Solid-State Cultivation of Aspergillus niger–Trichoderma reesei from Sugarcane Bagasse with Vinasse in Bench Packed-Bed Column Bioreactor

Solid-state cultivation (SSC) is microbial growth on solid supports under limited water conditions. Citric acid is a microbial aerobic metabolic product with several industrial applications, with production potential that can be obtained by SSF. Several wastes from agro-industries are used in SSF, such as sugarcane bagasse and vinasse. Cultures of mixed fungi or co-cultures are used in this SSF in order to complement the inoculum’s xylanolytic enzymes for action on the lignocellulosic material (bagasse). Thus, this study aims to evaluate the effect of inoculum (Aspergillus niger and Trichoderma reesei consortium) in the production of citric acid from sugarcane bagasse impregnated with vinasse using bench packed-bed reactors (PBR). The results show the importance of T. reesei and A. niger in inoculum at a ratio of 50:50 and 25:75, suggesting the use of solid support due to the complementation of the hydrolytic enzymes. The highest concentration of citric acid, approximately 1000 mg L−1, was obtained for 100 mm of bed height in 48 and 72 h, with maximum glucose yield in citric acid (2.2 mg citric acid mg glucose−1). kLa indicates that maintaining solid moisture and liquid film thickness is important to keep the oxygen transfer in SSC.


Introduction
Solid-state cultivation (SSC) is a process in which microbial growth occurs in solid support in the presence of limited water conditions [1]. This low-cost technique is useful in producing new biocatalysts and bioproducts using wastes from agro-industries [2,3]. In general, agro-https://doi.org/10.1007/s12010-021-03579-9 industrial byproducts act as physical support, which are usually impregnated with a nutrient supply capable of sustaining microbial growth [4].
Brazil, being the largest producer of sugarcane in the world, produces 25% of the global production. Sugarcane bagasse, a byproduct of sugar processing, contains cellulose, hemicellulose, lignin, simple sugars, and ashes in lower concentrations. Due to these characteristics, sugarcane bagasse is considered important solid support to produce several bioproducts by SSC [8][9][10][11][12][13][14]. Similarly, vinasse is the main wastewater in sugarcane processing, and it is generated during the ethanol distillation-fermentation in high volumes and is rich in organic matter and potassium. The major components of vinasse are organic matter, including nonfermentable substances, fermented products (glycerol and organic acids), and yeast residues that cannot be separated by distillation [15]. A wide range of organic compounds found in vinasse includes alcohols, aldehydes, ketones, esters, acids, and sugars. Therefore, using vinasse to impregnate bagasse as a nutrient solution for SSC could minimize the production cost by adding two byproducts generated in the sugarcane processing.
Citric acid is an important organic acid and has many applications in the food, beverage, chemical, and metallurgical industries [16]. The demand for citric acid increases by 4% each year; therefore, it is important to look for alternative ways of its production. A new approach in stimulating the microbial production of citric acid is by using lower alcohols (ethanol or methanol). The reason behind this approach is increasing the cell permeability or altering the activity of the citrate synthase and aconitase. Also, Aspergillus niger converts the available ethanol into acetyl-CoA, a key precursor molecule in the Krebs cycle [17]. Dhillon et al. [18] utilized lower alcohol to moisten the substrate for the production of citric acid. Thus, the use of vinasse in impregnating bagasse is an interesting approach.
Several types of bioreactors used for solid-state fermentation are Petri dishes, jars, wide-mouth Erlenmeyer flasks, Roux, roller bottles [19], and other types of stirred bench bioreactors [6]. An ORSTOM team developed lab-scale equipment composed of small columns filled with a previously inoculated medium placed in a thermoregulated water bath, while water-saturated air passes through each column [20]. These bioreactors known as "Raimbault Columns" are small packed-bed reactors (PBR) and are used by many researchers for proper aeration of the culture, optimization of the medium components, and gaseous transfer. The main feature of a PBR is forced aeration through a static bed, which helps in the replenishment of O 2 and moisture, and mitigates the accumulation of heat and CO 2 [21]. Moreover, in packed-bed bioreactors, the injection of humidified air through the low-cost solid substrates controls temperature, oxygen, and moisture, important aspects for the scale-up study of the SSC [2].
In this context, this research aimed to evaluate the synergistic effect of fungus (Aspergillus niger and Trichoderma reesei) in the production of citric acid from sugarcane bagasse impregnated with vinasse in bench PBR.

