sugar production was 419.25 mg/g for pretreated turfgrass pruning. Yu et al. reported that the high sugar recovery and VFA production simultaneously obtained after aqueous ammonia pretreatment of lawn grass [14]. Compared with the control, the enhancement of reducing sugar production by carbide slag pretreatment was mainly attributed to the improvement of contact capacity between cellulose and enzymes [18]. Based on above results, it is desirable to use carbide slag pretreatment to improve enzymatic hydrolysis and subsequent anaerobic fermentation of grass biomasses.

2.6. Kinetic analysis of VFA production In this study, the first order kinetic model (Eq. (1)), the cone model (Eq. (2)) and the modified Gompertz model (Eq. (3)) were applied to analyze VFA production kinetics during anaerobic fermentation of grass biomasses.

H = P[1 − exp(−khyd t )]

(1)

P 1 + (khyd t )−n

(2)

H=

H = P exp{ - exp[(λ - t) Rm e/P + 1]}

3.3. Effects of carbide slag pretreatment on anaerobic fermentation of grass biomasses

(3)

3.3.1. VFA production Fig. 3 shows the VFA production from four grass biomasses before and after carbide slag pretreatment. Clearly, the VFA production from pretreated grass biomasses was much higher than that from raw grass biomasses during anaerobic fermentation. The possible reason was that the solubilization of lignin and hemicellulose from grass biomasses and the disruption of grass structure by carbide slag pretreatment made the cellulose in solid fraction more accessible for enzymes and microbes [14]. The maximum VFA production of 8803.87 mg/L was achieved from pretreated turfgrass pruning, which increased by 33.02%, compared with that from raw turfgrass pruning. After carbide slag pretreatment, VFA production of 7547.11, 6186.65 and 8154.96 mg/L was achieved from panicum virgatum, triarrhena lutarioriparia, medicago sativa, increasing by more than 124.12%, 123.07% and 88.07%, respectively. Similarly, Wang et al. reported that the VFA production increased by about 100% after ultrasound-Ca(OH)2 pretreatment [13]. In addition, it was found that the VFA was rapidly produced in the initial 6 days at all fermentation systems, then the VFA concentration slowly increased until the highest value. Similar finding was also observed in the sludge anaerobic fermentation for VFA production [28]. The reducing sugar is the main product of enzymatic hydrolysis, while the VFA are the dominant biochemical product of acidogenic fermentation. According to the previous study, the enzymatic hydrolysis is similar to the hydrolysis process during anaerobic fermentation, and the products in hydrolysis stage are further converted to VFA [14]. There were few reports on the relationship between enzymatic hydrolysis and anaerobic fermentation. Fig. 4 shows the relationship between reducing sugar production and VFA production from both two stages. It

where H is the cumulative VFA production (mg/L) at a certain anaerobic fermentation time (t), P represents the cumulative VFA production potential (mg/L), Khyd is the hydrolysis rate constant (1/d), t is the anaerobic fermentation time (d), n is the shape factor, λ is the lag time (d), Rm is the maximum VFA production rate (mg/L·d), and e is 2.718 28. 3. Results and discussion 3.1. Effects of carbide slag pretreatment on compositions and CrI of grass biomasses It is generally considered that the compositions of biomasses influence its subsequent utilization [24]. The composition changes of grass biomasses after carbide slag pretreatment are illustrated in Fig. 1A. Carbide slag pretreatment improved the cellulose and protein content, but decreased the hemicellulose and lignin content in solid fraction, especially the cellulose content of turfgrass pruning increased by 76.4%. Similarly, Gu et al. and Wang et al. found that alkaline pretreatment using Ca(OH)2 effectively improved cellulose content in pretreated lignocellulosic biomasses [13,24]. The main reason was that the hemicellulose and lignin were solubilized from grass biomasses in an alkaline solution [13]. The CrI is related to the compositions of biomasses, representing the ratio of crystalline cellulose to amorphous region (often lignin and hemicellulose). Crystalline cellulose in lignocellulosic biomasses is highly recalcitrant because of the intermolecular hydrogen bonding 3

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Fig. 1. Compositions (A) and CrI (B) of four grass biomasses before and after carbide slag pretreatment (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

Fig. 4. Correlation of enzymatic hydrolysis and anaerobic fermentation of grass biomasses.

