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Article

Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi

by
Analdi Farniga
1,
Phimrak Khaokhajorn
2 and
Songsak Wattanachaisaereekul
1,*
1
School of Food Industry, King Mongkut’s Institute of Technology Ladkrabang, 1 Chalong krung Rd., Ladkrabang, Bangkok 10520, Thailand
2
Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, 49 Soi Thianthale 25, Bangkhunthian-Chaithale Rd., Thakham, Bangkhunthian, Bangkok 10150, Thailand
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(1), 14; https://doi.org/10.3390/fermentation10010014
Submission received: 24 November 2023 / Revised: 15 December 2023 / Accepted: 19 December 2023 / Published: 22 December 2023
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
Cassava bagasse has great potency as a substrate in the biorefinery industry. This paper proposes the valorisation of cassava bagasse into organic acids by cellulase through the co-cultivation of Aspergillus violaceofuscus and Trichoderma reesei RUT-C30 at the ratio 1:1. The optimised conditions for β-glucosidase production under submerged fermentation were pH 4.50, a tween 80 concentration of 0.05% (v/v), and a spore concentration of 7.18 × 107 spores·mL−1. We found base steam cassava bagasse (BSCB) to have high cellulose content, making it possible to replace avicel phosphoric acid swollen cellulose (PASC) as a substrate. The co-cultivation with the BSCB substrate had higher levels of β-glucosidase (1.72-fold), cellobiohydrolase (2.83-fold), and endoglucanase (2.82-fold) activity compared to that of the avicel PASC substrate. Moreover, acetic acid (7.41 g·L−1), citric acid (3.54 g·L−1), gluconic acid (0.30 g·L−1), and malic acid (0.37 g·L−1) were detected in the BSCB crude extract. These results demonstrate the considerable prospects of the A. violaceofuscus and T. reesei RUT-C30 co-cultivation approaches in the biorefinery industry.

Graphical Abstract

1. Introduction

Fibrous agricultural waste is a sustainable substrate used to produce numerous chemicals through enzymatic reactions. Cellulase is a consortium of the enzymes endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL), which work in a synergistic manner to break down cellulose [1]. Cellulase is an attractive prospect for the biorefinery of cellulose-based materials because of its low energy consumption, but nevertheless, it accounts for ~40% of the total costs of the biorefinery industry [2]. The development of a low-cost source of cellulase is therefore essential to the bio-recovery process and its growing applications.
BGL is the limiting factor in cellulose hydrolysis due to its role in converting cellobiose into glucose; when BGL is lacking, cellobiose accumulates and acts as an inhibitor of EG and CBH [3,4]. Thus, balanced proportions of these enzymes are required to maximise cellulose saccharification. Desirable saccharification yields from lignocellulose material, particularly cellulose, can be achieved by applying a combination of cellulase-producing microorganisms, known as the co-cultivation technique. The efficiency of co-cultivation in elevating the level of cellulase activity has previously been reported [5,6,7,8]. Filamentous fungi have been reported as being capable of producing cellulase, and fungi from Trichoderma genus are commonly used as cellular factories in cellulase manufacturing; however, this genus is incompetent at producing high yields of BGL and CBH [9,10]. Aspergillus species are well-known BGL producers [11,12,13], and hence, are candidates for co-cultivation with Trichoderma, specifically, T. reesei RUT-C30, which is a potent cellulase hyper-producers and the best-studied cellulolytic fungus available in the public domain [14,15]. However, in addition to finding suitable mixed cultures of cellulase producers, the selection of an economical cellulose substrate is also an essential consideration [16].
Thailand has a large agricultural sector, and cassava is one of its most important commodities; indeed, Thailand is the world’s biggest exporter of cassava products, accounting for 66% of the worldwide exports of native starch, 59% of cassava chips exports, and 31% of exports of modified starch [17]. Despite cassava being highly utilised in Thailand, the by-products of cassava starch production are rarely used as biorefinery substrates. The major by-product is cassava bagasse, a solid waste with a high cellulose content [18]. Fermentable sugars can be produced from cassava bagasse using fungal cellulase, and then further converted into valuable chemicals, e.g., organic acids [19,20].
Organic acids have been applied for numerous commercial purposes, including as food preservatives and precursors of biodegradable polymers [21]. Many studies have demonstrated the production of organic acids from cassava bagasse, but there have been no reports to date that used fungal co-cultivation. The valorisation of cassava bagasse through fermentation with filamentous fungi is a worthwhile strategy for producing organic acids; moreover, cassava bagasse is considered capable of replacing synthetic sugars as the starting material of organic acid production [22,23].
This study was carried out to determine both the suitable and ideal conditions for cellulase production through the co-cultivation of filamentous fungi. The optimal cultures and conditions were then used to produce organic acids from cassava bagasse.

