The objective of this work was to degrade lignocellulosic components in un-pretreated vinegar residue (VR) using a fungal consortium. Consortium-29, consisting of P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23, was constructed using orthogonal design combined with two-way interaction analysis. After seven days of cultivation, the reducing sugar yield reached 35.57 mg per gram of dry substrate (gds-1), which was 108.01% higher than the control (17.10 mg gds-1). Additionally, the xylanase and CMCase activity reached 439.07 U gds-1 and 8.15 U gds-1, which were 432.08% and 243.88% higher than that of pure cultures of A. niger (82.52 U gds-1) and P. chrysosporium (2.37 U gds-1), respectively. The cellulose, hemicellulose, and lignin contents decreased by 17.11%, 68.61%, and 14.44%, respectively, compared with that of the raw VR. The optimal fermentation conditions of consortium-29 were as follows: incubation temperature 25 °C, initial pH 6, initial moisture content 70%, inoculum size 1 ´ x 106 spores/mL, incubation time 5 days, urea/VR 1%, and MnSO4×H2O/VR 0.03%. This study suggests that consortium-29 is an efficient fungal consortium for un-pretreated VR degradation and has a potential application in lignocellulosic waste utilization with a low cost of operation.
Degradation of Lignocellulosic Components in Un-pretreated Vinegar Residue Using an Artificially Constructed Fungal Consortium
Yaoming Cui, Xiaofang Dong,* Jianming Tong, and Song Liu
The objective of this work was to degrade lignocellulosic components in un-pretreated vinegar residue (VR) using a fungal consortium. Consortium-29, consisting of P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23, was constructed using orthogonal design combined with two-way interaction analysis. After seven days of cultivation, the reducing sugar yield reached 35.57 mg per gram of dry substrate (gds-1), which was 108.01% higher than the control (17.10 mg gds-1). Additionally, the xylanase and CMCase activity reached 439.07 U gds-1and 8.15 U gds-1, which were 432.08% and 243.88% higher than that of pure cultures of A. niger (82.52 U gds-1) and P. chrysosporium (2.37 U gds-1), respectively. The cellulose, hemicellulose, and lignin contents decreased by 17.11%, 68.61%, and 14.44%, respectively, compared with that of the raw VR. The optimal fermentation conditions of consortium-29 were as follows: incubation temperature 25 °C, initial pH 6, initial moisture content 70%, inoculum size 1 106 spores/mL, incubation time 5 days, urea/VR 1%, and MnSO4H2O/VR 0.03%. This study suggests that consortium-29 is an efficient fungal consortium for un-pretreated VR degradation and has a potential application in lignocellulosic waste utilization with a low cost of operation.
Keyword: Lignocellulose degradation; Un-pretreated vinegar residue; Fungal consortia; Culture conditions optimization
Contact information: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; *Corresponding author: email@example.com
Vinegar residue (VR), a waste byproduct, is generated during the process of vinegar production by solid state fermentation (SSF) (Wang et al. 2011), and more than 2 million tons of VR are generated annually in China. VR is of poor nutritive value and low digestibility because of its high content of lignocellulosic components (Song et al. 2013). In fact, it is estimated that 10 to 50 billion tons of lignocellulosic materials per year, accounting for about 50% of the biomass in the world, could be sustainably harvested (Claassen et al. 1999). The renewability of lignocellulosic materials makes them the focus of high-level research because of the enormous economic, environmental, and social benefits.
In recent studies, lignocellulosic resources have been used to produce value-added products, including biofuels, biopolymers, chemicals, fertilizer, and animal feeds (Yang et al. 2004; Kausar et al. 2010; Zhang et al. 2011; Kalyani et al. 2013). The microbial strategy has attracted more and more attention due to the simplicity and low capital investment (Yang et al. 2011). The critical step in all the bioconversion processes is the degradation of recalcitrant carbohydrate polymers into monomer sugar.
As the low efficiency is the primary hindrance in current biodegradation processes, researchers have been paying more attention to fungi in order to improve the degradation efficiency. Fungi are efficient degraders because of their extracellular enzymatic system, hyphal penetration power, and the ability to produce an enormous number of spores, which can invade substrates quickly (Kausar et al. 2010; Sharma and Arora 2013). Because no single strain could produce all the enzymes necessary for lignocellulose degradation, current research is focused on fungal consortia with advantages of avoiding feedback regulation and metabolite repression (Wongwilaiwalin et al. 2010). Compared with traditional pure culture fermentations, mixed culture fermentations can allow the use of cheap and impure wastes, overcome the limitation of nutrition, achieve higher product yield and growth rate, and strengthen the protection of the culture from contamination (Yang et al. 2004; Lin et al. 2011). Although traditional fungal consortia obtained directly from nature usually have strong lignocellulose degradation abilities, their capabilities are easy to degenerate because of their unknown and complicated composition, adversely affecting the potential for practical application (Kato et al. 2005; Wongwilaiwalin et al. 2010). Since more and more fungi capable of lignocellulose degradation have been isolated and purified, there are increased opportunities for artificial construction of fungal consortia to improve the degradation efficiency.
