Bacterial cellulose production by Acetobacter xylinum ATCC 23767 using tobacco waste extract as culture medium

Bioresource Technology 274 (2019) 518–524

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Bacterial cellulose production by Acetobacter xylinum ATCC 23767 using tobacco waste extract as culture medium

T

Jianbin Yea, Shanshan Zhenga, Zhan Zhangb, Feng Yangc, Ke Maa, Yinjie Fengb, Jianqiang Zhenga, ⁎ Duobin Maoa, Xuepeng Yanga, a School of Food and Biological Engineering, Henan Provincial Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Dongfeng Road 5#, Zhengzhou 450002, Henan Province, China b Technology Center, China Tobacco Henan Industrial Co., Ltd, Zhengzhou 450000, China c Henan Cigarette Industrial Tobacco Sheet Co, Ltd, Henan, Xuchang 461000, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial cellulose Tobacco waste extract Acetobacter xylinum Nicotine Fermentation

In this study, bacterial cellulose (BC) was synthesized by Acetobacter xylinum ATCC 23767 using tobacco waste extract (TWE) as a carbon source. Nicotine was found to be an inhibitory factor for BC synthesis, but it can be removed at pH 9.0 by steam distillation. After removing nicotine, the BC production was 2.27 g/L in TWE prepared with solid-liquid (S-L) ratio at 1:10. To further enhance the BC production, two fermentation stages were performed over 16 days by re-adjusting the pH to 6.5 at 7 days, after the first fermentation stage was completed. Using this two-stage fermentation, the BC production could reach 5.2 g/L. Structural and thermal analysis by FE-SEM, FT-IR, XRD and TGA showed the properties of BC obtained from TWE were similar to that from Hestrin-Schramm (HS) medium. Considering the huge disposal tobacco waste in China, the present study provides an alternative methodology to synthesize BC.

1. Introduction Cellulose is one of the most abundant natural polymers on earth and has been widely used in many fields (Cacicedo et al., 2016; Jahan et al., 2018). It is a polymer of glucose units linked together by β-1, 4-

⁎

Corresponding author. E-mail address: [email protected] (X. Yang).

https://doi.org/10.1016/j.biortech.2018.12.028 Received 5 November 2018; Accepted 9 December 2018 Available online 10 December 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

glycosidic bonds. Traditionally, cellulose is extracted from the lignocellulosic biomass of plants, but it can also be synthesized by some bacteria, such as Gluconacetobacter, Acetobacter, Agrobacterium, and Rhizobium (Uzyol and Sacan, 2017). The latter material is called bacterial cellulose (BC). Although it has similar chemical composition to

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liquid pretreatment in the previous studies (Cheng et al., 2017; Hong et al., 2012). However, only the free sugar was extracted and utilized for the BC production in the present study, as the rest part of solid was transferred to the Reconstituted Tobacco Company of Xuchan (Henan Province, China) for the production of reconstituted tobacco. Acetobacter xylinum ATCC 23767 was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Nicotine was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Plant cellulose standard (CAS, 9004-34-6) and other chemicals were obtained from Shanghai Aladdin Bio-chem Technology Co., Ltd (Shanghai, China).