2. Materials and Methods

2.1. Microorganisms

A total of 24 fungal isolates from the Food Industry KMITL culture collection were examined for their ability to produce cellulase (Table S1). The cultures were grown on a potato dextrose agar plate for five days, after which, spore suspensions were prepared by harvesting the mycelia using 10 mL of sterile 0.1% (v/v) tween 80. Spore suspensions were kept in 25% glycerol at −80 °C for further use. The spore concentration was determined with a Neubauer haemocytometer under a microscope (Hirschmann Laborgeräte, Eberstadt, Germany).

2.2. Preparation of Avicel Phosphoric Acid Swollen Cellulose (PASC)

Avicel PASC was prepared according to the standard procedure with a slight modification [24]. One gram of avicel was combined with 3 mL of distilled water in a 50 mL centrifuge tube. Then, 10 mL of cold 86.2% phosphoric acid (Merck, Darmstadt, Germany) was added to the moist avicel, and the mixture was incubated at 4 °C for one hour with stirring every ten minutes. After an hour of incubation, 20 mL of ethanol was added to the slurry, and the resulting pellet was separated by centrifugation (Eppendorf: 5804R, Hamburg, Germany) at 3000× g and 4 °C for ten minutes. The supernatant was discarded after centrifugation. The cloudy, white PASC pellet was washed with ethanol twice and distilled water thrice. For temporary storage, the PASC was kept at 4 °C, and for long-term storage, it was kept at −20 °C.

2.3. Cellulase Production

2.3.1. Screening of Cellulase-Producing Fungi

The initial selection of cellulase-producing fungi was performed using the cellulase enzymatic index of fungi cultured on an agar plate containing avicel PASC. The composition of the media was adopted from [25,26], with modification of the agar and PASC contents (20 g·L−1 and 10 g·L−1, respectively). Twenty microlitres of spore suspension were inoculated on a surface of sterile media and incubated at 30 °C for 5–7 days. To obtain the hydrolysis zone, the growing fungi were stained with Congo Red solution, and then discoloured with 1 M NaCl [27]. The enzymatic index (EI) was determined as the ratio of clear zone diameter to colony diameter.

2.3.2. BGL Optimisation

Optimal conditions were determined for BGL production by the co-cultivation of FI-15 (identified as Aspergillus violaceofuscus) and Trichoderma reesei RUT-C30 under submerged fermentation (SMF). These cultures were selected based on the BGL activity assessment of cellulase-producing fungi under SMF using the avicel PASC substrate (Table S2). The optimised fermentation condition was evaluated by the response surface methodology (RSM). A three-level three-factor Box–Behnken design (BBD) was used to examine the variables that influence BGL production. According to the preliminary results and review articles, the key variables for producing cellulase with avicel PASC as the sole cellulose source are spore concentration, pH, and the tween 80 concentration in the media. A. violaceofuscus and T. reesei RUT-C30 at a ratio of 1:1 (1% v/v) were inoculated into 100 mL sterile media in an Erlenmeyer 250 mL flask and incubated in a shaking incubator (N-biotek: NB20, Bucheon, Gyeonggi Province, Republic of Korea) at 150 rpm (28 °C). According to the preliminary result (Figure S1), the cultures were harvested after twelve days of fermentation and centrifuged at 3000× g for 60 s. RSM was performed using Statgraphics Centurion-19 (©Statgraphics Technologies, Inc., The Plains, VA, USA). Table 1 presents the factors and coding levels of the independent variables in the BBD experimental design matrix.

2.4. Cassava Bagasse Valorisation

2.4.1. Sample Preparation

Cassava bagasse was washed with tap water and dried in a hot air oven at 60 °C overnight. The dried bagasse was then steamed in an autoclave with 2% NaOH, 2% HCl, or deionised H2O at 121 °C for 15 min to yield base steam cassava bagasse (BSCB), acid steam cassava bagasse (ASCB), and steam cassava bagasse (SCB), respectively [28]. Subsequently, the bagasse was washed until it reached neutral pH, and then dried in an oven as before. The lignocellulose variance between the treated and untreated cassava bagasse (UCB) was determined by comparing the Fourier-transform infrared (FTIR) spectra of the samples. The quantity of cellulose, hemicellulose, and lignin in each sample was determined using the National Renewable Energy Laboratory (NREL) standard methodology [29].