Fig. 2. Reducing sugar production from four grass biomasses before and after carbide slag pretreatment (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

can be found that the VFA production increased linearly with the increase of reducing sugar production with a R2 of 0.9064. The results indicated that the utilization of carbohydrates from hydrolysis contributed to the enhancement of VFA production in anaerobic fermentation. The study of sugar platform from enzymatic hydrolysis was widely conducted, however, only the carbohydrates was involved for reducing sugar production. In contrast, the VFA platform for VFA production from anaerobic fermentation related to the utilization of carbohydrates, proteins and so on, which were the main components from grass biomasses (Fig. 1). Therefore, the study of VFA platform based on grass biomasses should be obtained more attention [6]. The VFA production from four grass biomasses was compared with that from other typical lignocellulosic biomasses, and the results are illustrated in Table 1. Turfgrass pruning pretreated by carbide slag presented excellent performances with a VFA production of 8803.87 mg/L in this study, which was possibly attributed to the relatively softer structure with lower cellulose crystallinity and higher cellulose content of turfgrass pruning [13]. Another possible reason was due to the serious structure disruption after carbide slag pretreatment, which improved the contact opportunities of celluloses and microbes [14]. Compared to other pretreatment methods, carbide slag pretreatment also significantly reduced the chemical cost and realized the utilization of wasted carbide slag. Therefore, carbide slag pretreatment is an efficient and feasible pretreatment method for enhancing fermentative performances of turfgrass pruning.

Fig. 3. Effects of carbide slag pretreatment on VFA production from four grass biomasses (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

4

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Table 1 Comparison of VFA production from different biomasses after different pretreatments. Biomasses

Pretreatment method

VFA production

Refs.

Grass clipping Lawn grass Sludge Panicum virgatum Triarrhena lutarioriparia Medicago sativa Turfgrass pruning

Ultrasound + Ca(OH)2 (0.75%), 30 min, 75 ℃ Ammonium hydroxide (28–30%), 24 h, 50 ℃ Ultrasonic + NaOH, 28 kHz, 60 min, pH 12 Carbide slag (1.75%), 40 min, 120 ℃ Carbide slag (1.75%), 40 min, 120 ℃ Carbide slag (1.75%), 40 min, 120 ℃ Carbide slag (1.75%), 40 min, 120 ℃

3320 (mg/L) 4500 (mg/L) 3700 (mg/L) 7547.11 (mg/L) 6186.65 (mg/L) 8154.96 (mg/L) 8803.87 (mg/L)

[13] [14] [29] This study This study This study This study

parameters could be used to analyze the cumulative VFA production (P) during fermentation. It can be clearly seen that the cone model showed a higher cumulative VFA production than the other two models. After carbide slag pretreatment, the simulated cumulative VFA production from panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning increased by 115.64%, 111.02%, 84.89% and 37.63%, respectively, compared with that from raw grass biomasses. After carbide slag pretreatment, the maximum VFA production rate (Rm) for panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning increased by157.8%, 114.85%, 51.39% and 30.9%, respectively, compared with that of raw grass biomasses. Previous study showed that the Rm for switchgrass fermentation increased by 50.2% after 1% NaOH pretreatment [34]. The turfgrass pruning presented the highest Rm, which might be attributed to the release of more organics and its relatively loose structure. The hydrolysis rate constant (khyd) for raw grass biomasses was higher than that of pretreated grass biomasses in this study. The possible reason might be the solid separation from the mixture after carbide slag pretreatment, which would remove a part of easily biodegraded small organics by filtration and washing with tap water [33]. The lag time (λ) of pretreated grass biomasses was longer than that of raw grass biomasses, which highlighted that its change pattern agreed with that of the khyd. The simulation results indicated that the carbide slag pretreatment significantly enhanced the cumulative VFA production potential and VFA production rate.