Although the decomposition of lignocellulosic waste has been studied extensively, almost all of these studies involve a variety of pretreatment methods, such as acid hydrolysis, alkaline hydrolysis, and steam explosion. These strategies can separate lignin from polysaccharides, demolish the special crystal structure, and break down cellulose, leading to a high degradation rate with a relatively short period of incubation. However, the high concentrations of acids are corrosive, toxic, and hazardous, and need costly equipment that can resist corrosion (Yang and Wyman 2008). Alkaline treatment is a relatively slow process, and the added alkali must be removed (Bjerre et al. 1996; Chang et al. 2001). The steam explosion process involving the application of high-pressure steam consumes a great deal of energy (Mosier et al. 2005). These processes also generate various by-products, such as phenolic compounds, furfurals, and organic acids (Palmqvist and Hahn-Hägerdal 2000; Panagiotou and Olsson 2007), which adversely affect the growth of microorganisms in the subsequent fermentation. Both the pretreatment process and removal of toxic compounds mean high costs and significant environmental risks.
By contrast, no toxic compounds are usually detected in the fermented products without pretreatment (Jwanny et al. 1995; Karunanandaa et al. 1995; Adamović et al. 1998). However, there were very few reports on the biodegradation of un-pretreated lignocellulosic materials. Moreover, these studies did not adopt solid-state fermentation (SSF).
Therefore, the aim of this work was to degrade the lignocellulose in un-pretreated VR using an artificial constructed fungal consortium by SSF. This study will help generate a better understanding of the advantages of consortia over pure cultures. It may also provide the experimental basis for highly efficient and cost-competitive decomposition of lignocellulose, which could have a significant impact on the understanding of lignocellulosic waste utilization.
Aspergillus niger ACCC 30557, Trichoderma viride ACCC 30552, and Phanerochaete chrysosporium ACCC 30414 were purchased from the Agricultural Culture Collection of China (ACCC). Trichoderma koningii CGMCC 3.2878 and Candida utilis CGMCC 2.1180 were obtained from the China General Microbiological Culture Collection Center (CGMCC). Aspergillus ficuum NTG-23 was a mutant strain acquired by our lab from Aspergillus ficuum CGMCC 3.4322 (Wang et al. 2011). Spore suspensions were prepared for these fungi by growing them on malt-agar, Czapek’s, and potato dextrose agar at 28 °C for two weeks. Then, the spores were washed off with sterile saline and scattered with glass beads for 30 min. The final spore concentration of the working spore suspensions was adjusted to 1 107 spores/mL.
Substrate and SSF
VR was obtained from the Shanxi province in China and was air-dried and utilized as a substrate for SSF without being milled. The chemicals (g/L) of Mandel’s medium (Lin et al. 2011) with minor modifications were as follows: polysorbate 80 (Tween-80®) 2, NaNO3 2, KH2PO4 1.5, CaCl2 0.3, MgSO47H2O 0.3, FeSO47H2O 0.005, MnSO4H2O 0.0016, ZnSO47H2O 0.0014, and CoCl26H2O 0.0005; the pH was adjusted to 6. The mineral medium (45 mL) was added to 30 g of well mixed VR containing 0.3 g of urea. After sterilization for 20 min at 121 °C in a 500-mL Erlenmeyer flask, 1 mL of spore suspension (1 107 spores/mL) was aseptically added to the substrate and mixed thoroughly. The incubation temperature was 28 °C, and the incubation time was limited to 7 days.
Sugar Extraction and Determination
Reducing sugar was extracted by suspending the fermented products in buffer and shaking for 4 h at 250 rpm. Following this, the mixture was separated by centrifugation (3,000 rpm for 3 min) to obtain a culture filtrate. The clarified supernatant was then collected and used as the source of reducing sugar and enzymes. Lignocellulose degradation capacity in the supernatant was investigated based on the amount of released reducing sugar, which was quantified colorimetrically as glucose equivalent using the 3, 5-dinitrosalicylic acid (DNS) method (Miller 1959).
Cellulase activity was detected using sodium carboxymethyl cellulose (CMC) as the substrate, which is a soluble cellulose derivative, according to the method of Feng et al. (2011). Xylanase activity was tested by the methodology described by Latif et al. (2006). β-glucanase activity was assayed using dextran from leuconostoc as the substrate (Li et al. 2009). α-amylase activity was estimated by measuring the maltose content using the method of DNS (Dahlqvist 1962).
