Bioresource Technology 151 (2014) 113–119
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Production of bacterial cellulose by Gluconacetobacter hansenii CGMCC 3917 using only waste beer yeast as nutrient source Dehui Lin a,1, Patricia Lopez-Sanchez b,2, Rui Li a,3, Zhixi Li a,⇑ a b
College of Food Science and Engineering, Northwest A&F University, No. 28 Xinong Road, 712100 Yangling, Shaanxi, China Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Australia
h i g h l i g h t s It was found a novel approach to convert cellulosic wastes to green biomaterials. A two-step pre-treatment was used to treat WBY to improve the reducing sugar yield. Ultrasonication combining mild acid hydrolysis was an effective pre-treatment. BC yield of WBY treated by ultrasonication was almost 6 times as that of untreated WBY. BC produced from WBY hydrolysates displayed good physicochemical properties.
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Article history: Received 23 July 2013 Received in revised form 14 October 2013 Accepted 15 October 2013 Available online 24 October 2013 Keywords: Bacterial cellulose Gluconacetobacter hansenii Waste beer yeast Pre-treatment Property
a b s t r a c t In order to improve the use of waste beer yeast (WBY) for bacterial cellulose production by Gluconacetobacter hansenii CGMCC 3917, a two-step pre-treatment was designed. First WBY was treated by 4 methods: 0.1 M NaOH treatment, high speed homogenizer, ultrasonication and microwave treatment followed by hydrolysis (121 °C, 20 min) under mild acid condition (pH 2). The optimal pre-treatment conditions were evaluated by the reducing sugar yield after hydrolysis. 15% WBY treated by ultrasonication for 40 min had the highest reducing sugar yield (29.19%), followed by NaOH treatment (28.98%), high speed homogenizer (13.33%) and microwaves (13.01%). Treated WBY hydrolysates were directly supplied as only nutrient source for BC production. A sugar concentration of 3% WBY hydrolysates treated by ultrasonication gave the highest BC yield (7.02 g/L), almost 6 times as that from untreated WBY (1.21 g/L). Furthermore, the properties of the BC were as good as those obtained from the conventional chemical media. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the most abundant natural biopolymer in the world and it has been widely studied during the past decades (Klemm et al., 2005). Traditionally cellulose is extracted from plant tissues, but it can also be produced by some species of bacteria including genera Acetobacter, Agrobacterium, Gluconacetobacter, Rhizobium, and Sarcina. Cellulose produced by these microorganisms is commonly referred to as bacterial cellulose or BC (Klemm et al., 2005; Lisdiyanti et al., 2006). Though BC contains sets of parallel chains composed of b-1, 4-D-glucopyranose units interlinked by intermolecular hydrogen bonds, which is identical in chemical composition to plant cellulose (Wan et al., 2006), it displays many ⇑ Corresponding author. Tel.: +86 13571939821. E-mail addresses: [email protected] (D. Lin), [email protected] (P. Lopez-Sanchez), [email protected] (R. Li), [email protected] (Z. Li). 1 Tel.: +61 420520907. 2 Tel.: +61 33467373. 3 Tel.: +86 15091597123.
unusual physicochemical and mechanical properties, including higher purity, higher crystallinity, higher degree of polymerization, higher water absorbing and holding capacity, higher tensile strength and stronger biological adaptability (Klemm et al., 2001; Ul-Islam et al., 2012). Therefore, BC represents a potential alternative to plant-derived cellulose for specific applications in bio-medicine, cosmetics, high-end acoustic diaphragms, papermaking, food industry and other applications (Czaja et al., 2007; Klemm et al., 2011; Shah et al., 2013). However, the low yield and the high production cost of BC are important drawbacks for its industrial production and broad range of applications (Vandamme et al., 1998). In this context, it is highly relevant to investigate bacterial strains with stronger ability to produce BC using lower cost culture media. Recently, various cellulosic wastes from renewable agro-forestry residues or industrial by-products have been investigated to be as carbon sources to improve the production yield and decrease the economic cost. These include food process wastes (Carreira et al., 2011), hydrolyzed hemicelluloses from waste liquor of atmospheric acetic acid
0960-8524/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.052
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pulping (Uraki et al., 2002), beet and sugar cane molasses (Bae and Shoda, 2005), various fruit wastes (Hungund et al., 2013), konjac powder (Hong and Qiu, 2008), rice bark (Goelzer et al., 2009), wheat straw (Chen et al., 2012), Cotton-based waste textiles (Hong et al., 2012), maple syrup (Zeng et al., 2011), coffee cherry husk (Usha and Appaiah, 2011), and dry olive mill residue (Gomes et al., 2013). Furthermore making use of such waste materials would not only improve the sustainability of cellulose production by microorganisms, but also decrease environmental pollution associated with the disposal of industrial wastes. Beer production is an important economic activity in many countries, especially in China. Consequently, plenty of waste beer yeasts (WBY), being the second major by-product from brewing industry, is generated and either thrown out or fed to livestock and fowl. This not only results in a series of environmental problems, but also causes an enormous waste of resources (Yang et al., 2006). In general, WBY is mainly composed of 48–55% protein, 23–28% carbohydrate, 6–8% RNA, 1% glutathione, and 2% vitamin B. Moreover, they are rich in P, K, Ca, Fe, P and Mg (Liu et al., 2012). Due to its high nutritional content, it might be used for the production of green products by microorganisms. However, it is difficult to use it directly as a nutrient source for microorganisms, due to that most of the protein and carbohydrate exists in the cell walls in the form of large polymers (Yang et al., 2006). Therefore, it is important to disrupt the beer yeast cell walls and depolymerize large polymers to facilitate its use by microorganisms (Yang et al., 2006). Currently chemical pre-treatments are commonly used for the disruption and hydrolysis of yeast cells, such as sulfuric acid, hydrochloric acid and alkaline solution pre-treatments (Guo et al., 2008). Although chemical pre-treatments are effective, they are rather harsh and they can damage the nutrients. Moreover they can easily cause a secondary environmental pollution. In this sense, some studies are focusing on the use of physical pre-treatments including microwaves, ultrasonication and mechanical comminution, which are generally regarded as non-hazardous to the environment because few or none chemicals need to be added (Vodenicˇarová et al., 2006). When particles in a liquid suspension are treated by microwaves, ultrasonication or mechanical comminution, they are subjected to either surface erosion or size reduction (due to cavitation, shear force, ‘‘thermal’’ or ‘‘athermal’’ effects) (García et al., 2012; Yu et al., 2010). Consequently, these processes can be used to physically disrupt and break microbial cells in order to release nutrients from them (Guo et al., 2008). They can also be used to depolymerize large polysaccharide molecules and generate a pre-treated substrate that is more easily hydrolyzed via increasing the accessible surface area (Vodenicˇarová et al., 2006; Wang et al., 2010). However, physical pre-treatments cannot hydrolyze polysaccharides into monosaccharides directly and only monosaccharides can be used by Gluconacetobacter hansenii to produce BC. In order to solve the existing problem, a two-step pre-treatment, namely physico– chemical pre-treatment combining different mild chemical and physical treatment options, could be used. This combination should be not only effective for disrupting cells and dispersing large polymer aggregates, but also for improving hydrolysis. In previous studies, only chemical pre-treatments including acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis have been used to hydrolyze the waste beer yeast cells to release the nutrients for the production of green products (Cui et al., 2009; Ha et al., 2011, 2008). However, few studies have reported a two-step pre-treatment, combining mild chemical and physical treatment options, to treat WBY as a source of nutrients for bacterial cellulose production. In the present work, two-step pretreatments were explored in order to find an effective method to enhance the reducing sugar yield of WBY. WBY was first treated by four methods including 0.1 M NaOH treatment, high speed homogenizer, ultrasonication
and microwave treatment followed by hydrolysis at 121 °C for 20 min under mild acid condition (pH 2) as a second step. The effects of the four pre-treatments and the optimum pre-treatment conditions were evaluated by the reducing sugar yield after hydrolysis. Furthermore, treated WBY hydrolysates were directly served as only nutrient source for BC production by G. hansenii CGMCC3917 and we investigated the influences of the four pretreatments and the WBY hydrolysate concentration on the BC yield. Additionally, water holding capacity (WHC), water release rate (WRR) and water absorption rate (WAR) of the bacterial cellulose were measured and its microstructure was determined using scanning electron microscopy (SEM).