Fig. 5. Effects of carbide slag pretreatment on the percentage of individual VFA from anaerobic fermentation of four grass biomasses (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

3.3.2. VFA compositions The application of VFAs not only depends on VFA production but also on the compositions [30]. Therefore, it is necessary to investigate the VFA compositions from different grass biomasses. The proportions of acetate, propionate, n-butyrate, iso-butyrate, n-valerate and iso-valerate from four grass biomasses are shown in Fig. 5. The first five individual VFAs were detected in all fermented systems, but the iso-valerate was not detected in the fermentation system with raw turfgrass pruning and raw panicum virgatum as feedstock. The acetate from pretreated panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning accounted for 63.79%, 73.06% 68.44% and 75.04% of the total VFAs, respectively, significantly higher than that from raw grass biomasses. And the order of individual VFA proportion was as follows: acetate > butyric > propionate > valeric. In the process of nutrient removal from wastewater, the order of organic acid utilization is acetate acid > butyric acid > valeric acid > propionate acid [31]. Therefore, the fermentation broth of grass biomasses after carbide slag pretreatment was a promising choice for carbon source in wastewater treatment plant.

3.4. Substrate variation during fermentation 3.4.1. SCOD dynamic change Lignocellulosic biomass solubilization is the first step and also a main rate-limiting step of anaerobic fermentation. The solubilization of grass biomasses was expressed in terms of SCOD, and higher SCOD meant that more organic matters were released, providing for further VFA production in acidogenesis fermentation [35]. Fig. 6(A) shows the SCOD dynamic change during anaerobic fermentation. The initial SCOD was about 5833–9139 mg/L in all systems. The SCOD greatly increased in the first four days, and then slowly increased or remained steady till the end of anaerobic fermentation. In this study, the increased SCOD was possibly attributed to the high hydrolysis efficiency of cellulose, hemicellulose and protein in grass biomasses. The fermentation system of pretreated grass biomasses showed higher SCOD, indicating higher solubilization ability of particulate organics after carbide slag pretreatment than that of raw grass biomasses. The highest SCOD concentration of 16035 mg/L was obtained from the fermentation system of pretreated turfgrass pruning. The ratio of VFAs to SCOD is an important index, as it shows how much soluble organics can be bioconverted into VFAs [35,36]. As shown in Fig. 6(B), after anaerobic fermentation, the VFAs/SCOD of pretreated grass biomasses was higher than that of raw grass biomasses. Further calculation presented that the maximal VFAs/SCOD of 56.76% was achieved from the pretreated turfgrass pruning, 65.21% higher than that from the raw sample. The increased soluble organics, the loose and broken structure of turfgrass pruning after carbide slag pretreatment (Fig. 1) could improve the contact opportunities between

3.3.3. Kinetics analysis In most previous studies, the first order kinetic model and the modified Gompertz model were used to analyze the kinetics of VFA production [32,33]. Yang et al. introduced the cone model to assess hydrogen production during anaerobic fermentation, and found the cone model could better simulate hydrogen production [15]. Therefore, the first order kinetic model and the modified Gompertz model, in together the cone model, were applied to simulate the VFA production and the specific kinetic parameters are shown in Table 2. As expected, these three models fitted well the cumulative VFA production, and the R2 was higher than 0.96 for all situations. Thus, the simulation 5

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Table 2 Kinetic analysis for VFA production from grass biomasses simulated by the first-order kinetic model, the cone model and the modified Gompertz model (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)). Model

Parameter

PV-control

PV-CS

TL-control

TL-CS

MS-control

MS-CS

TP-control

TP-CS

First order

P (mg/L) Khyd (mg/L·d) R2 P (mg/L) Khyd (mg/L·d) R2 P (mg/L) Rm (mg/L·d) λ (d) R2

3504 0.3513 0.9827 3714 0.3352 0.9842 3352 513.2 0.0886 0.9952

7556 0.3106 0.9902 9566 0.3198 0.9946 7331 1323 0.5513 0.9771

2914 0.3556 0.9673 2919 0.3522 0.975 2786 450.4 0.2891 0.994

6149 0.3022 0.9766 9578 0.1817 0.986 6015 967.7 0.8768 0.965

4352 0.3981 0.9878 4557 0.5319 0.9866 4270 979.6 0.2957 0.9918

8092 0.3335 0.9851 9578 0.1817 0.986 7895 1483 0.8891 0.9696

6534 0.3593 0.9851 7049 0.476 0.9873 6331 1449 0.734 0.9793

8993 0.3518 0.9929 11,270 0.4678 0.9881 8769 1885 0.8037 0.9855

Cone

Modified Gompertz

Fig. 7. VS removal of four grass biomasses with and without carbide slag pretreatment (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