One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 mol of product from the substrates in 0.1 M acetic buffer at pH 5.50 (0.05 M phosphate buffer, pH 6.90 for α-amylase) per min at 37 °C. The activities of the enzyme were expressed as units per gram of dry substrate (U gds-1).
Construction of Consortia using Orthogonal Design
A standard orthogonal array L32 (231) was employed to construct the consortia. Six fungi were chosen as the six factors, and the influence of two-way interactions were also evaluated. The spore concentrations of each fungus in 1 mL of working spore suspensions were selected as levels. Each factor was assigned 0 spore/mL of spore concentration as the low level and 1 107spores/mL as the high level. Subsequently, the data were subjected to analysis of variance (ANOVA) to evaluate the main and interaction effects of different fungi on VR degradation.
Optimization of Culture Conditions
Various process parameters influencing the reducing sugar release and enzyme activity during SSF were identified. These included initial pH, moisture content, temperature, harvest time, inoculum size, urea content, and MnSO4 content. Moisture content was adjusted using various volumes of mineral medium, and initial pH was adjusted with 5 M NaOH or 1 M HCl (Latifian et al. 2007).
Determination of Fiber
Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and ash were determined using filter bags (ANKOM) as described by Van Soest et al. (1991). The fiber fractions were calculated as follows: hemicellulose = NDF ― ADF, cellulose = ADF ― ADL, lignin = ADL ― ash (El-Zoghbi 1994).
Fungal Populations Analysis
The amount of fungi was determined by the dilution method of plate counting. Plate counts were performed by the spread plate method. The counting media were solidified using 18 g per liter of agar powder. After thorough dispersion, the sample was diluted serially and plated in duplicate to obtain the viable count.
All fermentation experiments were carried out in triplicate. Statistical analysis was performed using SAS version 9.2. The data were analyzed using ANOVA, and significant means were tested with Duncan’s Multiple Range Test.
RESULTS AND DISCUSSION
Fungal Consortia Construction for VR Degradation using Orthogonal Design
VR consists primarily of rice chaff (Fig. 1), which cannot be degraded as much as straw. The lack of nutrients does not make VR a good natural culture medium, which requires the degraders to survive in this medium and then initiate the process of degradation. In the present work, the viable counts of P. chrysosporium, T. koningii, Trichoderma viride, A. niger, A. ficuum NTG-23, and Candida utilis increased over time (Fig. 2) in their individual pure cultures. These results show that all the selected fungi could grow well in this natural culture medium.
Fig. 1. The raw vinegar residue on an air-dry basis
Fig. 2. Data (mean) were calculated from three replicates on a dry matter (DM) basis; the results are expressed as log10CFU
SSF of lignocellulosic wastes has several advantages over submerged fermentation (SmF), such as decreased operational costs, low capital costs for equipment, and high volumetric productivity (Villas-Bôas et al. 2002; Yang et al. 2004). Hence, it was used in this study. Reducing sugar primarily contains glucose, fructose, galactose, lactose, and maltose, all of which are monosaccharides or disaccharides and very easily digested. Hence, microbial degradation of highly ordered carbohydrate polymers into reducing sugar not only intenerates VR, increasing the palatability, but also enhances the content of digestible carbohydrates and hydrolytic enzymes, which might make the product a potential renewable feedstuff and avoid environmental pollution.
Table 1. Orthogonal Design of Fungal Consortia for Raw VR Degradation
Among the 32 consortia constructed using the orthogonal design (Table 1), consortium 29, consisting of P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23, demonstrated the strongest VR decomposition ability, with a reducing sugar yield of 35.57 mg gds-1. Analysis of variance results in Table 2 show that all of the six strains significantly (p ＜ 0.01) affected the VR decomposition ability of the fungal consortia. Interaction impact investigation of strains strains on the VR decomposition was carried out to construct an effective degradation fungal consortium. According to an estimation of margin means in Table 2, the interactions of Trichoderma viride P. chrysosporium, Trichoderma viride T. koningii, Trichoderma viride A. niger, Trichoderma viride Candida utilis, and Trichoderma viride A. ficuum NTG-23 on the VR degradation were negative. Similarly, the interactions of Candida utilis P. chrysosporium, Candida utilis T. koningii, Candida utilis A. niger, Candida utilis Trichoderma viride, and Candida utilis A. ficuum NTG-23 on the VR degradation were also negative. The mixed cultivation of Trichoderma viride or Candida utilis with any of the other five fungi presented an inhibitory effect on the VR degradation. These results suggested that Trichoderma viride and Candida utilis might not cooperate well with the other five strains. Conversely, the interaction effects of P. chrysosporium T. koningii, P. chrysosporium A. niger, P. chrysosporium A. ficuum NTG-23, T. koningii A. niger, T. koningii A. ficuum NTG-23, and A. niger A. ficuum NTG-23 were found to be positive. These results indicated that P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23 might act synergistically in a consortium; thus, the fungal consortium-29 was thought to be the optimal fungal consortium for VR degradation.