2.4.2. Submerged Fermentation

The valorisation of cassava bagasse followed the obtained optimised conditions for BGL production. The treated cassava bagasse with the highest cellulose content was used as a cellulose source to replace avicel PASC, and the cellulase activity levels were compared between avicel PASC and the cassava bagasse. The cellulase (BGL, CBH, and EG), glucose, ethanol, and organic acids contents were also analysed.

2.5. Assays

2.5.1. Cellulase Activity

An endoglucanase (EG) assay was conducted in a total reaction mixture of 100 µL containing 90 µL of 1% carboxymethyl cellulose (CMC) solution and 10 µL of crude enzyme. The reaction mixture was incubated at 50 °C for 30 min. The released reducing sugars were then quantified by the 3,5-dinitro salicylic acid (DNS) method using glucose as the reducing sugar standard [30,31]. To measure cellobiohydrolase (CBH) activity, 10 µL of the crude enzyme was combined with 90 µL of 5 mM p-nitrophenyl β-D-cellobioside (pNPC) (Sigma-Aldrich, Burlington, MA, USA) dissolved in 0.05 M citrate buffer at pH 6.0. The mixture was incubated at 50 °C for 30 min to release p-nitrophenol, after which 200 µL of 1 M Na2CO3 was added to stop the reaction. For the β-glucosidase (BGL) assay, the reaction mixture consisted of 10 µL crude enzyme and 90 µL of 5 mM p-nitrophenyl β-D-glucopyranoside (pNPG) (Sigma-Aldrich, Burlington, MA, USA). The reaction was incubated at 50 °C for 30 min, and then terminated with 200 µL of 0.4 M Na2CO3. The quantity of p-nitrophenol liberated from pNPC and pNPG was determined by measuring the absorbance at 405 nm. For all the enzyme assays, one unit of enzyme activity was equivalent to the amount of enzyme required to produce one µmol of glucose or p-nitrophenol from the appropriate substrate per minute under the assay conditions [32,33]. All the enzyme contents are expressed in units per gram dry substrate (IU·gds−1).

2.5.2. Fourier-Transform Infrared (FTIR) Analysis

Differences in lignocellulose content among ASCB, BSCB, SCB, and UCB were determined based on the comparison of functional group signals in the FTIR absorption spectra (Bruker: Invenio, Bremen, Germany). The spectrometer was accessorised with a diamond attenuated total reflectance (ATR) crystal for sample scanning. Spectra were recorded from 400 to 4000 cm−1 with a spectral resolution of 4 cm−1. Sixty-four scans were applied to two replications of each sample, and the spectra were corrected against the background.

2.5.3. Glucose, Ethanol, and Organic Acid Analysis

Glucose, ethanol, and organic acid concentrations were assessed by high-performance liquid chromatography (HPLC) (Agilent Technologies: Agilent 1260, Santa Clara, CA, USA). The HPLC instrument was equipped with an Aminex HPX-87H column (Bio-rad Laboratories, Inc., Hercules, CA, USA) and 5 mM H2SO4 was used as the mobile phase. The HPLC conditions were set at a flow rate of 0.6 mL·min−1 (isocratic) with a temperature of 50 °C. Glucose and ethanol were detected using a refractive index (RI) detector, while organic acids were detected based on the UV absorbance at 210 nm.

3. Results

3.1. Cellulase Activity

3.1.1. Congo Red Staining

Twenty-four fungal isolates were screened for cellulase production according to their ability to grow on an avicel PASC agar plate, with Congo Red staining used to obtain the cellulase enzymatic index. Congo Red staining is favoured for the early screening of cellulase-producing organisms due to its rapidness and sensitivity [34,35,36]. Twelve fungi were observed to grow in this medium, which indicated their ability to produce cellulase. The enzymatic indices were variable among the fungi, as illustrated in Figure 1.
Avicel is a crystalline cellulose with a short carbon chain, and hence, is more easily hydrolysed into cellobiose and further broken down into glucose by BGL. Isolates FI-01, FI-04, and FI-15 exhibited the top three highest enzymatic indices, of which the former two were identified as Aspergillus niger, and the last one was A. violaceofuscus. The ability of filamentous fungi from the Aspergillus genus to produce high titres of BGL and CBH is well documented [9,37]. The low activity level of T. reesei RUT-C30 on avicel PASC media reflects poor fitness due to its lack of BGL, while also having sufficient endoglucanase to get by [7]. Interestingly, mixed cultures of A. violaceofuscus and T. reesei RUT-C30 demonstrated elevated BGL activity levels under SMF with avicel PASC as the substrate. Therefore, these strains were selected for optimisation to determine the most suitable fermentation conditions for promoting cellulase activity.