Sugarcane Bagasse and Vinasse
Sugarcane bagasse and vinasse were collected from a sugarcane processing unit (Araras, São Paulo, Brazil). A set of Tyler Mesh with 14 and 28 sieves was used to separate the sugarcane bagasse particles of an average diameter between 0.59 and 1.17 mm, being selected for the experiments. These separated particles were sterilized in polypropylene bags in an autoclave.
Raw vinasse obtained from ethanol distillation was sterilized at 121°C for 20 min in an autoclave, and pH was maintained by potentiometry, glucose content was estimated by the enzymatic method (kit LABORLAB®), and carbon and total nitrogen were analyzed using SHIMADZU® TOC-LCPN analyzer.

Screening of Inoculum Condition
A small packed-bed column bioreactor of 30 mm in diameter and 60 mm in bed height, known as "Raimbault Columns" [20], was used for screening fungal inoculum. Continuous watersaturated airflow at 30°C was provided. The sterilized sugarcane bagasse (solid support) was impregnated using vinasse and inoculum suspension with different proportions of fungi until the initial moisture reached 80%.
To prepare the inoculum, the SSC was set up with a spore suspension of Aspergillus niger, Trichoderma reesei, and a consortium of these at different volume proportions (75% A. niger and 25% T. reesei; 25% A. niger and 75% T. reesei; and 50% of each in the inoculum suspension). These samples were incubated for 4 days, as per the previous study done by Campanhol et al. [10].
Gravimetric analysis was done to analyze the moisture of the solid support. Crude fungal extracts were obtained by the addition of 1:15 deionized water (solid solvent) for 45 min at 100 rpm/28°C, with the subsequent addition of acetone following the same conditions as proposed by Khosravi-Darani and Zoghi [12] and adapted by Bastos et al. [9]. Glucose content was determined by the enzymatic glucose oxidase-peroxidase method (LABORLAB kit). Citric acid was estimated colorimetrically with a pyridine-acetic anhydride method using a commercial kit from IN VITRO® and HPLC Ultimate 3000 Dionex®, according to the method proposed by Pereira et al. [22].
Maximum productivity of citric acid and the specific production rates were calculated considering the elemental formula CH 1.72 O 0.55 N 0.17 of the fungi biomass as reported by Nielsen et al. [23], using biomass nitrogen determined by SHIMADZU® TOC-LCPN. Moreover, the citric acid yield obtained in the SSC cultures (Y P/S ) is estimated to glucose consumption and compared with the stoichiometric maximum, that is, 0.8 mol of produced carbon (citric acid) per mole of carbon of the consumed substrate [24].

SSC in Bench Packed-Bed Column Bioreactor
SSC was set up in a bench bioreactor (Fig. 1), with the inoculum condition was selected previously (Screening of Inoculum Condition). Bench packed-bed column (50-mm diameter and 400 mm in bed height) filled with the sterilized sugarcane bagasse (solid support) impregnated with vinasse and inoculum suspension (50% A. niger 50% T. reesei and 75% A. niger 25% T. reesei), with initial solid moisture of 80%, and continuous water-saturated air and 30°C temperature (thermal jacket).
Oxygen demand and the overall gas-liquid oxygen transfer coefficient (k L a) were evaluated by the mass balance in the columns, and the radial dissolved oxygen profile was estimated, respectively [25]. The calculation of oxygen profile concentration in the liquid film formed on the surface of the solid supports was done according to Thibault et al. [26].