Table 2. ANOVA and Estimation of the Two-way Interactions between Strains
Evaluation of the Lignocellulose Degradation Ability of Consortium-29
The reducing sugar release in consortium-29 was prominently higher than that of the pure P. chrysosporium culture, which presented the highest reducing sugar yield among all the single fungus pure culture (Fig. 3a). In fact, the content of reducing sugar in the product increased by 108.01% as compared with the control. In previous reports, mixed fermentation of Trichoderma sp. and Aspergillus sp. has proven to be an excellent candidate for the production of cellulolytic enzymes with strong hydrolytic ability because of the complementary interactions of cellulases from Trichoderma sp. strains and Aspergillus sp. strains. Trichoderma strains can secrete both endo- and exo-glucanase with high activities, with very low β-glucosidases activity, while strains of Aspergillus show high activity of β-glucosidases (Brijwani et al. 2010). P. chrysosporium degrades lignin efficiently and selectively using its ligninolytic enzyme system, which primarily comprises lignin peroxidase, manganese peroxidase, and laccase (Arora et al. 2002).
In this study, remarkably stronger degradation capacities were displayed by P. chrysosporium, T. koningii, and A. ficuum NTG-23 compared with the other three fungi (Fig 3a), indicating the important roles of these three strains in effective degradation fungal consortia. A. niger presented notably higher xylanase activity (Fig 3b) than the other five strains, which was similar to previous reports (Pal and Khanum 2010). Therefore, consortium-29, consisting of P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23, showed strong degradation ability because of its lignocellulolytic enzyme system, with an appropriate composition and good catalyzing characteristics, which was in accordance with the co-cultivation of specific fungi (Fang et al. 2010). Additionally, the xylanase and CMCase activity reached 439.07 U gds-1 and 8.15 U gds-1, which were 432.08% and 243.88% higher than that of the pure culture of A. niger (82.52 U gds-1) and P. chrysosporium (2.37 U gds-1), respectively. These findings also indicate the strong degradation capacity of consortium-29.
Fig. 3. Evaluation of lignocellulolytic enzyme activities and reducing sugar production of consortium-29 and individual pure cultures (spore concentration of the inoculum = 1 107spores/mL). Error bars represent the standard deviation (n = 3). A-E Means values with different letters differ significantly (p ＜ 0.01). The strains of I, II, III, IV, V, VI, VII were Consortium-29, P. chrysosporium, T. koningii, Trichoderma viride, A. niger, A. ficuum NTG-23, and Candida utilis, respectively. (a) Reducing sugar yield and enzyme activity. (b) Xylanase activity.
The content of fiber components and ash in fermented products obtained by various fermentation strains is presented in Fig. 4. According to the analysis of fiber fractions, all the contents of NDF, ADF, ADL, cellulose, hemicellulose, and lignin in the products of consortium-29 were the lowest among all the treatments (Fig. 4a-f). These results show that consortium-29 had significantly stronger lignocellulose (cellulose, hemicellulose, and lignin) decomposition ability than any of the six pure strains. In fact, through the treatment of consortium-29, the contents of cellulose, hemicellulose, and lignin decreased by 17.11%, 68.61%, 14.44% respectively, as compared with that of the raw VR. The ratio of NDF degradation reached 22.03%, which was equal to that of mixed culture solid fermentation of NaOH pretreated rice chaff by Trichoderma reesei, Aspergillus niger, and Saccharomyces cerevisiae under the optimal conditions (Yong et al. 2004). This result showed the high efficiency of consortium-29. Moreover, the percentage of ash in the fermented products of consortium-29 was the highest (Fig. 4g), while that in the raw VR was the lowest. These findings imply that the dry matter losses during fermentation by consortium-29 were the highest. In this case, the content of lignocellulose (hemicellulose, cellulose, and lignin) was still the lowest (Fig. 4d-f), which further demonstrated the strong lignocellulose degradation ability of consortium-29.
Optimization of the Culture Conditions of Consortium-29 for VR Degradation
Determining optimal conditions
The culture conditions were optimized according to an orthogonal experiment (Tables 3 and 4). From the value of R (Table 5), it was implied that the effects of the factors on reducing sugar yield in order of importance were incubation temperature, incubation time, urea content, MnSO4H2O content, inoculum size, initial pH, and initial moisture content. The optimal conditions were an incubation temperature of 25 °C, initial pH of 6，inoculum size 1 106 or 1 107 spores/mL, incubation time 5 days, urea/VR 1% (w/w), and MnSO4H2O/VR 0.03% (w/w).