3.1.2. BGL Optimisation

To determine the ideal conditions for BGL production from a mixed culture of A. violaceofuscus and T. reesei RUT-C30, various combinations of pH (4.5, 5.0, and 5.5), spore concentration (1 × 105, 5.01 × 107, and 1 × 108), and tween 80 concentration (0.1%, 0.3%, and 0.5%) were tested. For analysis purposes, each parameter value was coded with the highest level as +1 and the lowest as −1. Table 2 displays the BBD experimental design with 15 runs and the corresponding observed BGL activity values; interestingly the activity values were divided into two distinct groups, >20 and <13 IU·gds−1.
ANOVA was then performed, and the mean square contrasted with estimated experimental error to determine the significance of the effect of each variable on BGL activity (Table 3). At 95% confidence, three models (A: pH; B: spore concentration; AA) had p-values of less than 0.05, indicating these variables to be statistically significant. The relationship between these variables and BGL activity was therefore visualised using a response surface graph (Figure 2).
The response surface graph predicted a maximum BGL activity level of 30.60 IU·gds−1 for the media containing 0.05% tween 80, 7.18 × 107 spore·mL−1, and pH 4.5. However, the actual BGL activity level obtained under these conditions was 39.73 IU·gds−1. The analysis demonstrated the initial pH (acidic media) to be a leading influence on our mixed cultures of T. reesei RUT-C30 and A. violaceofuscus. This finding concurs with the previous studies on cellulase-producing fungi, which determined them to favour an initial pH in the range of 3.0–8.0 [4,38,39,40,41].
A surfactant like tween 80 is frequently blended with cellulose-containing media to augment the cellulase activity. Tween 80 promotes saccharification by enhancing the solubility of crystalline cellulose, suppressing enzyme inhibitors, and increasing cellulase thermal stability [42,43,44]. Nevertheless, the range of tween 80 concentrations tested here were observed to have no significant effect, which indicated the requirements of initial screening to examine the concentration prior to the optimisation using RSM, such as the Plackett–Burman design [10,45].

3.1.3. Cellulase Activity of A. violaceofuscus and T. reesei RUT-C30 in Monoculture and Mixed Culture

This study aimed to investigate the effectiveness of a mixed culture in enhancing the cellulase activity compared to that of the corresponding monocultures. The results (Table 4) revealed that the mixed culture promotes BGL activity, with respective increases of 1.39-fold and 21.71-fold relative to the monocultures of A. violaceofuscus and T. reesei RUT-C30, respectively. This result is in concordance with a previous study on elevated BGL activity levels in mixed cultures [1]. Notable, T. reesei RUT-C30, which is the most used strain in cellulase manufacturing, is unable to compete with A. violaceofuscus for cellulase production under SMF when using avicel PASC as the substrate. However, the opposite result is observed when using a wheat bran substrate, with T. reesei having a higher activity level than the three species of Aspergillus [46]. Interestingly, the A. violaceofuscus monoculture showed competence in producing a high cellulase activity level, particularly with CBH, with a 1.13-fold increase compared to mixed culture. This supports that A. violaceofuscus can be used in the cellulase industry in conjunction with Trichoderma fungi. Additionally, our findings emphasise the importance of optimising the culture ratio of mixed cultures for maximum cellulase activity, along with the need for further research to improve the EG activity.