plant cellulose, BC has higher degrees of purity, tensile strength, crystallinity, porosity, polymerization, water-holding capacity, biodegradability, and biological adaptability (Campano et al., 2016; Wang et al., 2017). These superior physicochemical properties result in a wide range of application, such as in the biomedical, textile, audio device (Lima et al., 2017; Shah et al., 2013; Shi et al., 2014). Thus, BC synthesis has attracted the increasing interest of many researchers. However, the high production cost of BC has become one of the main drawbacks to its general application in industrial and academic fields (Hu et al., 2010). The typical substrate for BC production includes several types of carbon sources, such as glucose, sucrose, fructose, glycerol, mannitol, and arabitol (Hong et al., 2012; Mohammadkazemi et al., 2015). The traditional Hestrin–Schramm (HS) medium was commonly used for the BC production, which is expensive and requires additional products, such other carbon source, yeast or peptone, etc. Therefore, inexpensive raw materials containing high levels of reducing sugars are frequently considered promising substrates for BC production. Recently, various industrial by-products and agroforestry wastes have been utilized as carbon sources to improve the yield and reduce the cost of BC production. Thus far, several raw waste materials have been demonstrated to be potential substrates for BC production, such as distillery effluent (Jahan et al., 2018), corn steep liquor (Costa et al., 2017), fruit juice (Kim et al., 2017), corn stalks (Cheng et al., 2017), litchi extract (Yang et al., 2016), beverage industrial waste (Fan et al., 2016), corncob acid hydrolysate (Huang et al., 2015), and waste beer yeast (Lin et al., 2014). Tobacco (Nicotiana) is an economic crop cultivated worldwide (Wang et al., 2015). China is the largest consumer and producer of tobacco products in the world, with more than 2 million tons of fluecured tobacco used for cigarette production every year (Liu et al., 2015). During the production process, approximately 1 million tons of tobacco waste is produced, which includes unwanted tobacco leaves, tobacco stems, and scraps (Wang et al., 2013; Zhong et al., 2010). Unfortunately, this tobacco waste is commonly dumped into landfills or incinerated because of its high content of toxic nicotine (Okunola et al., 2016; Zhang et al., 2013). This has endangered human health and contributed to environmental pollution. Recently, tobacco waste has been used as a substrate to produce fertilizer, pectinase, and some medicine precursors, for example (Chaturvedi et al., 2008; Wang et al., 2015; Zheng et al., 2016, 2017). However, to our knowledge, there are no reports showing tobacco waste as a substrate for BC production. In fact, the high content of sugars present in tobacco waste, including glucose, sucrose, fructose, and other polysaccharides, would make tobacco waste a promising substrate for BC production. In this study, we first evaluated tobacco waste extract (TWE) as a substrate for BC production and demonstrated that nicotine in the medium is an inhibitor of this process. Then, a steam-distillation process was used to remove the nicotine and increase the BC yield. BC production was further increased by adjusting the pH after first-stage fermentation. Using this two-stage fermentation strategy, TWE was shown to be an ideal substrate for BC production, which has the potential to reduce the cost of BC production and to solve the problem of tobacco waste. This study provides a green and sustainable method to reuse tobacco waste.

2.2. Methods 2.2.1. Preparation of TWE and other culture media TWE was prepared as follows. (1) Tobacco waste was boiled with distilled water for 1.5 h at different solid-to-liquid ratios (SL ratios, w/v, 1:4, 1:6, 1:8, 1:10, or 1:12). (2) After cooling, insoluble solids were removed and the TWE was collected by filtering through muslin cloth under vacuum. (3) The pH of the TWE was adjusted to 6.5 by adding 0.2 M NaOH or HCl. The sugar yield (%) was defined as Eq. (1), and the volume (L) was according to the liquid used during the extraction.

Sugar yield (%) Total sugar concentration in TWE =

( ) ∗ volume (L) ∗ 1000 ( ) mg L

ml L

solid tobacco waste weight (mg) ∗ 100%

(1)

To investigate the effect of extra carbon sources on BC production, modified TWE media were prepared by adding 5 g/L glucose, fructose, sucrose, or mannose. To evaluate the effect of nicotine on BC production, Hestrin-Schramm (HS) medium was prepared as follows (g/L): glucose (20.0), peptone (5.0), yeast extract (5.0), Na2HPO4 (2.7), and citric acid monohydrate (1.15), pH adjusted to 6.5. Different concentrations of nicotine (0, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0, or 2.4 g/L) were added to the HS medium. Pre-culture medium was prepared to increase the population of bacteria before fermentation (g/L): glucose (20.0), peptone (5.0), and yeast extract (5.0), initial pH of 6.5. 2.2.2. Remove of nicotine by steam distillation The nicotine in TWE was removed by steam distillation at different pH values. After ensuring the optimal solid-to-liquid ratio, 1000 mL of prepared TWE was added to the distillation flask, and the pH adjusted to various values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0) by addition of NaOH or HCl. Then, 200 mL of liquid was removed by steam distillation to eliminate the nicotine from the TWE. After cooling, 200 mL deionized water was added to the flask to replenish the volume and the pH was returned to 6.5 by the addition of NaOH or HCl. The original and residual nicotine contents of the TWE were both detected by high performance liquid chromatography (HPLC), and the nicotine removal was calculated as equation (2):

Nicotine removal rate original nicotine content - residual nicotine content = ∗ 100% original nicotine content

2. Materials and methods

(2)

2.2.3. Strain incubation and BC production A. xylinum ATCC 23767 powder was activated in a 10-mL tube containing pre-culture medium as described above at 30 °C for 2 days with 150 rpm shaking. The activated bacteria were then cultured for 2 days in a 250-mL shaker bottle containing 100 mL HS medium to increase the number of bacteria. Ten percent seed culture was transferred into different types of fermentation medium, followed by a 7-day static fermentation at 30 °C with 150 rpm shaking to produce BC. BC production (mg/L) was defined as: weight of BC production by bacteria