2. Methods 2.1. Microorganisms and materials G. hansenii strain was used in this study. This strain has been isolated from inoculums of a strain screened in homemade vinegar and then induced by high hydrostatic pressure treatment. It has a strong ability to produce BC more than three times as that of its initial strain. It was deposited as CGMCC3917 at China General Microbiological Culture Collection, Beijing, China and was maintained on glucose agar slants containing: 2% glucose (w/v), 0.5% yeast extract (w/v), 0.1% K2HPO4 (w/v), 1.5% MgSO47H2O (w/v), 1.5% ethanol (v/v), and 1.7% agar (w/v). It was stored at 4 °C in a refrigerator and sub-cultured every 2 months for inoculum development or stored at 80 °C using 20% (v/v) glycerol instead of agar for long-time storage (Ge et al., 2011). For seed inoculum, a loop of G. hansenii CGMCC 3917 was transferred from a slant culture into an Erlenmeyer flask (250 mL) containing 100 mL of seed medium with the same components as glucose agar slants but without agar. Then it was cultivated at 30 °C in a rotary shaker incubator at 150 rpm for 12–18 h until it reached the logarithmic growth phase (Ge et al., 2011). WBY was obtained from TsingTao Brewery Xi’an Hans group co., Ltd. (Xi’an, Shaan xi, China).
2.2. Pre-treatments and hydrolysis of WBY Pre-treatment of dry waste beer yeast was performed by mixing WBY with 100 mL of distilled water in a 250 mL round-bottom flask to reach a final concentration of 5%, 10%, 15% and 20% (w/v) respectively, which were regarded as untreated WBY mixed liquor. Four methods were investigated to treat WBY mixed liquor and details of the treatment procedures were as follows: Pre-treatment 1: mixed liquor with different concentrations of WBY was treated with 0.1 M NaOH at 50 °C for 6, 12, 18, 24, 30 and 36 h respectively. Pre-treatment 2: mixed liquor with different concentrations of WBY was treated by high speed homogenizer at 15,000 rpm (XHF-D, Ningbo Xingzhi Biotechnology Co., Ltd., Zhejiang, China) for 5, 10, 15 and 20 min respectively. Pre-treatment 3: mixed liquor with different concentrations of WBY was treated by ultrasonication at supersonic power of 500 W (YQ-1003A, Ningbo Power Ultrasonic Equipment Co., Ltd., Zhejiang, China) for 10, 20, 30, 40, 50 and 60 min respectively. Pre-treatment 4: mixed liquor with different concentrations of WBY was treated by microwaves at microwave power of 600 W (Galanz P70D20P-TF, China) for 5, 10, 15 and 20 min respectively. After each pre-treatment, the samples were hydrolyzed at 121 °C for 20 min under mild acid condition (pH 2.0). Then they were centrifuged at 4000g for 15 min to remove sediments and sterilized water was added to the supernatant up to a volume of
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100 mL. For each pre-treatment a sample which was not centrifuged was used as control. 2.3. Determination of total reducing sugars The total reducing sugar concentration was determined after hydrolysis by PAHBAH reducing end assay described by Lever (1972) with D-glucose as standard. Sugar yield was calculated using the following equation:
Reducing sugar yield ð%Þ ¼
Reducing sugar weight ðgÞ 100 Waste yeast weight ðgÞ
2.4. BC production and harvest 2.4.1. BC production using WBY hydrolysates as nutrient source The prepared seed inoculum (9%, v/v) was transferred into a glass vessel (500 mL) containing different treated WBY hydrolysates as described above after adjusting their pH to 5 using 2 M NaOH and they were cultivated statically at 30 °C for 14 days. 2.4.2. BC production using WBY hydrolysates with different concentrations The pre-treatment with the highest reducing sugar yield was selected to further investigate the optimal sugar concentration for BC production. After pre-treatment, WBY was hydrolyzed at 121 °C for 20 min under mild acid condition (pH 2). Then it was centrifuged at 4000g for 15 min to remove sediments and the supernatant was collected and added with sterilized water or sterilized glucose solution (50%, w/v) from its initial sugar concentration of 4.38% (w/v) to reach a final concentration of 1%, 3%, 5% and 7% (w/v) respectively. Finally, the prepared seed inoculum (9%, v/v) was transferred into a glass vessel (500 mL) containing 100 mL of WBY hydrolysates prepared at different concentrations as described above after adjusting its pH to 5 using 2 M NaOH and they were cultivated statically at 30 °C for 14 days. 2.4.3. BC purification and quantification After cultivation, the BC membranes were rinsed with running water overnight, soaked in 0.1 M NaOH at 80 °C for 2 h to remove bacteria, and then washed with deionized water several times to completely remove alkali. The purified cellulose was dried at 105 °C for 12 h and weighted until constant weight. For each membrane, triplicate experiments were performed, and the mean values were calculated (Wu et al., 2010). 2.5. BC membrane analysis 2.5.1. Water holding capacity (WHC) For the determination of WHC, wet BC samples were removed from the storage container with tweezers. The samples were shaken twice quickly and then weighed, followed by drying at 60 °C for 48 h in order to completely remove water from them. WHC was calculated by the following formula (Shezad et al., 2010):
Water holding capacity ¼
Mass of water removed during drying ðgÞ Dry weight of cellulose ðgÞ
2.5.2. Water release rate (WRR) To determine WRR, Shezad et al.’s (2010) method was used with small modifications. The wet BC samples were cut into small pieces and dried using a freeze dryer (MCFD5508, SIM International CO.) for 48 h. The dried BC samples (1 g) were subsequently immersed in distilled water under shaking (100 rpm) condition at room temperature for rewetting. After complete rewetting
(stabilized wet weight), samples were analysed for WRR by continuously measuring their weights at various time intervals at room temperature until complete drying. The weights of the BC samples at different time intervals were plotted against time. 2.5.3. Water absorption rate (WAR) The dry BC sample was cut into four pieces and then the pieces were subsequently immersed in distilled water under shaking (100 rpm) condition at room temperature for rewetting. Samples were analysed for WAR by continuously measuring their weights at various time intervals at room temperature until complete absorption. WAR of dry BC membrane was calculated by the following equation:
Water absorption rate ð%Þ ¼
W1 W 100 W
where W (g) is the weight of dry BC sample; W1 (g) is the weight of BC after it absorbed water. The experiments were carried out for ten replicates. 2.5.4. SEM observation For scanning electronic microscope observation, the air-dried BC membrane was mounted on a copper stub using double adhesive carbon conductive tape and coated with platinum for 30 s using a platinum coating facility (Auto Fine Coater JFC-1300, Jeol, Japan). The SEM photographs were obtained by scanning electron microscope (JSM-6360LV, Jeol, Japan) at room temperature at 15 kV. 3. Results and discussion 3.1. Effects of pre-treatments on the reducing sugar yield of WBY In order to determine which pre-treatment led to a higher yield of reducing sugars, the reducing sugar content was assessed after hydrolysis. Two variables, namely the dosage of WBY and duration of the pre-treatment, were investigated. As shown in Fig. 1, the reducing sugar yield was significantly affected by the dosage of WBY and the pre-treatment time for all four pre-treatments. The reducing sugar yields of untreated WBY are represented by time 0 in the plots. The results of pre-treatment 1 using 0.1 M NaOH are described in Fig. 1A. During the first 6 h, the reducing sugar yield increased slowly for the four dosages. We suggest that this lag time could be due to the soaking time required for the NaOH to start working on the yeast cells. After 18 h pre-treatment, the reducing sugar yield (w/w) from 20% WBY reached the highest value of 20.48%. While all other WBY dosages reached a plateau after 30 h pretreatment. The maximum sugar yield values achieved were 20.59%, 22.26% and 28.98% for 5%, 10% and 15% WBY respectively. The reducing sugar yield increased with the WBY dosage with the exception of 20% WBY. When 20% WBY was treated, the highest value reached faster (18 h) but it was the lowest yield, similar to that obtained from 5% WBY. This suggests that the concentration of NaOH used was not enough to treat the 20% WBY and a higher concentration could potentially improve the reducing sugar yield of 20% WBY. Fig. 1B shows the results of pre-treatment 2 using a high speed homogenizer. It can be observed that a dosage of 20% WBY gave the highest reducing sugar yield after 10 min. While all other dosages reached their maximum values after 15 min. The maximum reducing sugar yield increased with the dosage of WBY with values of 9.83%, 11.22% and 16.17% for 5%, 10% and 15% respectively. Although the maximum yield from 20% WBY reached faster with a value of 13.26%, it was lower than that from 15% WBY. Similar trend was observed in pre-treatment 1 for the dosage of 20%.