According to the statistical analysis (Table 5), it was found that incubation temperature, inoculum size, incubation time, urea content, and MnSO4H2O content had very significant effects on the release of reducing sugars (p 0.01); initial pH had a significant effect on this release (0.01 p 0.05); and initial moisture content had no significant effect (p 0.05).
The values of CMCase activity were analyzed to obtain the R value (Table 5), which indicated that the effects of the factors on CMCase activity in order of importance were urea content, incubation time, MnSO4H2O content, incubation temperature, initial pH, inoculum size, and initial moisture content. The optimal conditions were as follows: incubation temperature of 30 or 35 °C, initial pH of 5 or 6, initial moisture content of 70% (w/w), inoculum size of 1 106 spores/mL, incubation time of 5 days, urea/VR 1% (w/w), and MnSO4H2O/VR 0.03% or 0.06% (w/w).
Fig. 4. Data (mean ± SD) for each treatment were calculated from three replicates on a dry matter (DM) basis. A-D Means values with different letters differ significantly (p 0.01); a-bMeans values with different letters differ significantly (p 0.05). The strains of I, II, III, IV, V, VI, VII, VIII are non-strains, P. chrysosporium, T. koningii, Trichoderma viride, A. niger, A. ficuum NTG-23, Candida utilis, and consortium-29, respectively.
Table 3. Factors and their Levels for Orthogonal Experiments
It was found that incubation temperature, initial pH, inoculum size, incubation time, urea content, and MnSO4H2O content had very significant effects on CMCase activity (p 0.01); and initial moisture content had a significant effect on it (0.01 p 0.05).
Effect of factor level
Temperature was the key physical variable in the SSF. In this work, the optimal temperature for the reducing sugar release and CMCase activity in the fermented product of consortium-29 was investigated. The maximum reducing sugar yield occurred at 25 °C (Table 5), which is similar to a large amount of fungi having relatively low optimal temperature in their cultures (Kim et al. 2005). It is well known that fungi favor a moist environment as they grow. The optimal moisture content during the SSF depends on the natural requirements of the microbe, the type of the end products, and the nature of the substrate (Kalogeris et al. 2003a). Hence, the influence of moisture content was investigated for VR degradation by consortium-29.
Table 4. Analyses of the Reducing Sugar Yield and CMCase Activity in the Orthogonal Experiment
The highest CMCase activity was obtained when the initial moisture content was 70% (Table 5), which might be due to the faster growth of fungi at high moisture content and the earlier initiation of enzyme production in the subsequent fermentation. Referring to previous reports, it was found that high moisture content enhanced the microbial growth and lignocellulolytic enzyme system production when lignocellulosic substrates were used as the carbon sources in the SSF (Kalogeris et al. 2003b).
The maximum reducing sugar production and the highest CMCase activity were acquired when the initial pH was 6 and 5 (Table 5), respectively. This property suggested that consortium-29 could be used to degrade lignocellulosic wastes and produce hydrolytic enzyme under weak acidic conditions.
An inoculum size that is too large leads to intense competition for oxygen and nutritive substances, which could decrease synthetic products. However, an inoculum size that is too small might result in longer incubation time and low fermentation productivity. Therefore, the optimal inoculum size was investigated in this study. The optimal reducing sugar production was obtained when the inoculum size was 1 107 or 1 106 spores/mL, and the optimal CMCase activity was exhibited when the inoculum size was 1 106 spores/mL.
The incubation time required to reach maximum levels of degradation might be affected by different ratios of amorphous to crystalline cellulose (Ögel et al. 2001). Therefore, the fermentation time was further analyzed. Both the highest reducing sugar release and CMCase activity were observed after five days (Table 5). These results are similar to previous reports (Yang et al. 2004; Feng et al. 2011). These findings showed that aerobic metabolism prevailed during the first five days, and that, in subsequent fermentation stages, oxygen was limited and the reducing sugar was converted to other products (Feng et al. 2011).
In previous reports, inorganic nitrogen sources were optimal for the acquisition of maximum lignocellulolytic enzyme activity (Kalogeris et al. 2003a). Urea, the most important inorganic nitrogen source, was used in this study. Both the maximum reducing sugar production and the highest CMCase activity were obtained when the content of urea was 1% (Table 5).
Manganese plays an important role in lignin biodegradation by white rot fungi, both as an active mediator for Mn peroxidase and as a regulator for lignin peroxidase, manganese peroxidase, and laccase secretion (Kerem and Hadar 1995). Therefore, the effect of Mn content was investigated. The maximum reducing sugar production and the highest CMCase activity were acquired when the MnSO4H2O contents were 0.03% and 0.06% (Table 5), respectively. The optimal Mn content of consortium-29 for CMCase activity was similar to the reported optimal MnSO4 content (600 μgg-1) for Pleurotus ostreatus (Kerem et al. 1995).