40.55% and 56.76%, respectively (Fig. 7). According to the VFA production (Fig. 3) and VS removal (Fig. 7), the most suitable substrate for VFA production was pretreated turfgrass pruning by carbide slag pretreatment. The maximum VS removal in this work was significantly higher in comparison to that of raw turfgrass pruning (34.35%) in this study, grass waste (25.6%) [2], grass clipping (31.1%) [13] and waste activated sludge (32.6%) [37] in the other studies. The high VS removal after carbide slag pretreatment might be due to the disruption of structure of pretreated grass biomasses and high contact capacity between organics and microbes [30]. The carbide slag pretreatment greatly promoted the biodegradability and utilization efficiency of grass biomasses. 3.5. Analysis of microbial community structure Fig. 6. Effects of carbide slag pretreatment on SCOD concentration (A) and VFAs/SCOD (B) during fermentation process (panicum virgatum (PV), triarrhena lutarioriparia (TL), medicago sativa (MS), turfgrass pruning (TP), and carbide slag pretreatment (CS)).

To further reveal the mechanisms of VFA production enhancement after carbide slag pretreatment, the microbial community biodiversity and community richness in fermentation systems of raw and pretreated turfgrass pruning are presented in Table 3. In two fermentation systems of raw and pretreated turfgrass pruning, 31,196 and 31,579 sequences

substrate and enzymes or microbes. Therefore, the carbide slag pretreatment could effectively improve the conversion of SCOD to VFAs in fermentation system.

Table 3 Comparison of microbial community biodiversity and richness indices in fermentation systems of raw and pretreated turfgrass pruning.

3.4.2. VS removal VS removal as a vital parameter is usually used to assess the utilization efficiency of substrate for VFA production during anaerobic fermentation. The VS removal of panicum virgatum, triarrhena lutarioriparia, medicago sativa and turfgrass pruning were 37.54%, 31.26%, 6

Samples

Sequences

OTUs

Chao 1

Shannon

Simpson

Coverage

TP-control TP-CS

31,196 31,579

438 402

475.66 361.06

4.19 4.34

0.03127 0.03006

0.998077 0.998337

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influence on VFA production (Fig. 3). The dominant microbes of both fermentation systems at phylum level mainly included three categories, Firmicutes, Proteobacteria and Bacteroidetes, which accounted for more than 74.8%, but their proportions were different. Firmicutes was the most abundant phylum, accounting for 35.65% and 42.68% of total effective bacterial sequences in fermentation system of raw turfgrass pruning and pretreated turfgrass pruning, respectively. The phylum Firmicutes contained several acidogenic bacteria to produce proteases, lipases, and several extracellular enzymes biodegrading various kinds of complex organics (such as proteins and carbohydrates) for VFA production [13]. As the positive contributors for hydrolysis and acidogenesis [13], the phylum Bacteroidetes was also detected. Compared with raw turfgrass pruning, pretreated turfgrass pruning promoted the growth of Bacteroidetes to some extents. Proteobacteria is a major microbe and main consumer of propionate, butyrate and acetate, and its existence plays a key role during the hydrolysis and acidogenesis of biomasses [41,42]. The proportion of phylum Proteobacteria decreased in the fermentation system of pretreated turfgrass pruning. Based on above analysis, the improvement of acidogenesis after carbide slag pretreatment might be attributed to the higher relative abundance of Firmicutes and Bacteroidetes and the lower relative abundance of Proteobacteria. Fig. 8(B) shows that in total 22 genera abundance > 1% in at least one fermentation system. The genus Clostridium belonging to the phylum Firmicutes had high relative abundance of 22.42% and 16.43% in fermentation system of pretreated and raw turfgrass pruning, respectively. Clostridium was found to be the dominant genus in anaerobic fermentation system and played significant role in the conversion of organics to VFAs [43,44]. The genus Bacteroides, affiliated to phylum Bacteroidetes, was another major VFA-producing genus in these fermentation systems, which contained several xylanolytic genes for the hydrolysis of complex substrate and could utilize various kinds of carbohydrates (e.g. cellulose and hemicellulose) for efficient VFA production. In fermentation system of pretreated turfgrass pruning the relative abundance of Bacteroides was 16.5% in comparison with that of raw turfgrass pruning (11.34%). Therefore, the high VFA production from pretreated turfgrass pruning might be as a result of efficient enrichment of both Clostridium and Bacteroides. Additionally, the genus Ruminiclostridium belonging to the phylum Firmicutes was observed in the fermentation system of pretreated turfgrass pruning, with a relevant abundance of 9.24% (Fig. 8B). The presence of more genus Ruminiclostridium might also be responsible for the high VFA production and high acetate formation in fermentation system of pretreated turfgrass pruning. Considering the results at phylum and class level (Fig. 8A and B), the carbide slag pretreatment promoted the growth of acidogens by providing massive available organics, thus improving the utilization/ conversion of these organics to form VFAs. Effective enrichment of phylum Firmicutes and Bacteroidetes and genus Clostridium and Bacteroides contributed to the significant improvement of VFA production after carbide slag pretreatment.