3.2. Cassava Bagasse Valorisation

3.2.1. Cellulose, Hemicellulose, and Lignin Analysis

FTIR analysis enables the prediction of a compound’s presence based on the characteristic infrared absorption spectra of functional groups. Diamond attenuated total reflection (ATR) is a convenient and non-destructive method for obtaining infrared spectra from solid materials, including lignocellulose. The infrared spectra compositions of ASCB, BSCB, SCB, and UCB are given in Figure 3.
All the samples were considered to contain cellulose, hemicellulose, and lignin based on the appearance of the fingerprint area of their infrared spectra, which have been described in numerous publications [47,48,49]. Three dominant peaks were observed at the wavenumbers 1015 cm−1, 1605 cm−1, and 3320 cm−1. The peak in the area of 800–1150 cm−1 indicates the C-O-C at the β-(1-4)-glycosidic bond, which is considered indicative of cellulose and hemicellulose. The peak in the area of 1500–1650 cm−1 is an aromatic ring vibration band characteristic of lignin. The 3320 cm−1 band is indicative of the hydroxyl groups present in cellulose, hemicellulose, and lignin. Moreover, the BSCB spectrum shows prominent peaks at 1007 cm−1 and 3309 cm−1, indicating abundant cellulose and hemicellulose.
On top of this qualitative analysis, the quantitative analysis of the component compounds is required for a well-presented value. The results from applying the NREL method for measuring cellulose, hemicellulose, and lignin contents are presented in Table 5.
Alkaline steam explosion is often used as a pre-treatment process to make lignocellulose materials more amenable to processing (i.e., biorefinery production by enzyme digestion). This pre-treatment involves first heating the lignocellulose material in an alkaline solution, and then performing rapid depressurisation, causing the material to undergo a form of explosion. As shown in Table 5, the highest cellulose content was observed for BSCB, followed by SCB, UCB, and finally, ASCB. The cellulose content in BSCB is relatively higher than those in rice bran [50], wheat straw [51], and corn stover [52]. Meanwhile, ASCB is probably susceptible to peeling off of the cellulose and hemicellulose parts, leading to the decreased content of those compounds. Ultimately, BSCB was selected for the assessment of cellulase enzyme functionality under the co-cultivation of A. violaceofuscus and T. reesei RUT-C30.

3.2.2. Cellulase Profiling on Cassava Bagasse and Avicel PASC

The cellulase profile of fungi cultivated on cassava bagasse substrate was evaluated using conditions similar to the avicel PASC substrate assessment. The results are listed in Table 6 and indicate that cassava bagasse provides a more idyllic substrate compared to that of avicel PASC, as the activity levels of BGL, CBH, and EG were all much greater (by 1.72-fold, 2.83-fold, and 2.82-fold, respectively). There are many possible factors that may affect the cellulase secretion ability of fungi, and so, explain this distinction of the substrates. The previous studies have found that catabolic repression, the presence of cellulase inhibitors, and cellulose accessibility all exert major influences [53,54]. Furthermore, fungi employ a large set of enzymes to degrade the lignocellulose biomass, which can cause their enzyme activity profiles to vary across different substrates [55]. Penicillium sp. have been reported to produce BGL when cultivated on several agricultural wastes [56], though with a lower activity level than observed here on BSCB. Therefore, growing a mixed culture of T. reesei RUT-C30 and A. violaceofuscus using BSCB as the substrate is a potential approach for providing sugar-based derived chemicals.

3.2.3. Organic Acid Production on BSCB

We further assayed the organic acids (acetic acid, citric acid, gluconic acid, lactic acid, malic acid, oxalic acid, succinic acid, and tartaric acid), ethanol, and glucose present in the SMF filtrate on day ten of co-cultivation. Some organic acids were detected (acetic acid, citric acid, gluconic acid, gluconic acid, and malic acid), as illustrated in Figure 4. Notably, organic acid production, particularly acetic acid, was inversely proportional to the cellulase activity of a similar filtrate likewise collected on day ten of mixed culturing. Greater amounts of acetic acid (1.24-fold), citric acid (5.21-fold), and malic acid (1.45-fold) were obtained when using avicel PASC rather than BSCB as the substrate. Interestingly, gluconic acid was only found in the BSCB-containing media (0.30 g·L−1).
Aspergillus fungi have been acclaimed for their potency in secreting a vast array of organic acids [57]. In our mixed cultures, the fungi might utilise the entirety of the glucose generated due to the low concentration of cellulose, which was only 1% (w/v) of the total media volume. This circumstance led to the absence of glucose on day 10 of fermentation. A high citric acid concentration was found in the PASC-containing media, alongside an absence of gluconic acid. The massive accumulation of citric acid probably led to the inactivation of the glucose oxidase enzyme, which is responsible for converting glucose into gluconic acid [58,59]. Moreover, glucose oxidase requires a high glucose concentration and proper aeration, which were probably unavailable in the avicel PASC-containing media.
All told, co-cultivated A. violaceofuscus and T. reesei RUT-C30 have a promising ability to produce organic acids, especially when provided with a low concentration of the cellulose substrate. These results support the prospect of developing cassava bagasse as a low-cost biomass feedstock in organic acid production, and thereby, promoting sustainable refining chemical factory processes.