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Fig. 1. Influences of four pre-treatments on the reducing sugar yield of WBY after hydrolysis. Pre-treatment 1:0.1 M NaOH treatment (A), pre-treatment 2: high speed homogenizer (B), pre-treatment 3: ultrasonication (C), pre-treatment 4: microwaves (D).
Furthermore the highest reducing sugar yield obtained from pretreatment 2 was relatively lower compared to that from pre-treatment 1. The reducing sugar yields obtained from pre-treatment 3 using ultrasonication (Fig. 1C) showed similar trends to pre-treatments 1 and 2. The dosage of 20% displayed the highest reducing sugar yield after ultrasonication for 30 min. While all other dosages reached their maximum values after 40 min. The maximum reducing sugar yield increased with the dosage of WBY with values of 16.97%, 21.53% and 29.19% for 5%, 10% and 15% WBY respectively. The highest reducing sugar yield obtained from pre-treatment 3 was clearly higher than that obtained from pre-treatment 2 and similar to that obtained from pre-treatment 1. After microwave pre-treatment for 10 min, the WBY dosage of 10%, 15% and 20% led to almost similar reducing sugar yield 9.46%, 9.77%, 9.96% respectively, while 5% WBY gave a relatively lower reducing sugar yield of 8.63% (Fig. 1D). After 15 min pretreatment, the four dosages reached their maximum values and the maximum reducing sugar yield increased with the dosage of WBY with values of 9.26%, 12.25% and 13.01% for 5%, 10% and 15% respectively, except for the dosage of 20%. In general the highest reducing sugar yield obtained from pre-treatment 4 was the lowest among the four pre-treatments. These results indicated that the optimal WBY dosage was 15% for the four pre-treatments. As discussed above the reducing sugar yield of the 20% WBY could be increased by a higher concentration of NaOH in pre-treatment 1. For the other pre-treatments the results suggest that 15% WBY might display enough specific surface area to subject to erosion before hydrolysis and the 20% WBY might be too viscous for the physical processes to work as efficient as that for lower concentrations. Changing the settings used in the instruments such as shear input, ultrasonication or microwave
power may assist in increasing the reducing sugar yield of the 20% WBY after hydrolysis. The highest reducing sugar yield obtained from ultrasonication (29.19%) was almost six times higher than that obtained from the untreated WBY (4.15%), followed by NaOH treatment (28.29%), high speed homogenizer (13.33%) and microwave treatment (13.01%). Although WBY treated by 0.1 M NaOH (pre-treatment 1) and ultrasonication (pre-treatment 3) had the similar reducing sugar yield, the best pre-treatment time of ultrasonication (40 min) was obviously shorter than that of 0.1 M NaOH pre-treatment (30 h). Furthermore ultrasonication without harsh chemicals used could reduce the environmental impact. Therefore, it can be concluded that ultrasonication in combination with mild acid hydrolysis was the most effective pre-treatment to improve the reducing sugar yield of WBY with an optimal dosage of 15% and a best pre-treatment time of 40 min. 3.2. BC production After hydrolysis, WBY hydrolysates were directly supplied to G. hansenii CGMCC 3917 as carbon and nutrient sources to produce BC without any extra nutrient added. In Fig. 2 it can be observed that the BC yields obtained using centrifuged WBY from pre-treatment 1and pre-treatment 3 were both higher than those from the WBY which were not centrifuged. This could be likely due to the fact that the un-centrifuged samples after these pre-treatments have a high sugar concentration (they showed the highest sugar yields) which could cause the inhibition of the BC production and reduce the supply of oxygen by the liquid medium. While in the centrifuged samples the reducing sugar concentration was decreased by diluting the supernatant with water, which could lead to a better concentration for BC production. In contrast, the centrifuged WBY from pre-treatment 2, pre-treatment 4 and the untreated
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8 1%
7
3%
6
BC yield (g/L)
WBY produced lower BC yields compared to those from the uncentrifuged WBY. Likely the cellulose production by G. hansenii CGMCC 3917 in this case was inhibited due to the low sugar concentration present in the centrifuged samples, which were further diluted from the already low sugar yields of WBY obtained from these pre-treatments. Additionally, BC production using WBY treated by ultrasonication (pretreatment 3) displayed the highest yield (3.89 g/L) of all four pre-treatments. Furthermore it was clearly higher than that from pre-treatment 1 using 0.1 M NaOH (2.33 g/L), despite the fact that they have almost the same sugar concentration after hydrolysis. It can be concluded that the harsh alkali used in pre-treatment 1 might degrade other nutrients in the waste beer yeast which are beneficial for G. hansenii CGMCC 3917 during BC production, while those nutrients are still present after ultrasonication pretreatment. In order to improve the BC production, the ultrasonication pretreatment with the highest reducing sugar yield was selected to further investigate the optimal sugar concentration. WBY hydrolysates treated by ultrasonication, with a reducing sugar yield of 29.19% (w/w), which corresponds to a reducing sugar concentration of 4.38% (w/v), were either mixed with sterilized water or sterilized glucose solution (50%, w/v) to reach a final sugar concentration of 1%, 3%, 5%, 7% (w/v) respectively. As described in Fig. 3, the reducing sugar concentration had a marked effect on the BC production. The BC yield using WBY hydrolysates with 1% sugar concentration was the lowest and it reached the maximum value of 2.18 g/L on day 7. When the sugar concentration was increased to 3%, the BC yield increased significantly showing a peak at 7.02 g/L on day 10. However a further increase in sugar concentration reduced dramatically the BC yield, 5% sugar led to a maximum of 5.73 g/L on day 8 and the maximum yield for 7% sugar concentration was only 2.88 g/L on day 7. Hence it was concluded that the highest sugar concentrations inhibited the BC production, which is in agreement with the findings from previous studies (Carreira et al., 2011). Additionally, it was also concluded that the sugar concentration would affect the fermentation cycle to some extent, due to the fact that the BC yields from the various sugar concentrations reached their maximum at different cultivation time. To sum up, after optimization, the highest BC yield (7.02 g/L) was almost 6 times as that obtained from the untreated WBY (1.21 g/L). The cellulose membrane was obviously thicker:
5%
5
7%
4 3 2 1 0 0
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9
10 11 12 13 14
Culture Time (Day) Fig. 3. The yield of BC produced by CGMCC 3917 using WBY hydrolysates with different concentrations.