2.1. Materials Tobacco waste, was collected from China Tobacco Henan Industrial Co., Ltd. (Zhengzhou, China). The waste was found to be composed of 25.4% cellulose, 22.3% hemicellulose, 3.1% lignin, 6.2% pectin, and ∼38.5% soluble substance and 5.1% ash. Among the soluble substance, 25.1% sugar, 9.2% protein, 2.2% nicotine were detected. In order to holistic valorize the crop waste, the cellulose or hemicellulose were first pre-hydrolysis by acetic acid or enzymatic saccharification by ionic 519

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(15.24 mg/mL). The low extraction efficiency at the SL ratio of 1:4 could have resulted from insufficient soaking of the tobacco waste. The BC production with different TWEs was not obviously related to the concentration of total sugar. Considering the relative total sugar concentrations, the TWE with the SL ratio of 1:6 was supposed to produce the highest BC production. However, the highest BC production (1.54 g/L) was observed with the SL ratio of 1:10, followed by SL ratios of 1:8 (1.28 g/L), 1:12 (1.27 g/L), 1:6 (0.84 g/L), and 1:4 (0.75 g/L). Previous studies have demonstrated that nicotine is one of the main bacteriotoxic chemical components when using tobacco waste as a carbon source for fermentation (Liu et al., 2015; Wang et al., 2009). In the present study, we also detected nicotine in different TWEs. Nicotine was extracted from the tobacco waste along with the sugars, and both nicotine and total sugar concentrations showed similar trends with respect to SL ratio. It seems that nicotine could be an inhibitor of BC production in our experiments. For example, the concentration of nicotine in the TWE at an SL ratio of 1:12 (1.06 mg/mL) was significantly lower than that at an SL ratio of 1:6 (2.21 mg/mL), which likely contributed to the higher BC production in the former (1.27 g/L). It is noteworthy that the highest BC production (1.54 g/L) was observed with the TWE at an SL ratio of 1:10. TWE with ratio 1:10 provides the highest sugar extraction yield, which is a best reason to select this value, if nicotine is removed in the final process. These results indicate that both the nicotine and sugar concentrations could be major factors affecting BC production when using TWEs as carbon sources, with the sugars acting as substrates and the nicotine acting as an inhibitor. Thus, TWE at an SL ratio of 1:10 was used further in this study for BC synthesis.

fermentation in 1 L TWE medium. 2.2.4. Harvest and structural analysis of BC After 7 days of fermentation, the cellulose pellicles were harvested and the pH of the fermented liquid was adjusted to 6.5 for the secondstage fermentation. The cellulose pellicles were harvested again after 16 days fermentation. The cellulose pellicles were washed with 0.2 M NaOH at 80 °C for 2 h to remove bacteria and other impurities, and then washed with distilled water several times to neutralize the pH. Thereafter, the purified BC was filtered and dried overnight at 80 °C to a constant weight to calculate the BC production. The morphology of BC was studied by using Field emission scanning electron microscope (FESEM). Prepared BC samples were sputtering with gold for 120 s using an ion sputter coater (SC-701 Quick coater, Japan) and then observed at 10,000× magnification with a field emission scanning electron microscope (JSM-7001F, Jeol, Japan) operated at 5 kV. BC samples were first mixed with spectroscopic-grade potassium bromide powder (1% w/w). Then, Fourier transform infrared (FT-IR) spectroscopy was carried out to measure the functional structure of BC samples, in the range of 400–4000 cm−1 wavelength with a FTIR spectrometer (Vertex70, Bruker, Germany). Plant cellulose was used as a standard to confirm the specific functional structure of cellulose. The thermal degradation behavior of BC was performed by using a simultaneous thermal analyzer (STA 449F3, Netzsch, Germany). 10 mg BC was subjected to the thermal treatment, and then scanned over a temperature range from 50°Cto 600 °C with a heating rate of 10 °C /min, under nitrogen atmosphere with a flow rate of 70 mL/min. The X-ray diffraction (XRD) was used to analyze the crystalline structure of BC. Samples were scanned from 5 to 60° (2θ range) at a scan speed of 0.5°/min, by using D/max-RAX ray diffractometer (D8 Advance, Bruker, Germany) with Cu Kα radiation (λ = 0.154 nm) operated at 40 kV and 30 mA. The crystal size of BC and crystallinity index (CI) was determined according to previous researches (Costa et al., 2017).