Results and Discussion
Vinasse is used in the experiments as a moistening and nutrient solution of sugarcane bagasse particles. The C/N ratio of vinasse is around 20, with an average composition of total organic carbon (10,360 mg L −1 ), total nitrogen (502.7 mg L −1 ), zinc (0.69 mg L −1 ), copper Fig. 1 Schematic of bench lab-scale column bioreactor used in SSC of fungal consortium from sugarcane bagasse (adapted from [19]) (0.035 mg L −1 ), iron (14.5 mg L −1 ), and manganese (3.11 mg L −1 ). In addition, the bagasse had a total organic carbon content of 27.6 g 100 g −1 . Table 1 shows the production of citric acid in 4 days from bagasse impregnated with vinasse with different proportions between the fungi Aspergillus niger and Trichoderma reesei in "Raimbault Columns" bioreactors (PBR with 200-mm height and 30-mm diameter). For screening of inoculum condition, only A. niger produced the highest yield, followed by a fungal consortium with 50% of each fungus. In fact, A. niger is used for the industrial production of citric acid [13,24]. However, there is no significant difference between these two conditions and the condition of 75% A. niger and 25% T. reesei was observed. The obtained yield shows that the 50:50 fungal consortium yield a high concentration of citric acid by glucose, suggesting better use of the substrate due to the complementation of the hydrolytic enzymes of the fungi.
Since some part of the microbial citric acid produced is intracellular (mostly released out of the cells as citrate), the inoculum can cause high concentrations of the product. Thus, we obtained concentrations of 141.10 ± 56.101 mg L −1 on using inoculum suspension containing T. reesei; 1202.59 ± 54.86 mg L −1 on using A. niger inoculum and 568.97 ± 36.58 mg L −1 on using inoculum suspensions containing 50:50 consortium, respectively. Inoculum suspension of A. niger greatly affected the production of citric acid. The initial amount of citric acid precipitated from the inoculum is negligible. Thus, in such conditions, inoculum suspensions containing 50:50 consortium yielded the highest concentration of citric acid by SSC.
The highest concentrations of approximately 1000 mg L −1 of citric acid yield are obtained using 100 mm of bed height in 48 and 72 h (Fig. 2). The use of a column bioreactor helps in controlling temperature and forced aeration better and maintaining the optimal relationship between airflow and particle bed volume, which will help in the adequate development of fungi [21]. Thus, increased production of citric acid was observed using Raimbault Columns, which indicates the usage of this process in bench scale [9,10,14]. However, one limitation of this study is the bed height. We noticed that citric acid production is dependent on the bed height, especially in 48 h, suggesting a limitation in the oxygen transfer from heights greater than 100 mm. Therefore, bed height governs the phenomena of mass transfer and microbial growth in SSC in column bioreactors.
According to the axial glucose profiles (Fig. 3), a reduction in levels over time and at different bed heights is observed. However, at a bed height of 100 mm in 48 and 72 h, a high amount of citric acid is produced.
A considerable degree of microbial activity is observed in the first 24 h due to the consumption of the structural polysaccharides of the bagasse and the release of hexoses. In this case, a high amount of glucose is produced, which is used in other processes. Therefore, Raimbault Columns is proposed to scale up to bench PBR to accommodate both objectives in 113.25 ± 3.24 0.08 a single bioreaction system, and also economic feasibility should be considered. Previous studies prove that the production of citric acid by A. niger is dependent on the availability of oxygen [24]. Also, forced aeration is the main highlight in the packed-bed reactor; therefore, it is a suitable system for the large-scale production of citric acid [21]. The yield obtained using different inoculum conditions has been compared and showed in Table 2. The presence of T. reesei in the inoculum (50-50%) is responsible for the higher production of citric acid. Although A. niger generates a higher amount of citric acid, the release of glucose for this conversion seems to be more linked to the growth of T. reesei. Moreover, the yield on using this inoculum condition was higher than that reported by Campanhol et al. [10] and shown in Table 1. The PBR bench is the most feasible process as it supports aerobic microbial production. The maximum yield from glucose to citric acid (2.2 mg of citric acid per mg of glucose) was obtained at 100 mm of bed height in 48 and 72 h, suggesting that Fig. 2 Axial profiles of citric acid for SSF in bench packed-bed bioreactor. Inoculum 50% A. niger 50% T. reesei (a) and 75% A. niger 25% T. reesei (b) Fig. 3 Axial profiles of glucose for SSF in bench packed-bed bioreactor. Inoculum 50% A. niger 50% T. reesei (a) and 75% A. niger 25% T. reesei (b) maintaining solid moisture and, consequently, liquid film thickness, is important to maintain the efficiency of oxygen transfer in SSF.