15.4 mm compared to 2.3 mm obtained from the untreated WBY and 5.7 mm from the un-centrifuged WBY (Fig. 4, see Supplementary figure). Moreover, the 100 mL of WBY hydrolysates was almost completely used by G. hansenii CGMCC 3917 to produce BC (Fig. 4C, see Supplementary figure) and its yield doubled that obtained from the conventional chemical media (3.58 g/L) (Ge et al., 2011). Although some researchers have investigated various cellulosic wastes from renewable agro-forestry residues or industrial by-products to produce BC, some extra nutrients are added to media to improve the BC yield. For instance, Hong and Qiu (2008) have obtained an increase of BC production with Ca2+ introduced into the medium of konjac powder hydrolysates, Gomes et al. (2013) have enhanced BC production with supplemented nitrogen and phosphate sources in olive mill residue hydrolysates, Ha et al. (2008) have improved BC yield with adding glucose into waste beer yeast hydrolysates as supplement nutrient. In the present study no extra nutrient was added. Moreover, BC production using WBY pretreated by ultrasonication displayed a high yield and WBY hydrolysates could totally substitute the conventionally used chemical media. Our findings could not only overcome the high BC production cost, but also reduce the waste of resources and environmental pollution. Therefore, it has been not only demonstrated that waste beer yeast has great potential to produce BC, but also that ultrasonication is an effective pre-treatment tool in biomass pre-treatment technology.
4.5 4 3.5
uncentrifuge
BC yield (g/L)
3
centrifuge
2.5 2 1.5 1 0.5 0 1
2
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5
Fig. 2. BC production from WBY treated by different methods. Pre-treatment 1:0.1 M NaOH treatment (1), pre-treatment 2: high speed homogenizer (2), pre-treatment 3: ultrasonication (3), pre-treatment 4: microwaves (4), un-treatment (5).
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Water content (%)
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untreated WBY and nearly doubled that from the conventional chemical media. Additionally, the properties and microstructure of BC produced by WBY hydrolysates were as good as those obtained from the conventionally used chemical media. Acknowledgements This work was financially supported by the Fundamental Research Funds for the Central Universities (QN2009072). The technical support of Dr. Shuangkui Du, associate professor of the College of Food Science and Engineering, Northwest A&F University, is also gratefully acknowledged.
100
Time (h)
Appendix A. Supplementary data Fig. 5. Evaluation of WRR and WAR of BC produced by CGMCC 3917 using WBY hydrolysates.
3.3. Characterization of BC 3.3.1. WHC, WRR and WAR WHC, WRR and WAR are some of the most important properties measured to determine the possible applications of BC in medical areas (Ciechanska, 2004) and food industries (Vandamme et al., 1998). WHC of wet BC from WBY hydrolysates by G. hansenii CGMCC 3917 was found to be 104 times its dry weight, similar to that obtained from a conventional chemical medium (105). As can be seen in Fig. 5, BC displayed slow water release ability, it needed nearly 80 h to completely lose all the water, and quick water absorbing ability, it only took 40 h to fully absorb water to reach 89.93% water content again, in agreement with the values previously obtained from other researches (Ul-Islam et al., 2012). All these characteristics are the result of the bacterial cellulose microstructure, specifically the porosity and surface area of the BC matrix (Ul-Islam et al., 2012). 3.3.2. SEM observation Material properties are always linked to the structure at all levels which is the result of chemical composition, arrangement, and processing conditions (Ciechanska, 2004). The surface and inner matrix of BC sheets were evaluated through SEM analysis, as shown in Fig. 6, see Supplementary figure. The microstructure of the bacterial cellulose produced under the optimal conditions selected in this work is similar to the one previously presented in the literature (Klemm et al., 2001). The micrograph of the cross section shows that the micro-fibrils of BC membranes are randomly and loosely arranged with plenty of spaces among them (Fig. 6A, see Supplementary figure). This arrangement of fibrils results in the formation of pores with different diameters on the surface (Fig. 6B, see Supplementary figure) and through the entire matrix of the BC sheets (Fig. 6C and D, see Supplementary figure). In this microstructure the water molecules can be sandwiched between the pores of the thick fibrils, and these fibrils can act as a shield for water molecules. Hence, these fibrils display high water holding capacity, slow water release ability and quick water absorbing ability. 4. Conclusions It has been demonstrated that WBY has great potential to be used as nutrient source for G. hansenii CGMCC 3917 to produce BC. Ultrasonication in combination with mild acid hydrolysis is an effective pre-treatment to improve the reducing sugar yield of WBY and BC yield. Moreover, the highest BC yield obtained from this pretreated WBY was almost six times as that from the