3.2. Effect of nicotine on BC production and nicotine removal from TWE In order to investigate the effect of nicotine on BC production, nicotine was added exogenously to the HS medium at different concentrations and the BC production were compared. Fig. 1 shows that the BC production was reduced with increasing nicotine content in the HS medium. Specifically, when the nicotine concentration increased from 0 to 1.2 mg/mL, the BC production was slightly reduced from 3.26 to 2.49 g/L. However, the BC production was reduced dramatically (1.37 g/L) when the nicotine concentration reached 1.6 mg/mL. After adding nicotine to a final concentration of 2.4 mg/mL, the BC production was only 0.51 g/L, which was almost 1/6 that observed in HS medium. Nicotine toxicity to bacteria has been reported (Wang et al., 2009; Yuan et al., 2006). In the present study, nicotine is an obvious inhibitor of BC production at high concentrations. Although some previous studies have suggested that extra nitrogen sources could enhance BC production (Huang et al., 2015; Nguyen et al., 2008), nicotine was not included. Thus, nicotine should be removed when using TWEs as carbon sources to improve BC production. So far, two main methods were reported to remove nicotine from the TWE, one is degradation by microbe and the other one is steam distillation. The former one mainly focus one the biodegradation by the bacteria, such as Rhodococcus, Pseudomonas, Acinetobacter, ect. (Gong et al., 2016; Wang et al., 2013; Zhong, et al., 2010). Nevertheless, the free sugar in the TWE will also be utilized by these microbe during the biodegradation process, which may affect the BC production in the present study. Thus, to remove the nicotine from the TWE, steam distillation was performed at different pH values in this study. The nicotine removal rate, total residual sugars, and BC production were obtained (Fig. 2). The results show that the nicotine removal rate increased with increasing pH. The removal rate of nicotine was 52.9% at pH 7.0, 63.9% at pH 8.0, and 88.7% at pH 9.0. Nevertheless, the removal rate was only slightly increased to 90.9% at pH 10.0, which indicates that increasing the pH beyond this point is unnecessary. The nicotine removal rate was remarkably decreased at acidic pH. For example, a nicotine removal rate of only 1.0% was observed at pH 3.0. Steam distillation under alkaline conditions has been commonly used to

2.2.5. Analysis methods The total sugar and reducing sugar concentrations of the TWE media were assayed using the dinitrosalicylic acid (DNS) method (Cakar et al., 2014). Glucose, xylose, arabinose, galactose, mannose, and sucrose were determined by ion chromatography, using an ICS-3000 from Dionex (Sunnyvale, CA, USA) with an electrochemical detector and a CarboPac™ PA20 (3 × 150 mm) separation column equipped with a CarboPac PA20 (3 × 30 mm) guard column (Dionex). Elution was performed with 2 mM NaOH for 25 min, followed by regeneration for 5 min with 100 mM NaOH and equilibration for 15 min with 2 mM NaOH. The flow rate was 0.4 mL/min. The nicotine in TWE was detected by HPLC, as described in a previous report (Zhong et al., 2010). 2.2.6. Statistical analysis All experiments were repeated in triplicate. The means and standard deviation (SD) values are shown in the figures and tables. The different extraction efficiency of the TWEs were compared by the t-student test using EPI info7.0. 3. Results and discussion 3.1. Chemical composition of different TWE and BC production Table 1 shows the chemical composition of the TWEs at different solid-to-liquid (SL) ratios (w/v, SL ratio = 1:4, 1:6, 1:8, 1:10, and 1:12). In order to select an optimal SL ratio, the sugar yield (%) was used to evaluate the extraction efficiency. The extraction efficiency of the TWE increased as the SL ratio increased from 1:4 to 1:10, and then decreased slightly at 1:12 (P > 0.05 compared to the 1:10 SL ratio value). However, the concentration of total sugar in the TWE was highest when the SL ratio was 1:6 (22.43 mg/mL), followed by the SL ratios of 1:8 (21.07 mg/mL), 1:10 (18.67 mg/mL), 1:4 (16.39 mg/mL), and 1:12 520

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Table 1 Chemical composition of TWEsa with different solid to liquid ratios. Chemical composition

Nicotine (mg/L) Glucose (mg/L) Arabinose (mg/L) Galactose (mg/L) Fructose (mg/L) Mannose (mg/L) Xylose (mg/L) reducing sugar (mg/L) Sucrose (mg/L) Total sugar (mg/L) Sugar yield (%,1 L) Sugar/nicotineb (mg/L) BC production (mg/L)