Taking both reducing sugar yield and CMCase activity into account, the optimal fermentation conditions for lignocellulosic component degradation were as follows: incubation temperature of 25 °C, initial pH of 6, initial moisture content of 70% (w/w), inoculum size of 1 106 spores/mL, incubation time of 5 days, urea/VR ratio 1% (w/w), and MnSO4H2O/VR ratio 0.03% (w/w).
Table 5. Analyses of the Effect of Factors on the Reducing Sugar Yield and CMCase Activity
All the optimal levels of temperature, inoculum size, incubation time, urea content and MnSO4 content were the lowest that were considered, which means the lowest cost. The optimal pH is easily controlled. The moisture content of fresh VR is about 70%, in accordance with the optimal moisture content, which implies that the moisture of VR do no need to adjust before fermentation. Moreover, about 40 to 45% of total projected cost for hydrolysis of lignocellulosic biomass is attributed to the process of sugar release from lignocellulosic materials, including pretreatment, enzyme production and enzymatic hydrolysis (Wooley et al. 1999; Yang and Wyman 2008; Guo et al. 2010; Hui et al. 2013). Besides, the cost of SmF is more than 4 times of that of SSF. SSF was adopted in this technology and VR substrate could directly use without being milled and pretreated. All the above mention bring down the cost, and make this technology a cost-competitive strategy for large-scale practical utilization of lignocellulosic wastes.
- Fungal consortium-29 was constructed using orthogonal design combined with two-way interaction analysis based on the reducing sugar yield. It is composed of P. chrysosporium, T. koningii, A. niger, and A. ficuum NTG-23 and is an efficient candidate for lignocellulosic component degradation in un-pretreated vinegar residue.
- Degradation of lignocellulosic components in un-pretreated vinegar residue using fungal consortium-29 by SSF is more efficient than using the individual pure culture and represents a cost-competitive strategy for lignocellulosic waste utilization.
- The optimal fermentation conditions of consortium-29 for un-pretreated VR degradation were as follows: incubation temperature of 25 °C, initial pH of 6, initial moisture content of 70%, inoculum size of 1 106 spores/mL, incubation time of 5 days, urea/VR ratio 1%, and MnSO4H2O/VR ratio 0.03%.
This study was financially supported by a Grant (2011BAD26B01-2) from the National Key Technology RD Program for the 12th Five-year Plan of China and the Special Fund for the Innovative Team of the Chinese Academy of Agricultural Sciences (ASTIP-IAS08).
Adamović, M., Grubić, G., Milenković, I., Jovanović, R., Protić, R., Sretenović, L., and Stoićević, L. (1998). “The biodegradation of wheat straw by Pleurotus ostreatus mushrooms and its use in cattle feeding,” Anim. Feed Sci. Technol. 71(3), 357-362. DOI: 10.1016/S0377-8401(97)00150-8
Arora, D. S., Chander, M., and Gill, P. K. (2002). “Involvement of lignin peroxidase, manganese peroxidase and laccase in degradation and selective ligninolysis of wheat straw,” Int. Biodeterior. Biodegrad. 50(2), 115-120. DOI: 10.1016/S0964-8305(02)00064-1
Bjerre, A. B., Olesen, A. B., Fernqvist, T., Plöger, A., and Schmidt, A. S. (1996). “Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose,” Biotechnol. Bioeng. 49(5), 568-577. DOI: 10.1002/(SICI)1097-0290(19960305)49:5-568–AID-BIT10-3.0
Brijwani, K., Oberoi, H. S., and Vadlani, P. V. (2010). “Production of a cellulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran,” Process Biochem. 45(1), 120-128. DOI: 10.1016/j.procbio.2009.08.015
Chang, V. S., Nagwani, M., Kim, C.-H., and Holtzapple, M. T. (2001). “Oxidative lime pretreatment of high-lignin biomass,” Appl. Biochem. Biotechnol. 94(1), 1-28. DOI: 10.1385/ABAB:94:1:01
Claassen, P., Van Lier, J., Contreras, A. L., Van Niel, E., Sijtsma, L., Stams, A., De Vries, S., and Weusthuis, R. (1999). “Utilisation of biomass for the supply of energy carriers,” Appl. Microbiol. Biotechnol. 52(6), 741-755. DOI: 10.1007/s002530051586
Dahlqvist, A. (1962). “A method for the determination of amylase in intestinal content,” Scand. J. Clin. Lab. Invest. 14(2), 145-151. DOI: 10.3109/00365516209079686
El-Zoghbi, M. (1994). “Biochemical changes in some tropical fruits during ripening,” Food Chem. 49(1), 33-37. DOI: 10.1016/0308-8146(94)90229-1
Fang, H., Zhao, C., and Song, X.-Y. (2010). “Optimization of enzymatic hydrolysis of steam-exploded corn stover by two approaches: Response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02,” Bioresour. Technol. 101(11), 4111-4119. DOI: 10.1016/j.biortech.2010.01.078
Feng, Y., Yu, Y., Wang, X., Qu, Y., Li, D., He, W., and Kim, B. H. (2011). “Degradation of raw corn stover powder (RCSP) by an enriched microbial consortium and its community structure,” Bioresour. Technol. 102(2), 742-747. DOI: 10.1016/j.biortech.2010.08.074
Guo, P., Zhu, W., Wang, H., Lue, Y., Wang, X., Zheng, D., and Cui, Z. (2010). “Functional characteristics and diversity of a novel lignocelluloses degrading composite microbial system with high xylanase activity,” J. Microbiol. Biotechnol. 20(2), 254-264.