Oxygen transfer in SSC depends on the moisture and thickness of the liquid film. Solid moisture is maintained over time in these conditions. As reported by Bastos et al. [9], bed height and airflow help in maintaining moisture in SSC. The moisture content can alter the liquid film thickness formed on the surface of the solid particles, directly affecting the global gas-liquid oxygen transfer coefficient (K L a). Thus, optimum and constant moisture helps in reaching the highest values of K L a, and therefore, it is important to maintain water content in solids, regardless of microbial activity. According to Arora et al. [21], only lower reactor volume allows the absence of free water in SSC. However, the absence of free water in the solid medium is observed in bench PBR [1,26].
The scaling up of batch bioreactors inevitably involves swelling. In these cases, the availability of oxygen at different bed heights is monitored in SSC [9]. These studies also show a relationship between the volume of the bioreactor and the airflow. Shorter bed heights have shorter residence time, which may limit the availability of oxygen. Also, the highest oxygen consumption (the largest difference between the values of the column inlet and outlet) can be observed at bed heights of 100 and 200 mm, especially in the period between 48 and 72 h, agreeing with the greater production of citric acid, as shown in Fig. 4.
The industrial production of citric acid is extremely dependent on aeration, and even short intervals of reduction in dissolved oxygen can cause irreversible losses. Aspergillus niger presents two respiratory models during the accumulation of citric acid [24]. Thus, the limitations of oxygen transfer in SSC can completely affect the production of microbial Table 2 Maximum yields glucose to citric acid (Y P/S ), maximum productivities (P rod max), and specific production rates (μ P ) for SSC in bench column bioreactor at different inoculum conditions Inoculum Y P/S max (mg citric acid mg glucose −1 ) P rod max (mg citric acid L −1 h −1 ) μ P (mg citric acid g biomass − metabolites. However, it is difficult to understand the interaction between fungal growth and physical mass transfer phenomena. The nature of the solid support used, its porosity and moisture content, and oxygen, moisture, and temperature gradients in column bioreactors are important for oxygen transfer [27,28]. In addition, due to the production of metabolic water, the initial value of moisture content in SSC rises in most cases, which changes the properties of solid support. However, moisture in the bagasse bed remains constant, indicating that the variations in oxygen demand (Fig. 4) are associated with the metabolic activity of the fungi. Figure 5 shows the characteristic profile of K L a as a function of the thickness of the liquid film present on the surface of the bagasse [25,26]. Similar conditions are observed for all bed heights, being calculated from experimental values (oxygen demand, particle bed volume, volumetric airflow) and assumed values (thickness variation, oxygen diffusivity in the liquid film).
According to Doran [29], critical K L a is estimated in which the minimum value to leading non-limiting oxygen conditions is around 0.75 s −1 , leading to an unfavorable situation starting at 150 μm of liquid film thickness. However, if the moisture content is maintained, variation in liquid film thickness can be neglected, keeping the K L a in the appropriate range.

Conclusions
This study concludes that the production of citric acid using bagasse with vinasse in bench packed-bed bioreactors can be improved by the optimization of the inoculum condition of the fungal consortium.
Availability of Data and Material (Data Transparency) All data were generated from experimental tests in the laboratory coordinated by the advisor.
Code Availability Not applicable.