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 10.052. References Bae, S., Shoda, M., 2005. Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor. Appl. Microbiol. Biotechnol. 67, 45–51. Carreira, P., Mendes, J.A., Trovatti, E., Serafim, L.S., Freire, C.S., Silvestre, A.J., Neto, C.P., 2011. Utilization of residues from agro-forest industries in the production of high value bacterial cellulose. Bioresour. Technol. 102, 7354–7360. Chen, L., Hong, F., Yang, X.x., Han, S.f., 2012. Biotransformation of wheat straw to bacterial cellulose and its mechanism. Bioresour. Technol. 135, 464–468. Ciechanska, D., 2004. Multifunctional bacterial cellulose/chitosan composite materials for medical applications. Fibres Text. East. Eur. 12, 69–72. Cui, M., Yuan, Z., Zhi, X., Shen, J., 2009. Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria. Int. J. Hydrog. Energy 34, 7971– 7978. Czaja, W.K., Young, D.J., Kawecki, M., Brown, R.M., 2007. The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 8, 1–12. García, A., Alriols González, M., Labidi, J., 2012. Evaluation of the effect of ultrasound on organosolv black liquor from olive tree pruning residues. Bioresour. Technol. 108, 155–161. Ge, H.J., Du, S.K., Lin, D.H., Zhang, J.N., Xiang, J.L., Li, Z.X., 2011. Gluconacetobacter hansenii subsp. nov., a high-yield bacterial cellulose producing strain induced by high hydrostatic pressure. Appl. Biochem. Biotechnol. 165, 1519–1531. Goelzer, F., Faria-Tischer, P., Vitorino, J., Sierakowski, M.R., Tischer, C., 2009. Production and characterization of nanospheres of bacterial cellulose from Acetobacter xylinum from processed rice bark. Mater. Sci. Eng. C 29, 546–551. Gomes, F.P., Silva, N.H., Trovatti, E., Serafim, L.S., Duarte, M.F., Silvestre, A.J., Neto, C.P., Freire, C.S., 2013. Production of bacterial cellulose by Gluconacetobacter sacchari using dry olive mill residue. Biomass Bioenergy, 1–7. Guo, L., Li, X.-M., Bo, X., Yang, Q., Zeng, G.-M., Liao, D.-x., Liu, J.-J., 2008. Impacts of sterilization, microwave and ultrasonication pretreatment on hydrogen producing using waste sludge. Bioresour. Technol. 99, 3651–3658. Ha, J.H., Shehzad, O., Khan, S., Lee, S.Y., Park, J.W., Khan, T., Park, J.K., 2008. Production of bacterial cellulose by a static cultivation using the waste from beer culture broth. Korean J. Chem. Eng. 25, 812–815. Ha, J.H., Shah, N., Ul-Islam, M., Park, J.K., 2011. Potential of the waste from beer fermentation broth for bio-ethanol production without any additional enzyme, microbial cells and carbohydrates. Enzym. Microb. Technol. 49, 298–304. Hong, F., Qiu, K., 2008. An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter acetisubsp. xylinus ATCC 23770. Carbohydr. Poly. 72, 545–549. Hong, F., Guo, X., Zhang, S., Han, S.f., Yang, G., Jönsson, L.J., 2012. Bacterial cellulose production from cotton-based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment. Bioresour. Technol. 104, 503–508. Hungund, B., Prabhu, S., Shetty, C., Acharya, S., Prabhu, V., 2013. Production of bacterial cellulose from Gluconacetobacter persimmonis GH-2 using dual and cheaper carbon sources. J. Microb. Biochem. Technol. 5, 031–033. Klemm, D., Schumann, D., Udhardt, U., Marsch, S., 2001. Bacterial synthesized cellulose – artificial blood vessels for microsurgery. Prog. Polym. Sci. 26, 1561– 1603. Klemm, D., Heublein, B., Fink, H.P., Bohn, A., 2005. Cellulose: fascinating biopolymer and sustainable raw material. Angew. Chem. Int. Ed. 44, 3358–3393. Klemm, D., Kramer, F., Moritz, S., Lindström, T., Ankerfors, M., Gray, D., Dorris, A., 2011. Nanocelluloses: a new family of nature-based materials. Angew. Chem. Int. Ed. 50, 5438–5466. Lever, M., 1972. A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47, 273–279. Lisdiyanti, P., Navarro, R.R., Uchimura, T., Komagata, K., 2006. Reclassification of Gluconacetobacter hansenii strains and proposals of Gluconacetobacter
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of bacterial cellulose by Gluconacetobacter hansenii CGMCC 3917 using only waste beer yeast as nutrient source Dehui Lin a,1, Patricia Lopez-Sanchez b,2, Rui Li a,3, Zhixi Li a,⇑ a b
College of Food Science and Engineering, Northwest A&F University, No. 28 Xinong Road, 712100 Yangling, Shaanxi, China Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Australia
h i g h l i g h t s It was found a novel approach to convert cellulosic wastes to green biomaterials. A two-step pre-treatment was used to treat WBY to improve the reducing sugar yield. Ultrasonication combining mild acid hydrolysis was an effective pre-treatment. BC yield of WBY treated by ultrasonication was almost 6 times as that of untreated WBY. BC produced from WBY hydrolysates displayed good physicochemical properties.
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 14 October 2013 Accepted 15 October 2013 Available online 24 October 2013 Keywords: Bacterial cellulose Gluconacetobacter hansenii Waste beer yeast Pre-treatment Property
a b s t r a c t In order to improve the use of waste beer yeast (WBY) for bacterial cellulose production by Gluconacetobacter hansenii CGMCC 3917, a two-step pre-treatment was designed. First WBY was treated by 4 methods: 0.1 M NaOH treatment, high speed homogenizer, ultrasonication and microwave treatment followed by hydrolysis (121 °C, 20 min) under mild acid condition (pH 2). The optimal pre-treatment conditions were evaluated by the reducing sugar yield after hydrolysis. 15% WBY treated by ultrasonication for 40 min had the highest reducing sugar yield (29.19%), followed by NaOH treatment (28.98%), high speed homogenizer (13.33%) and microwaves (13.01%). Treated WBY hydrolysates were directly supplied as only nutrient source for BC production. A sugar concentration of 3% WBY hydrolysates treated by ultrasonication gave the highest BC yield (7.02 g/L), almost 6 times as that from untreated WBY (1.21 g/L). Furthermore, the properties of the BC were as good as those obtained from the conventional chemical media. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the most abundant natural biopolymer in the world and it has been widely studied during the past decades (Klemm et al., 2005). Traditionally cellulose is extracted from plant tissues, but it can also be produced by some species of bacteria including genera Acetobacter, Agrobacterium, Gluconacetobacter, Rhizobium, and Sarcina. Cellulose produced by these microorganisms is commonly referred to as bacterial cellulose or BC (Klemm et al., 2005; Lisdiyanti et al., 2006). Though BC contains sets of parallel chains composed of b-1, 4-D-glucopyranose units interlinked by intermolecular hydrogen bonds, which is identical in chemical composition to plant cellulose (Wan et al., 2006), it displays many ⇑ Corresponding author. Tel.: +86 13571939821. E-mail addresses: [email protected] (D. Lin), [email protected] (P. Lopez-Sanchez), [email protected] (R. Li), [email protected] (Z. Li). 1 Tel.: +61 420520907. 2 Tel.: +61 33467373. 3 Tel.: +86 15091597123.