Solid-to-liquid ratio 1/4

1/6

1/8

1/10

1/12

1.86 ± 0.10 6.85 ± 0.29 0.17 ± 0.04 0.28 ± 0.07 4.36 ± 0.14 1.73 ± 0.10 0.43 ± 0.09 15.23 ± 0.46 2.68 ± 0.18 16.39 ± 1.12 6.55 ± 0.45 8.81 0.75 ± 0.05

2.21 ± 0.11 10.70 ± 1.01 0.16 ± 0.04 0.23 ± 0.04 4.29 ± 0.25 1.80 ± 0.24 0.32 ± 0.08 18.67 ± 0.50 2.94 ± 0.15 22.43 ± 1.76 13.46 ± 1.05 10.1 0.84 ± 0.10

1.63 ± 0.11 9.94 ± 0.84 0.16 ± 0.04 0.19 ± 0.05 4.43 ± 0.25 1.66 ± 0.23 0.25 ± 0.05 16.56 ± 1.13 2.72 ± 0.18 21.07 ± 1.17 16.85 ± 0.93 12.9 1.28 ± 0.21

1.24 ± 0.11 8.92 ± 0.50 0.13 ± 0.02 0.17 ± 0.04 3.70 ± 0.37 1.46 ± 0.12 0.13 ± 0.02 14.40 ± 0.92 2.78 ± 0.30 18.67 ± 0.45 18.67 ± 0.45 15.1 1.54 ± 0.31

1.06 ± 0.05 7.44 ± 0.72 0.10 ± 0.02 0.12 ± 0.03 2.39 ± 0.19 1.36 ± 0.15 0.08 ± 0.02 12.33 ± 0.94 2.45 ± 0.11 15.24 ± 1.38 18.30 ± 1.65 14.3 1.27 ± 0.14

BC, bacterial cellulose. All data are presented with mean ± SD (n = 3). a TWE, tobacco waste extract. b The ratio of total sugar to nicotine.

(18.07 mg/mL) was observed at pH 8.0, with residual sugar levels of 17.33 mg/mL at pH 9.0 and 17.05 mg/mL at pH 10.0. The lowest residual sugar content (16.10 mg/mL) occurred at pH 4.0. Fig. 2 shows that the sugar content was maintained when nicotine was removed at different pH values. As shown in Tables 1 and 2, glucose, fructose, sucrose, and mannose were the four main sugars detected in the TWEs and NR TWEs. For example, the NR TWE (nicotine removed at pH = 9.0) with an SL ratio of 1:10, contained 7.08 mg/mL glucose, 3.37 mg/mL fructose, 2.42 mg/mL sucrose, and 1.06 mg/mL mannose (Table 2), which were similar to the original TWE (Table 1). The results in the present study suggest that steam distillation is an efficient method to remove nicotine while preserving sugar content. The BC production increased with increased nicotine removal (Fig. 2), when the sugar content was maintained in the TWEs. After removing 90.9% of the nicotine in the TWE at pH 9.0, the highest BC production (2.27 g/ L) was observed. Compared with that of the original TWE (Table 1, 1.54 g/L), the BC production (Fig. 2, 2.27 g/L) increased ∼47.4% after removing the nicotine. A similar BC production was observed at pH 10.0, which was attributed to similar nicotine removal rates. In contrast, the residual sugar content was 16.83 mg/mL at pH 3.0, but the BC production was only 1.35 g/L due to the higher nicotine content (1.23 g/L). This result is consistent with that observed with the HS medium, in that the BC yield was obviously inhibited when the nicotine concentration was higher than 1.2 g/L (Fig. 1).

Fig. 1. Effect of nicotine on bacterial cellulose (BC) production yield in the Hestrin-Schramm medium. Means ± SDs (n = 3) are plotted.

3.3. Effect of adding extra carbon sources on BC production using TWE medium Compared to using other renewable agroforestry residues as substrates for BC synthesis (Supplementary), the BC production (2.27 g/L) was relatively low using NR TWE. This could be attributed to the lower total sugar in TWE than in other renewable cellulosic wastes (Cheng et al., 2017; Huang et al., 2015; Yang et al., 2016). Thus, extra carbon sources were added into the NR TWE to increase its BC production yield. To make the total sugar content exceed 20 mg/mL, 5 g of each sugar (glucose, fructose, sucrose, or mannose) was respectively added to 1 L NR TWE medium. As shown in Fig. 3, the BC production increased to 2.56 g/L after adding 5 g fructose, which was only slightly increased by 16.4% from the original NR TWE (2.27 g/L). Previous studies have demonstrated that fructose can enhance BC synthesis because the bacteria prefer to use it as a substrate (Dahman et al., 2010). After supplementing the TWE with glucose, the BC production was only slightly increased to 2.36 g/L. This lower increase in BC production could be