Fig. 8. Relative abundance of microbial community during fermentation of raw and pretreated turfgrass pruning (TP) by carbide slag (CS): (A) phylum level and (B) genus level.

were respectively obtained, which suggested that both fermentation systems possessed abundant microbial community [2]. The number of operational taxonomic units (OTUs) was 438 and 402 in fermentation systems of raw and pretreated turfgrass pruning, respectively, which indicated that the carbide slag pretreatment did not greatly change the microbial community biodiversity. Chao 1 representing the microbial community richness had same trend with the OTUs in both fermentation systems. Shannon and Simpson index represent the microbial diversity, and higher Shannon index or lower Simpson index mean higher microbial community diversity [2]. The fermentation system of pretreated turfgrass pruning had the relatively high microbial diversity compared with that of raw turfgrass pruning. In addition, both systems had a high coverage of bacteria sequences of more than 0.998, which demonstrated that the sequences could cover the microbial community biodiversity in both fermentation systems [38,39]. The VFA production performances were closely associated with microbial community structure in the fermentation systems [40]. In this study, the effects of carbide slag pretreatment on the microbial community structure and compositions at phylum and genus levels are illustrated in Fig. 8. As depicted in Fig. 8(A), there were 15 phyla with a relative abundance > 1% in at least one fermentation system, and other phyla were grouped as “others”. Clearly, both fermentation systems had similar microbial species, therefore, the carbide slag pretreatment did not change the metabolic pathway of anaerobic microbes, unlike the

3.6. Overall implications Introducing carbide slag as an alkaline waste into the pretreatment of grass biomasses to improve VFA production in anaerobic fermentation is a promising technology. The detailed mechanisms of influencing enzymatic hydrolysis and anaerobic fermentation by carbide slag pretreatment were revealed for the first time. Carbide slag benefits the disruption of biomass structure and promotes the biodegradability of pretreated grass biomasses, thereby provided more biodegradable substrates for subsequent reducing sugar and VFA production. It is well known that insufficient carbon source in wastewater treatment plant was a common problem [37]. Therefore, the fermentation broth containing abundant VFAs and high proportion of acetate is promising to be selected as carbon resource in wastewater treatment plant. In 7

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addition, grass biomasses treatment and carbide slag utilization can be realized simultaneously. The pilot-scale and large-scale applications of this technology should be valued in future research.

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4. Conclusions

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Carbide slag pretreatment improved the cellulose content and CrI value of four grass biomasses, and resulted in significantly high reducing sugar production from enzymatic hydrolysis and VFA production from anaerobic fermentation. Pretreated turfgrass pruning obtained the maximum reducing sugar production of 419.25 mg/g and VFA production of 8803.87 mg/L. There was a positive relationship between enzymatic hydrolysis and anaerobic fermentation. Enrichment of phylum Firmicutes and genus Clostridium was responsible for the significant enhancement of VFA production in the fermentation system of pretreated turfgrass pruning. In summary, the grass biomasses are suitable as feedstocks of VFA production after carbide slag pretreatment, especially turfgrass pruning, which broadens the application of grass biomasses. And carbide slag is potential and promising in the pretreatment of biomasses for high added-value product recovery due to the high efficiency and low cost.

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Declaration of Competing Interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements [29]

This research was funded by National Natural Science Foundation of China (51578068) and the Fundamental Research Funds for the Central Universities (2017CGP017).

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