unusual physicochemical and mechanical properties, including higher purity, higher crystallinity, higher degree of polymerization, higher water absorbing and holding capacity, higher tensile strength and stronger biological adaptability (Klemm et al., 2001; Ul-Islam et al., 2012). Therefore, BC represents a potential alternative to plant-derived cellulose for specific applications in bio-medicine, cosmetics, high-end acoustic diaphragms, papermaking, food industry and other applications (Czaja et al., 2007; Klemm et al., 2011; Shah et al., 2013). However, the low yield and the high production cost of BC are important drawbacks for its industrial production and broad range of applications (Vandamme et al., 1998). In this context, it is highly relevant to investigate bacterial strains with stronger ability to produce BC using lower cost culture media. Recently, various cellulosic wastes from renewable agro-forestry residues or industrial by-products have been investigated to be as carbon sources to improve the production yield and decrease the economic cost. These include food process wastes (Carreira et al., 2011), hydrolyzed hemicelluloses from waste liquor of atmospheric acetic acid
0960-8524/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.052
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pulping (Uraki et al., 2002), beet and sugar cane molasses (Bae and Shoda, 2005), various fruit wastes (Hungund et al., 2013), konjac powder (Hong and Qiu, 2008), rice bark (Goelzer et al., 2009), wheat straw (Chen et al., 2012), Cotton-based waste textiles (Hong et al., 2012), maple syrup (Zeng et al., 2011), coffee cherry husk (Usha and Appaiah, 2011), and dry olive mill residue (Gomes et al., 2013). Furthermore making use of such waste materials would not only improve the sustainability of cellulose production by microorganisms, but also decrease environmental pollution associated with the disposal of industrial wastes. Beer production is an important economic activity in many countries, especially in China. Consequently, plenty of waste beer yeasts (WBY), being the second major by-product from brewing industry, is generated and either thrown out or fed to livestock and fowl. This not only results in a series of environmental problems, but also causes an enormous waste of resources (Yang et al., 2006). In general, WBY is mainly composed of 48–55% protein, 23–28% carbohydrate, 6–8% RNA, 1% glutathione, and 2% vitamin B. Moreover, they are rich in P, K, Ca, Fe, P and Mg (Liu et al., 2012). Due to its high nutritional content, it might be used for the production of green products by microorganisms. However, it is difficult to use it directly as a nutrient source for microorganisms, due to that most of the protein and carbohydrate exists in the cell walls in the form of large polymers (Yang et al., 2006). Therefore, it is important to disrupt the beer yeast cell walls and depolymerize large polymers to facilitate its use by microorganisms (Yang et al., 2006). Currently chemical pre-treatments are commonly used for the disruption and hydrolysis of yeast cells, such as sulfuric acid, hydrochloric acid and alkaline solution pre-treatments (Guo et al., 2008). Although chemical pre-treatments are effective, they are rather harsh and they can damage the nutrients. Moreover they can easily cause a secondary environmental pollution. In this sense, some studies are focusing on the use of physical pre-treatments including microwaves, ultrasonication and mechanical comminution, which are generally regarded as non-hazardous to the environment because few or none chemicals need to be added (Vodenicˇarová et al., 2006). When particles in a liquid suspension are treated by microwaves, ultrasonication or mechanical comminution, they are subjected to either surface erosion or size reduction (due to cavitation, shear force, ‘‘thermal’’ or ‘‘athermal’’ effects) (García et al., 2012; Yu et al., 2010). Consequently, these processes can be used to physically disrupt and break microbial cells in order to release nutrients from them (Guo et al., 2008). They can also be used to depolymerize large polysaccharide molecules and generate a pre-treated substrate that is more easily hydrolyzed via increasing the accessible surface area (Vodenicˇarová et al., 2006; Wang et al., 2010). However, physical pre-treatments cannot hydrolyze polysaccharides into monosaccharides directly and only monosaccharides can be used by Gluconacetobacter hansenii to produce BC. In order to solve the existing problem, a two-step pre-treatment, namely physico– chemical pre-treatment combining different mild chemical and physical treatment options, could be used. This combination should be not only effective for disrupting cells and dispersing large polymer aggregates, but also for improving hydrolysis. In previous studies, only chemical pre-treatments including acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis have been used to hydrolyze the waste beer yeast cells to release the nutrients for the production of green products (Cui et al., 2009; Ha et al., 2011, 2008). However, few studies have reported a two-step pre-treatment, combining mild chemical and physical treatment options, to treat WBY as a source of nutrients for bacterial cellulose production. In the present work, two-step pretreatments were explored in order to find an effective method to enhance the reducing sugar yield of WBY. WBY was first treated by four methods including 0.1 M NaOH treatment, high speed homogenizer, ultrasonication
and microwave treatment followed by hydrolysis at 121 °C for 20 min under mild acid condition (pH 2) as a second step. The effects of the four pre-treatments and the optimum pre-treatment conditions were evaluated by the reducing sugar yield after hydrolysis. Furthermore, treated WBY hydrolysates were directly served as only nutrient source for BC production by G. hansenii CGMCC3917 and we investigated the influences of the four pretreatments and the WBY hydrolysate concentration on the BC yield. Additionally, water holding capacity (WHC), water release rate (WRR) and water absorption rate (WAR) of the bacterial cellulose were measured and its microstructure was determined using scanning electron microscopy (SEM).
2. Methods 2.1. Microorganisms and materials G. hansenii strain was used in this study. This strain has been isolated from inoculums of a strain screened in homemade vinegar and then induced by high hydrostatic pressure treatment. It has a strong ability to produce BC more than three times as that of its initial strain. It was deposited as CGMCC3917 at China General Microbiological Culture Collection, Beijing, China and was maintained on glucose agar slants containing: 2% glucose (w/v), 0.5% yeast extract (w/v), 0.1% K2HPO4 (w/v), 1.5% MgSO47H2O (w/v), 1.5% ethanol (v/v), and 1.7% agar (w/v). It was stored at 4 °C in a refrigerator and sub-cultured every 2 months for inoculum development or stored at 80 °C using 20% (v/v) glycerol instead of agar for long-time storage (Ge et al., 2011). For seed inoculum, a loop of G. hansenii CGMCC 3917 was transferred from a slant culture into an Erlenmeyer flask (250 mL) containing 100 mL of seed medium with the same components as glucose agar slants but without agar. Then it was cultivated at 30 °C in a rotary shaker incubator at 150 rpm for 12–18 h until it reached the logarithmic growth phase (Ge et al., 2011). WBY was obtained from TsingTao Brewery Xi’an Hans group co., Ltd. (Xi’an, Shaan xi, China).
2.2. Pre-treatments and hydrolysis of WBY Pre-treatment of dry waste beer yeast was performed by mixing WBY with 100 mL of distilled water in a 250 mL round-bottom flask to reach a final concentration of 5%, 10%, 15% and 20% (w/v) respectively, which were regarded as untreated WBY mixed liquor. Four methods were investigated to treat WBY mixed liquor and details of the treatment procedures were as follows: Pre-treatment 1: mixed liquor with different concentrations of WBY was treated with 0.1 M NaOH at 50 °C for 6, 12, 18, 24, 30 and 36 h respectively. Pre-treatment 2: mixed liquor with different concentrations of WBY was treated by high speed homogenizer at 15,000 rpm (XHF-D, Ningbo Xingzhi Biotechnology Co., Ltd., Zhejiang, China) for 5, 10, 15 and 20 min respectively. Pre-treatment 3: mixed liquor with different concentrations of WBY was treated by ultrasonication at supersonic power of 500 W (YQ-1003A, Ningbo Power Ultrasonic Equipment Co., Ltd., Zhejiang, China) for 10, 20, 30, 40, 50 and 60 min respectively. Pre-treatment 4: mixed liquor with different concentrations of WBY was treated by microwaves at microwave power of 600 W (Galanz P70D20P-TF, China) for 5, 10, 15 and 20 min respectively. After each pre-treatment, the samples were hydrolyzed at 121 °C for 20 min under mild acid condition (pH 2.0). Then they were centrifuged at 4000g for 15 min to remove sediments and sterilized water was added to the supernatant up to a volume of