Fig. 2. Residual total sugar and BC production of NR TWEs under different nicotine removal rates at different pH values. BC, bacterial cellulose; NR TWE, nicotine removed tobacco waste extract. Means ± SDs (n = 3) are plotted. A, B, C, D, E, F, G, H represented the NR TWEs at pH 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, respectively. The pH of all NR TWEs was adjusted back to 6.5 for BC production.

collect the nicotine from tobacco (Jones et al., 2001; Wang et al., 2006). The results in the present study indicate that the nicotine in TWEs can be removed efficiently at an appropriate alkaline pH. After removing the nicotine, water was added back and the sugar content and BC production were investigated. Very small amounts of sugar were lost during nicotine removal. The highest content of sugar 521

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Table 2 Chemical compositions of NR TWEsa at different pH values. Chemical compositionb

pH = 10.0

pH = 9.0

pH = 8.0

pH = 7.0

pH = 6.0

pH = 5.0

pH = 4.0

pH = 3.0

Nicotine (mg/L) Glucose (mg/L) Fructose (mg/L) Mannose (mg/L) Reducing sugar (mg/L) Sucrose (mg/L) Total sugar (mg/L)

0.11 ± 0.03 6.49 ± 1.01 3.27 ± 0.42 1.07 ± 0.19 13.93 ± 1.26 2.10 ± 0.23 17.05 ± 0.87

0.14 ± 0.02 7.08 ± 0.60 3.37 ± 0.45 1.06 ± 0.18 13.20 ± 1.10 2.42 ± 0.22 17.33 ± 1.16

0.45 ± 0.02 7.85 ± 0.77 3.40 ± 0.70 1.32 ± 0.12 15.10 ± 1.70 2.62 ± 0.36 18.07 ± 1.60

0.58 ± 0.02 7.15 ± 0.84 3.43 ± 0.15 1.01 ± 0.2 12.77 ± 1.45 2.08 ± 0.24 16.80 ± 1.60

0.84 ± 0.02 7.60 ± 0.70 3.30 ± 0.70 1.19 ± 0.1 13.37 ± 1.02 2.31 ± 0.31 17.28 ± 1.05

1.09 ± 0.37 7.29 ± 2.60 3.20 ± 1.17 1.11 ± 0.4 13.37 ± 4.94 2.73 ± 0.86 17.47 ± 1.20

1.19 ± 0.02 7.11 ± 0.47 2.50 ± 0.33 1.22 ± 0.17 12.50 ± 1.12 1.87 ± 0.25 16.10 ± 0.42

1.23 ± 0.01 7.12 ± 0.43 3.23 ± 0.47 1.13 ± 0.23 12.40 ± 0.92 2.78 ± 0.31 16.83 ± 1.44

All data are presented with mean ± SD (n = 3). a NR TWEs, nicotine-removed tobacco waste extracts. b Galactose, arabinose, and xylose were not detected.

Fig. 3. Effect of various extra carbon sources on BC production yield. Five grams of the indicated extra carbon source was added into the NR TWE. BC, bacterial cellulose; TWE, tobacco waste extract; NR, nicotine-removed. Means ± SDs (n = 3) are plotted.

Fig. 4. BC production yield using two-stage fermentation strategy. The pH was adjusted after 7 days of fermentation (first fermentation stage). BC, bacterial cellulose. Means ± SDs (n = 3) are plotted.

attributed to the formation of gluconic acid when the bacteria use glucose as a substrate for growth (Masaoka et al., 1993). No obvious changes in BC production yield were observed after adding sucrose or mannose, although prior research had demonstrated that sucrose and mannose could be ideal carbon sources for BC synthesis (Mohammadkazemi et al., 2015; Tsouko et al., 2015). Collectively, our results indicate that extra carbon sources may not be a useful strategy to improve BC production by using NR TWE as culture medium.

Table 3 The chemical composition of NR-TWE* at different fermentation stages.