4. Conclusions

Optimised conditions for SMF with co-cultivated A. violaceofuscus and T. reesei RUT-C30 were determined using BBD with avicel PASC as the substrate. It was observed that BGL is effectively secreted under a low pH. Both the treated and untreated cassava bagasse in this study were high in cellulose content, indicating cassava bagasse to be a promising substrate for the cellulose-based industry. In addition, the BSCB-containing media were able to compete with the avicel PASC media in the production of cellulase and organic acids. These findings confirm the proposed strategy for utilising cassava bagasse as a substrate for the co-cultivation of A. violaceofuscus and T. reesei RUT-C30, which has potential applications in the biomass-based biorefinery industry.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation10010014/s1, Figure S1: BGL production from Trichoderma reesei RUT-C30 and Aspergillus violaceofuscus co-cultivation under SMF using avicel PASC substrate; Table S1: List of fungal isolates and their cellulase enzymatic indices on avicel PASC substrate; Table S2: Cellulase activity of monocultures and mixed-cultures under SMF using avicel PASC substrate.

Author Contributions

A.F.: Methodology; validation; formal analysis; investigation; data curation; writing—original draft. P.K.: Methodology; validation; formal analysis; investigation; data curation. S.W.: Conceptualization; methodology; validation; investigation; supervision; writing—review and editing; project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This research is a result of the project entitled “Innovation of lignocellulolytic enzyme production by microbial co-cultivation Grant NO.RE-KRIS/FF65/40” by King Mongkut’s Institute of Technology Ladkrabang (KMITL), which has been received funding support from the NSRF (FRB650028/0258).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available on request to the corresponding author.