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100 mL. For each pre-treatment a sample which was not centrifuged was used as control. 2.3. Determination of total reducing sugars The total reducing sugar concentration was determined after hydrolysis by PAHBAH reducing end assay described by Lever (1972) with D-glucose as standard. Sugar yield was calculated using the following equation:
Reducing sugar yield ð%Þ ¼
Reducing sugar weight ðgÞ 100 Waste yeast weight ðgÞ
2.4. BC production and harvest 2.4.1. BC production using WBY hydrolysates as nutrient source The prepared seed inoculum (9%, v/v) was transferred into a glass vessel (500 mL) containing different treated WBY hydrolysates as described above after adjusting their pH to 5 using 2 M NaOH and they were cultivated statically at 30 °C for 14 days. 2.4.2. BC production using WBY hydrolysates with different concentrations The pre-treatment with the highest reducing sugar yield was selected to further investigate the optimal sugar concentration for BC production. After pre-treatment, WBY was hydrolyzed at 121 °C for 20 min under mild acid condition (pH 2). Then it was centrifuged at 4000g for 15 min to remove sediments and the supernatant was collected and added with sterilized water or sterilized glucose solution (50%, w/v) from its initial sugar concentration of 4.38% (w/v) to reach a final concentration of 1%, 3%, 5% and 7% (w/v) respectively. Finally, the prepared seed inoculum (9%, v/v) was transferred into a glass vessel (500 mL) containing 100 mL of WBY hydrolysates prepared at different concentrations as described above after adjusting its pH to 5 using 2 M NaOH and they were cultivated statically at 30 °C for 14 days. 2.4.3. BC purification and quantification After cultivation, the BC membranes were rinsed with running water overnight, soaked in 0.1 M NaOH at 80 °C for 2 h to remove bacteria, and then washed with deionized water several times to completely remove alkali. The purified cellulose was dried at 105 °C for 12 h and weighted until constant weight. For each membrane, triplicate experiments were performed, and the mean values were calculated (Wu et al., 2010). 2.5. BC membrane analysis 2.5.1. Water holding capacity (WHC) For the determination of WHC, wet BC samples were removed from the storage container with tweezers. The samples were shaken twice quickly and then weighed, followed by drying at 60 °C for 48 h in order to completely remove water from them. WHC was calculated by the following formula (Shezad et al., 2010):
Water holding capacity ¼
Mass of water removed during drying ðgÞ Dry weight of cellulose ðgÞ
2.5.2. Water release rate (WRR) To determine WRR, Shezad et al.’s (2010) method was used with small modifications. The wet BC samples were cut into small pieces and dried using a freeze dryer (MCFD5508, SIM International CO.) for 48 h. The dried BC samples (1 g) were subsequently immersed in distilled water under shaking (100 rpm) condition at room temperature for rewetting. After complete rewetting
(stabilized wet weight), samples were analysed for WRR by continuously measuring their weights at various time intervals at room temperature until complete drying. The weights of the BC samples at different time intervals were plotted against time. 2.5.3. Water absorption rate (WAR) The dry BC sample was cut into four pieces and then the pieces were subsequently immersed in distilled water under shaking (100 rpm) condition at room temperature for rewetting. Samples were analysed for WAR by continuously measuring their weights at various time intervals at room temperature until complete absorption. WAR of dry BC membrane was calculated by the following equation:
Water absorption rate ð%Þ ¼
W1 W 100 W
where W (g) is the weight of dry BC sample; W1 (g) is the weight of BC after it absorbed water. The experiments were carried out for ten replicates. 2.5.4. SEM observation For scanning electronic microscope observation, the air-dried BC membrane was mounted on a copper stub using double adhesive carbon conductive tape and coated with platinum for 30 s using a platinum coating facility (Auto Fine Coater JFC-1300, Jeol, Japan). The SEM photographs were obtained by scanning electron microscope (JSM-6360LV, Jeol, Japan) at room temperature at 15 kV. 3. Results and discussion 3.1. Effects of pre-treatments on the reducing sugar yield of WBY In order to determine which pre-treatment led to a higher yield of reducing sugars, the reducing sugar content was assessed after hydrolysis. Two variables, namely the dosage of WBY and duration of the pre-treatment, were investigated. As shown in Fig. 1, the reducing sugar yield was significantly affected by the dosage of WBY and the pre-treatment time for all four pre-treatments. The reducing sugar yields of untreated WBY are represented by time 0 in the plots. The results of pre-treatment 1 using 0.1 M NaOH are described in Fig. 1A. During the first 6 h, the reducing sugar yield increased slowly for the four dosages. We suggest that this lag time could be due to the soaking time required for the NaOH to start working on the yeast cells. After 18 h pre-treatment, the reducing sugar yield (w/w) from 20% WBY reached the highest value of 20.48%. While all other WBY dosages reached a plateau after 30 h pretreatment. The maximum sugar yield values achieved were 20.59%, 22.26% and 28.98% for 5%, 10% and 15% WBY respectively. The reducing sugar yield increased with the WBY dosage with the exception of 20% WBY. When 20% WBY was treated, the highest value reached faster (18 h) but it was the lowest yield, similar to that obtained from 5% WBY. This suggests that the concentration of NaOH used was not enough to treat the 20% WBY and a higher concentration could potentially improve the reducing sugar yield of 20% WBY. Fig. 1B shows the results of pre-treatment 2 using a high speed homogenizer. It can be observed that a dosage of 20% WBY gave the highest reducing sugar yield after 10 min. While all other dosages reached their maximum values after 15 min. The maximum reducing sugar yield increased with the dosage of WBY with values of 9.83%, 11.22% and 16.17% for 5%, 10% and 15% respectively. Although the maximum yield from 20% WBY reached faster with a value of 13.26%, it was lower than that from 15% WBY. Similar trend was observed in pre-treatment 1 for the dosage of 20%.
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Fig. 1. Influences of four pre-treatments on the reducing sugar yield of WBY after hydrolysis. Pre-treatment 1:0.1 M NaOH treatment (A), pre-treatment 2: high speed homogenizer (B), pre-treatment 3: ultrasonication (C), pre-treatment 4: microwaves (D).
Furthermore the highest reducing sugar yield obtained from pretreatment 2 was relatively lower compared to that from pre-treatment 1. The reducing sugar yields obtained from pre-treatment 3 using ultrasonication (Fig. 1C) showed similar trends to pre-treatments 1 and 2. The dosage of 20% displayed the highest reducing sugar yield after ultrasonication for 30 min. While all other dosages reached their maximum values after 40 min. The maximum reducing sugar yield increased with the dosage of WBY with values of 16.97%, 21.53% and 29.19% for 5%, 10% and 15% WBY respectively. The highest reducing sugar yield obtained from pre-treatment 3 was clearly higher than that obtained from pre-treatment 2 and similar to that obtained from pre-treatment 1. After microwave pre-treatment for 10 min, the WBY dosage of 10%, 15% and 20% led to almost similar reducing sugar yield 9.46%, 9.77%, 9.96% respectively, while 5% WBY gave a relatively lower reducing sugar yield of 8.63% (Fig. 1D). After 15 min pretreatment, the four dosages reached their maximum values and the maximum reducing sugar yield increased with the dosage of WBY with values of 9.26%, 12.25% and 13.01% for 5%, 10% and 15% respectively, except for the dosage of 20%. In general the highest reducing sugar yield obtained from pre-treatment 4 was the lowest among the four pre-treatments. These results indicated that the optimal WBY dosage was 15% for the four pre-treatments. As discussed above the reducing sugar yield of the 20% WBY could be increased by a higher concentration of NaOH in pre-treatment 1. For the other pre-treatments the results suggest that 15% WBY might display enough specific surface area to subject to erosion before hydrolysis and the 20% WBY might be too viscous for the physical processes to work as efficient as that for lower concentrations. Changing the settings used in the instruments such as shear input, ultrasonication or microwave