Jwanny, E., Rashad, M., and Abdu, H. M. (1995). “Solid-state fermentation of agricultural wastes into food through Pleurotus cultivation,” Appl. Biochem. Biotechnol. 50(1), 71-78. DOI: 10.1007/BF02788041
Kalogeris, E., Christakopoulos, P., Katapodis, P., Alexiou, A., Vlachou, S., Kekos, D., and Macris, B. (2003a). “Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus under solid state cultivation of agricultural wastes,” Process Biochem. 38(7), 1099-1104. DOI: 10.1016/S0032-9592(02)00242-X
Kalogeris, E., Iniotaki, F., Topakas, E., Christakopoulos, P., Kekos, D., and Macris, B. (2003b). “Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw,” Bioresour. Technol. 86(3), 207-213. DOI: 10.1016/S0960-8524(02)00175-X
Kalyani, D., Lee, K.-M., Kim, T.-S., Li, J., Dhiman, S. S., Kang, Y. C., and Lee, J.-K. (2013). “Microbial consortia for saccharification of woody biomass and ethanol fermentation,” Fuel. 107, 815-822. DOI: 10.1016/j.fuel.2013.01.037
Karunanandaa, K., Varga, G., Akin, D., Rigsby, L., and Royse, D. (1995). “Botanical fractions of rice straw colonized by white-rot fungi: Changes in chemical composition and structure,” Anim. Feed Sci. Technol. 55(3), 179-199. DOI: 10.1016/0377-8401(95)00805-W
Kato, S., Haruta, S., Cui, Z. J., Ishii, M., and Igarashi, Y. (2005). “Stable coexistence of five bacterial strains as a cellulose-degrading community,” Appl. Environ. Microbiol. 71(11), 7099-7106. DOI: 10.1128/AEM.71.11.7099-7106.2005
Kausar, H., Sariah, M., Mohd Saud, H., Zahangir Alam, M., and Razi Ismail, M. (2010). “Development of compatible lignocellulolytic fungal consortium for rapid composting of rice straw,” Int. Biodeterior. Biodegrad. 64(7), 594-600. DOI: 10.1016/j.ibiod.2010.06.012
Kerem, Z., and Hadar, Y. (1995). “Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation,” Appl. Environ. Microbiol. 61(8), 3057-3062.