Acknowledgments

The authors thank the staff and members of the Fungal Biotechnology Laboratory, Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi (KMUTT). We thank the School of Food Industry, King Mongkut’s Institute of Technology Ladkrabang (KMITL), and the NSFC-NRCT Collaboration Project for their facilities and support.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 10 00014 g001
Figure 1. Cellulase enzymatic indices of fungi that grew on avicel PASC. Different letters above bars indicate statistically significant difference in means based on Tukey’s HSD at a 95% confidence level.
Figure 1. Cellulase enzymatic indices of fungi that grew on avicel PASC. Different letters above bars indicate statistically significant difference in means based on Tukey’s HSD at a 95% confidence level.
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Figure 2. Response surface graph of the impact of independent factors spore concentration and pH on BGL activity from the co-cultivation of A. violaceofuscus and T. reesei RUT-C30 under SMF with avicel PASC substrate.
Figure 2. Response surface graph of the impact of independent factors spore concentration and pH on BGL activity from the co-cultivation of A. violaceofuscus and T. reesei RUT-C30 under SMF with avicel PASC substrate.
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Figure 3. FTIR spectra of ASCB, BSCB, SCB, and UCB.
Figure 3. FTIR spectra of ASCB, BSCB, SCB, and UCB.
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Figure 4. Acetic acid, citric acid, gluconic acid, and malic acid produced after ten days of co-cultivation of A. violaceofuscus and T. reesei RUT-C30 on BSCB (horizontal line fill) and avicel PASC (vertical line fill) substrates. Yields are expressed in g per L of media. All experiments were performed in triplicate, and error bars indicate the standard deviation (n.d. = not detected).
Figure 4. Acetic acid, citric acid, gluconic acid, and malic acid produced after ten days of co-cultivation of A. violaceofuscus and T. reesei RUT-C30 on BSCB (horizontal line fill) and avicel PASC (vertical line fill) substrates. Yields are expressed in g per L of media. All experiments were performed in triplicate, and error bars indicate the standard deviation (n.d. = not detected).
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Table 1. Factor and coding levels of independent variables in the BBD experimental design.
Table 1. Factor and coding levels of independent variables in the BBD experimental design.
FactorCoding Level
−101
A: pH4.555.5
B: Spore concentration (spores·mL−1)1 × 1055.01 × 1071 × 108
C: Tween 80 (%)0.010.030.05
Table 2. RSM experimental matrix and BGL activity results.
Table 2. RSM experimental matrix and BGL activity results.
Run No.ABCBGL
(IU·gds−1)
14.50 (−1)5.01 × 107 (0)0.05 (+1)31.460
24.50 (−1)1 × 105 (−1)0.03 (0)20.071
35.00 (0)1 × 105 (−1)0.01 (−1)6.513
45.00 (0)1 × 108 (+1)0.01 (−1)9.316
55.00 (0)5.01 × 107 (0)0.03 (0)11.238
65.50 (+1)1 × 105 (−1)0.03 (0)4.416
75.00 (0)1 × 105 (−1)0.05 (+1)9.860
85.50 (+1)5.01 × 107 (0)0.01 (−1)7.631
95.00 (0)1 × 108 (+1)0.05 (+1)10.476
104.50 (−1)5.01 × 107 (0)0.01 (−1)27.426
115.50 (+1)1 × 108 (+1)0.03 (0)11.089
125.00 (0)5.01 × 107 (0)0.03 (0)8.357
135.50 (+1)5.01 × 107 (0)0.05 (+1)12.101
145.00 (0)5.01 × 107 (0)0.03 (0)11.545
154.50 (−1)1 × 108 (+1)0.03 (0)28.207
A, pH; B, spore concentration; C, tween 80 concentration.
Table 3. ANOVA of response surface models.
Table 3. ANOVA of response surface models.
ModelSum of SquaresDFMean SquareF-Ratiop-Value
A (pH)646.6871646.687109.6500.000
B (spore concentration)41.533141.5337.0400.045
C (tween 80 concentration)21.161121.1613.5900.117
AA241.6251241.62540.9700.001
AB0.53510.5350.0900.775
AC0.04810.0480.0100.932
BB23.518123.5183.9900.102
BC1.19611.1960.2000.671
CC5.18515.1850.8800.392
R-squared = 97.1127%
Table 4. Cellulase activity of monocultures and mixed cultures.
Table 4. Cellulase activity of monocultures and mixed cultures.
Organism1 BGL
(IU·gds−1)		1 CBH
(IU·gds−1)		1 EG
(IU·gds−1)
T. reesei RUT C-30 (TR)1.83 ± 0.1421.28 ± 0.0280.010 ± 0.003
A. violaceofuscus (AV)28.48 ± 0.2954.42 ± 0.8530.023 ± 0.005
Mixed culture (TR + AV)39.73 ± 3.9103.89 ± 0.3800.017 ± 0.002
1 Enzyme activity analysis was carried out in triplicate.
Table 5. Percent cellulose, hemicellulose, and lignin in different treatments of cassava bagasse.
Table 5. Percent cellulose, hemicellulose, and lignin in different treatments of cassava bagasse.
BiomassCellulose
(% w/w)		Hemicellulose
(% w/w)		Acid Soluble Lignin
(% w/w)		Acid Insoluble Lignin
(% w/w)
ASCB58.01 ± 1.101.29 ± 0.022.77 ± 0.1015.65 ± 0.34
BSCB72.91 ± 0.770.90 ± 0.022.44 ± 0.036.41 ± 0.23
SCB66.77 ± 1.572.17 ± 0.102.54 ± 0.074.48 ± 0.21
UCB63.82 ± 0.832.18 ± 0.022.53 ± 0.084.92 ± 0.49
Table 6. Cellulase activity on avicel PASC and BSCB substrates.
Table 6. Cellulase activity on avicel PASC and BSCB substrates.
Substrate1 BGL
(IU·gds−1)		1 CBH
(IU·gds−1)		1 EG
(IU·gds−1)
Avicel PASC39.73 ± 3.913.89 ± 0.380.017 ± 0.002
BSCB68.30 ± 2.2211.01 ± 1.070.048 ± 0.002
1 Enzyme activity analysis was carried out in triplicate.
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Farniga, A.; Khaokhajorn, P.; Wattanachaisaereekul, S. Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi. Fermentation 2024, 10, 14. https://doi.org/10.3390/fermentation10010014

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Farniga A, Khaokhajorn P, Wattanachaisaereekul S. Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi. Fermentation. 2024; 10(1):14. https://doi.org/10.3390/fermentation10010014

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Farniga, Analdi, Phimrak Khaokhajorn, and Songsak Wattanachaisaereekul. 2024. "Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi" Fermentation 10, no. 1: 14. https://doi.org/10.3390/fermentation10010014

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