power may assist in increasing the reducing sugar yield of the 20% WBY after hydrolysis. The highest reducing sugar yield obtained from ultrasonication (29.19%) was almost six times higher than that obtained from the untreated WBY (4.15%), followed by NaOH treatment (28.29%), high speed homogenizer (13.33%) and microwave treatment (13.01%). Although WBY treated by 0.1 M NaOH (pre-treatment 1) and ultrasonication (pre-treatment 3) had the similar reducing sugar yield, the best pre-treatment time of ultrasonication (40 min) was obviously shorter than that of 0.1 M NaOH pre-treatment (30 h). Furthermore ultrasonication without harsh chemicals used could reduce the environmental impact. Therefore, it can be concluded that ultrasonication in combination with mild acid hydrolysis was the most effective pre-treatment to improve the reducing sugar yield of WBY with an optimal dosage of 15% and a best pre-treatment time of 40 min. 3.2. BC production After hydrolysis, WBY hydrolysates were directly supplied to G. hansenii CGMCC 3917 as carbon and nutrient sources to produce BC without any extra nutrient added. In Fig. 2 it can be observed that the BC yields obtained using centrifuged WBY from pre-treatment 1and pre-treatment 3 were both higher than those from the WBY which were not centrifuged. This could be likely due to the fact that the un-centrifuged samples after these pre-treatments have a high sugar concentration (they showed the highest sugar yields) which could cause the inhibition of the BC production and reduce the supply of oxygen by the liquid medium. While in the centrifuged samples the reducing sugar concentration was decreased by diluting the supernatant with water, which could lead to a better concentration for BC production. In contrast, the centrifuged WBY from pre-treatment 2, pre-treatment 4 and the untreated
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8 1%
7
3%
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WBY produced lower BC yields compared to those from the uncentrifuged WBY. Likely the cellulose production by G. hansenii CGMCC 3917 in this case was inhibited due to the low sugar concentration present in the centrifuged samples, which were further diluted from the already low sugar yields of WBY obtained from these pre-treatments. Additionally, BC production using WBY treated by ultrasonication (pretreatment 3) displayed the highest yield (3.89 g/L) of all four pre-treatments. Furthermore it was clearly higher than that from pre-treatment 1 using 0.1 M NaOH (2.33 g/L), despite the fact that they have almost the same sugar concentration after hydrolysis. It can be concluded that the harsh alkali used in pre-treatment 1 might degrade other nutrients in the waste beer yeast which are beneficial for G. hansenii CGMCC 3917 during BC production, while those nutrients are still present after ultrasonication pretreatment. In order to improve the BC production, the ultrasonication pretreatment with the highest reducing sugar yield was selected to further investigate the optimal sugar concentration. WBY hydrolysates treated by ultrasonication, with a reducing sugar yield of 29.19% (w/w), which corresponds to a reducing sugar concentration of 4.38% (w/v), were either mixed with sterilized water or sterilized glucose solution (50%, w/v) to reach a final sugar concentration of 1%, 3%, 5%, 7% (w/v) respectively. As described in Fig. 3, the reducing sugar concentration had a marked effect on the BC production. The BC yield using WBY hydrolysates with 1% sugar concentration was the lowest and it reached the maximum value of 2.18 g/L on day 7. When the sugar concentration was increased to 3%, the BC yield increased significantly showing a peak at 7.02 g/L on day 10. However a further increase in sugar concentration reduced dramatically the BC yield, 5% sugar led to a maximum of 5.73 g/L on day 8 and the maximum yield for 7% sugar concentration was only 2.88 g/L on day 7. Hence it was concluded that the highest sugar concentrations inhibited the BC production, which is in agreement with the findings from previous studies (Carreira et al., 2011). Additionally, it was also concluded that the sugar concentration would affect the fermentation cycle to some extent, due to the fact that the BC yields from the various sugar concentrations reached their maximum at different cultivation time. To sum up, after optimization, the highest BC yield (7.02 g/L) was almost 6 times as that obtained from the untreated WBY (1.21 g/L). The cellulose membrane was obviously thicker:
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15.4 mm compared to 2.3 mm obtained from the untreated WBY and 5.7 mm from the un-centrifuged WBY (Fig. 4, see Supplementary figure). Moreover, the 100 mL of WBY hydrolysates was almost completely used by G. hansenii CGMCC 3917 to produce BC (Fig. 4C, see Supplementary figure) and its yield doubled that obtained from the conventional chemical media (3.58 g/L) (Ge et al., 2011). Although some researchers have investigated various cellulosic wastes from renewable agro-forestry residues or industrial by-products to produce BC, some extra nutrients are added to media to improve the BC yield. For instance, Hong and Qiu (2008) have obtained an increase of BC production with Ca2+ introduced into the medium of konjac powder hydrolysates, Gomes et al. (2013) have enhanced BC production with supplemented nitrogen and phosphate sources in olive mill residue hydrolysates, Ha et al. (2008) have improved BC yield with adding glucose into waste beer yeast hydrolysates as supplement nutrient. In the present study no extra nutrient was added. Moreover, BC production using WBY pretreated by ultrasonication displayed a high yield and WBY hydrolysates could totally substitute the conventionally used chemical media. Our findings could not only overcome the high BC production cost, but also reduce the waste of resources and environmental pollution. Therefore, it has been not only demonstrated that waste beer yeast has great potential to produce BC, but also that ultrasonication is an effective pre-treatment tool in biomass pre-treatment technology.
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Fig. 2. BC production from WBY treated by different methods. Pre-treatment 1:0.1 M NaOH treatment (1), pre-treatment 2: high speed homogenizer (2), pre-treatment 3: ultrasonication (3), pre-treatment 4: microwaves (4), un-treatment (5).
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untreated WBY and nearly doubled that from the conventional chemical media. Additionally, the properties and microstructure of BC produced by WBY hydrolysates were as good as those obtained from the conventionally used chemical media. Acknowledgements This work was financially supported by the Fundamental Research Funds for the Central Universities (QN2009072). The technical support of Dr. Shuangkui Du, associate professor of the College of Food Science and Engineering, Northwest A&F University, is also gratefully acknowledged.
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Appendix A. Supplementary data Fig. 5. Evaluation of WRR and WAR of BC produced by CGMCC 3917 using WBY hydrolysates.
3.3. Characterization of BC 3.3.1. WHC, WRR and WAR WHC, WRR and WAR are some of the most important properties measured to determine the possible applications of BC in medical areas (Ciechanska, 2004) and food industries (Vandamme et al., 1998). WHC of wet BC from WBY hydrolysates by G. hansenii CGMCC 3917 was found to be 104 times its dry weight, similar to that obtained from a conventional chemical medium (105). As can be seen in Fig. 5, BC displayed slow water release ability, it needed nearly 80 h to completely lose all the water, and quick water absorbing ability, it only took 40 h to fully absorb water to reach 89.93% water content again, in agreement with the values previously obtained from other researches (Ul-Islam et al., 2012). All these characteristics are the result of the bacterial cellulose microstructure, specifically the porosity and surface area of the BC matrix (Ul-Islam et al., 2012). 3.3.2. SEM observation Material properties are always linked to the structure at all levels which is the result of chemical composition, arrangement, and processing conditions (Ciechanska, 2004). The surface and inner matrix of BC sheets were evaluated through SEM analysis, as shown in Fig. 6, see Supplementary figure. The microstructure of the bacterial cellulose produced under the optimal conditions selected in this work is similar to the one previously presented in the literature (Klemm et al., 2001). The micrograph of the cross section shows that the micro-fibrils of BC membranes are randomly and loosely arranged with plenty of spaces among them (Fig. 6A, see Supplementary figure). This arrangement of fibrils results in the formation of pores with different diameters on the surface (Fig. 6B, see Supplementary figure) and through the entire matrix of the BC sheets (Fig. 6C and D, see Supplementary figure). In this microstructure the water molecules can be sandwiched between the pores of the thick fibrils, and these fibrils can act as a shield for water molecules. Hence, these fibrils display high water holding capacity, slow water release ability and quick water absorbing ability. 4. Conclusions It has been demonstrated that WBY has great potential to be used as nutrient source for G. hansenii CGMCC 3917 to produce BC. Ultrasonication in combination with mild acid hydrolysis is an effective pre-treatment to improve the reducing sugar yield of WBY and BC yield. Moreover, the highest BC yield obtained from this pretreated WBY was almost six times as that from the
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