3.4. Increasing BC production by adjust pH during fermentation In order to further increasing the BC production, the effect of fermentation parameters on BC production using NR TWE medium were also investigate (data not shown). The optimal fermentation parameters for BC production was as following: initial pH was 6.5, temperature was 30 °C, fermentation time was 7 days, and inoculum size was 10%, which were similar to the former research focus on the BC synthesis using corn stalk extracts as substrates (Cheng et al., 2017). However, the BC production was only 2.56 g/L under the optimal fermentation parameters after 7 days fermentation (Fig. 4). Since the sugar substrate content was 17.33 g/L in the NR TWE, a BC production that was greater than 2.56 g/ L was anticipated. Several previous reports focus on BC producing from other natural carbon resources were summarized (Supplementary). BC production from these raw waste materials was almost larger than 4.0 g/L, which could lead them to be an ideal carbon source for the BC production. Comparing to using other material, the utilized rate of sugar by using TWE was rarely lower after 7 days fermentation. After 7 days fermentation, the BC production in NR-TWE was not increased along with the fermentation went on. In order to investigate the utilized rate of sugar after 7 days fermentation, the main chemical composition of several sugar was detected and listed in the Table 3. Results showed that more than half of the carbon resource were still exist in the culture, indicated that BC production was potential to be increased by suitable strategies. It was worth to note that the pH was only 2.65 after 7 days fermentation. Nevertheless, BC pellicles rather than irregularly

Chemical composition

Initial

After first stage

After second stage

nicotine (mg/L) Glucose (mg/L) Fructose (mg/L) Mannose (mg/L) Reducing sugar (mg/L) Sucrose (mg/L) total sugar (mg/L) pH

0.14 ± 0.02 7.08 ± 0.60 3.37 ± 0.45 1.06 ± 0.18 13.20 ± 1.10 2.42 ± 0.22 17.33 ± 1.16 6.5

0.13 ± 0.03 4.04 ± 0.23 2.33 ± 0.06 0.89 ± 0.22 7.93 ± 0.23 2.20 ± 0.10 10.20 ± 0.26 2.6

0.12 1.07 0.14 0.04 1.83 0.77 2.30 3.7

± ± ± ± ± ± ±

0.01 0.15 0.01 0.06 0.15 0.35 0.20

All data are presented with mean ± SD (n = 3). NR-TWE*: nicotine removed TWE at pH 9.0.

cellulose particles was anticipated to form during the fermentation process in the present study, which suggested that agitating or stirring is not allowed (Tanskul et al., 2013). Under this situation, controlling the pH throughout the whole fermentation may not be an optional choice for increasing BC production. In the other side, the BC production is limited when the pellicles grows downward, which may due to the insufficient oxygen after forming pellicles in the surface of culture medium (Borzani and Souza, 1995). Thus, we tried to increase BC production further by adjusting the pH to 6.5 after 7 days fermentation and continuing with a second stage fermentation. Specifically, after 7 days of fermentation, the BC was first harvested to remove the limitation of insufficient oxygen, then the pH was adjusted to 6.5 by adding 0.1 M NaOH, and BC synthesis was allowed to proceed. Fig. 4 shows that the BC production sharply increased during the second stage of fermentation starting from 8 days; the final BC production reached 5.2 g/L and most of the carbon source were utilized again (Table 3). This result indicates that BC synthesis was inhibited by the acidic pH resulting from the intermediate acid metabolism in the fermented liquid. Although the bacterium A. xylinum prefers a slightly acidic

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Table 4 Characteristics peaks obtained for BC samples and plant cellulose. Medium

Plant cellulose HS medium original TWE NR TWE of first stage NR TWE of second stage

Crystallinity (%)

96.23 86.00 91.70 92.70 93.95

Crystallinity Index (%)

Size of Crystals(A°)

Segal equation

1Ī0

110

28 54 61 56 72

*

77.47 67.04 85.58 83.39 77.75

/ 106 107 123 92

Crystallite structure 200

004

44 52 59 52 61

41 /* /* /* /*

Cellulose Cellulose Cellulose Cellulose Cellulose

I I I I I

*: No peak was observed at this crystal plane.

using Segal method (Costa et al., 2017). Results were listed in the Table 4, showed all three BC samples obtained from TWE mediums have similar CI value and indicates similar crystalline morphology of the polymers and the physical characteristics of the membranes existing in these BC samples. The Plant cellulose was also identified as cellulose I, with peaks at 2 ϴ angles of 15.1, 22.8 and 34.8°, corresponding to the (1 I¯ 0), (2 0 0), and (0 0 4) crystal planes. To summarize, after removing nicotine and using a two-stage fermentation strategy, the BC production was 5.2 g/L, which maximized the sugar substrate utilization. BC production from the NR TWE was nearly 1.6 times as that from using HS medium, which suggested that TWEs are ideal substrates for BC production. Structural analysis showed that the BC obtained from NR TWE belongs to the cellulose I, and the properties were similar to that from the HS medium.