Kim, H., Lim, J., Joo, J., Kim, S., Hwang, H., Choi, J., and Yun, J. (2005). “Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea,” Bioresour. Technol. 96(10), 1175-1182. DOI: 10.1016/j.biortech.2004.09.021
Latif, F., Asgher, M., Saleem, R., Akrem, A., and Legge, R. (2006). “Purification and characterization of a xylanase produced by Chaetomium thermophile NIBGE,” World J. Microbiol. Biotechnol. 22(1), 45-50. DOI: 10.1007/s11274-005-5745-4
Latifian, M., Hamidi-Esfahani, Z., and Barzegar, M. (2007). “Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions,” Bioresour. Technol. 98(18), 3634-3637. DOI: 10.1016/j.biortech.2006.11.019
Li, Y., Yin, Q., Ding, M., and Zhao, F. (2009). “Purification, characterization and molecular cloning of a novel endo-β-1, 4-glucanase AC-EG65 from the mollusc Ampullaria crossean,” Comp. Biochem. Physiol B: Biochem. Mol. Biol. 153(2), 149-156. DOI: 10.1016/j.cbpb.2009.02.011
Lin, H., Wang, B., Zhuang, R., Zhou, Q., and Zhao, Y. (2011). “Artificial construction and characterization of a fungal consortium that produces cellulolytic enzyme system with strong wheat straw saccharification,” Bioresour. Technol. 102(22), 10569-10576. DOI: 10.1016/j.biortech.2011.08.095
Miller, G. L. (1959). “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Anal. Chem. 31(3), 426-428. DOI: 10.1021/ac60147a030
Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., and Ladisch, M. (2005). “Features of promising technologies for pretreatment of lignocellulosic biomass,” Bioresour. Technol. 96(6), 673-686. DOI: 10.1016/j.biortech.2004.06.025
Ögel, Z., Yarangümeli, K., Dündar, H., and Ifrij, I. (2001). “Submerged cultivation of scytalidium thermophilum on complex lignocellulosic biomass for endoglucanase production,” Enzyme Microb. Technol. 28(7), 689-695. DOI: 10.1016/S0141-0229(01)00315-5
Pal, A., and Khanum, F. (2010). “Production and extraction optimization of xylanase from Aspergillus niger DFR-5 through solid-state-fermentation,” Bioresour. Technol. 101(19), 7563-7569. DOI: 10.1016/j.biortech.2010.04.033
Palmqvist, E., and Hahn-Hägerdal, B. (2000). “Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition,” Bioresour. Technol. 74(1), 25-33. DOI: 10.1016/S0960-8524(99)00161-3
Panagiotou, G., and Olsson, L. (2007). “Effect of compounds released during pretreatment of wheat straw on microbial growth and enzymatic hydrolysis rates,” Biotechnol. Bioeng. 96(2), 250-258. DOI: 10.1002/bit.21100
Sharma, R. K., and Arora, D. S. (2013). “Fungal degradation of lignocellulosic residues: An aspect of improved nutritive quality,” Crit. Rev. Microbiol (0), 1-9. DOI: 10.3109/1040841X.2013.791247
Song, Z., Dong, X., Tong, J., and Wang, Z. (2013). “In sacco evaluation of ruminal degradability of waste vinegar residue as a feedstuff for ruminants,” Anim. Prod. Sci. 53(4), 292-298. DOI: 10.1071/AN12116
Van Soest, P. V., Robertson, J., and Lewis, B. (1991). “Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition,” J. Dairy Sci. 74(10), 3583-3597. DOI: 10.3168/jds.S0022-0302(91)78551-2
Villas-Bôas, S. G., Esposito, E., and Mitchell, D. A. (2002). “Microbial conversion of lignocellulosic residues for production of animal feeds,” Anim. Feed Sci. Technol. 98(1), 1-12. DOI: 10.1016/S0377-8401(02)00017-2
Wang, Z.-H., Dong, X.-F., Zhang, G.-Q., Tong, J.-M., Zhang, Q., and Xu, S.-Z. (2011). “Waste vinegar residue as substrate for phytase production,” Waste Manage Res. 29(12), 1262-1270. DOI: 10.1177/0734242X11398521
Wongwilaiwalin, S., Rattanachomsri, U., Laothanachareon, T., Eurwilaichitr, L., Igarashi, Y., and Champreda, V. (2010). “Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system,” Enzyme Microb. Technol. 47(6), 283-290. DOI: 10.1016/j.enzmictec.2010.07.013
Wooley, R., Ruth, M., Glassner, D., and Sheehan, J. (1999). “Process design and costing of bioethanol technology: A tool for determining the status and direction of research and development,” Biotechnol. Prog. 15(5), 794-803. DOI: 10.1021/bp990107u
Yang, B., and Wyman, C. E. (2008). “Pretreatment: The key to unlocking low-cost cellulosic ethanol,” Biofuels Bioprod. Biorefin. 2(1), 26-40. DOI: 10.1002/bbb.49
Yang, Y., Wang, B., Wang, Q., Xiang, L., and Duan, C. (2004). “Research on solid-state fermentation on rice chaff with a microbial consortium,” Colloids Surf. B 34(1), 1-6. DOI: 10.1016/j.colsurfb.2003.10.009
Yang, X., Wang, J., Zhao, X., Wang, Q., and Xue, R. (2011). “Increasing manganese peroxidase production and biodecolorization of triphenylmethane dyes by novel fungal consortium,” Bioresour. Technol 102(22), 10535-10541. DOI: 10.1016/j.biortech.2011.06.034
Zhang, Q., He, J., Tian, M., Mao, Z., Tang, L., Zhang, J., and Zhang, H. (2011). “Enhancement of methane production from cassava residues by biological pretreatment using a constructed microbial consortium,” Bioresour. Technol. 102(19), 8899-8906. DOI: 10.1016/j.biortech.2011.06.061
Article submitted: January 7, 2015; Peer review completed: March 22, 2015; Revisions received and accepted: April 16, 2015; Published: April 22, 2015.