environment to synthesize its BC due to the acidic environments to which the bacterium is accustomed (Ha et al., 2011). Further increasing the pH dramatically decreased BC production, which could be attributed to the low growth of A. xylinum as well as the inhibition of enzyme participated in the BC producing process. Thus, two stage fermentation by adjusting the pH is an alternative method to increase the BC production in NR TWE. 3.5. Morphological and structural analysis of BC FE-SEM results showed that BC produced by using TWE as carbon source owns a dense network of interwoven ultrafine fibrils, which is similar to the observations of previous studies by using other raw waste materials (Costa et al., 2017; Yang et al., 2016; Lin et al., 2014). Compared to the HS medium, BC produced by TWE medium owned denser network and shorter width of fibrils (Supplementary). BC produced by using NR TWE as medium had better morphology than that produced by using original TWE, such as smoother surface and denser network. It seems that the width of fibrils of BC produced by the second stage fermentation is slightly shorter than the first stage fermentation. This may be contributed to lower contents of carbon source in the second stage. Overall, the FE-SEM results showed the BC produced by using TWE owns smooth surface of the fibers. FT-IR spectroscopy results showed both BC samples from HS medium and TWE mediums own characteristic peaks of cellulose (Supplementary). The distinguish peaks of 3340 cm−1 indicates O–H stretching, 2900 cm−1 indicates C–H stretching, 1645 cm−1 indicates C–O–C stretching, 1064 cm−1 indicates C–O stretching and 900 cm−1 indicates γ (COC) in plane, symmetric stretching. The plant cellulose is also observed as the standard product, which confirms the chemical structure of the BC. Similar results have been reported in the previous studies (Yang et al., 2016; Jahan et al., 2018). No obvious difference is observed between BC samples obtained from HS medium and TWE medium, indicating that no influent on the function group of BC. TGA results revealed that BC samples obtained from TWE and HS medium own similar degradation curve (Supplementary). BC samples are stable until temperature increased up to 250 °C, and then start to decompose beyond this temperature. At a range of 250–340 °C, about 60% of BC was decomposed and ∼10% to ∼20% of BC was left undecomposed up to 500 °C. The degradation curve of plant cellulose sample was also observed, 100% plant cellulose was decomposed at a range of 250–340 °C. These results were similar to the previous reports (Jahan et al., 2018), which indicated that BC is more thermostable than the plant cellulose. However, no obvious difference is observed between different medium. X-ray diffraction (XRD) patterns were used to evaluate the crystalline structure as well as the changes in crystallinity of the BC produced from different mediums (Supplementary). All BC samples were diffraction profiles characteristic of cellulose I, with peaks at 2ϴ angles of 14.8, 16.8 and 22.8° for BC obtained from HS medium, 2 ϴ angles of 14.4, 16.6 and 22.6° for BC obtained from three different TWE mediums, corresponding to the (1 I¯ 0), (1 1 0), and (2 0 0) crystal planes. The crystallinity index (CI) was calculated based on peak intensity

4. Conclusions We have provided evidence supporting TWE as a potential carbon source for BC synthesis. The nicotine in TWE was demonstrated to inhibit BC production and could be removed by steam distillation at pH 9.0. Under these conditions, the nicotine removal was 90%, increasing the BC production from 1.54 to 2.27 g/L. Using two-stage fermentation, the BC production could be further increased to 5.2 g/L by adjusting the pH to 6.5 after 7 days static fermentation. Structural analysis revealed the similar properties of BC obtained from TWEs medium and HS medium. Declaration of interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Acknowledgements The research was supported by the Key Technologies R&D Program of Henan Province (Grant No. 172102410038) and National Natural Science Foundation of China (Grant No. 41807401, No. 31571778 and No. 21476217), Doctor Support Grants of Zhengzhou University of Light Industry (BSJJ2014066, Zhengzhou University of Light Industry). We also thank the editors and reviewers for their suggestions on the manuscript writing. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2018.12.028. References Borzani, W., Souza, S.J., 1995. Mechanism of the film thickness increasing during the bacterial production of cellulose on non-agitated liquid media. Biotechnol. Lett. 17, 1271–1272. Cacicedo, M.L., Castro, M.C., Servetas, I., Bosnea, L., Boura, K., Tsafrakidou, P., Dima, A., Terpou, A., Koutinas, A., Castro, G.R., 2016. Progress in bacterial cellulose matrices

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