Solid-state Fermentation Bioreactors Research Articles

Changes in vinegar quality and microbial dynamics during fermentation using a self-designed drum-type bioreactor.

The bioreactor based on solid-state fermentation technology has been developed for vinegar production, standardization of fermentation process and stabilization of vinegar quality. The microbial community diversity, and volatile compounds of six cultivars of vinegar samples fermented in a self-designed solid-state fermentation bioreactors were investigated using Illumina MiSeq platform and gas chromatography mass spectrometry (GC-MS) technology. The correlations between the richness and diversity of microbiota and volatile profiles, organic acids, as well as physicochemical indicators were explored by R software with the coplot package. The findings indicated that Acetobacter, norank-c-Cyanobacteria, and Weissella played key roles during fermentation process. Norank-f-Actinopolyporaceae, norank-c-Cyanobacteria, Pediococcus, and Microbacterium had significant correlations with the physicochemical characteristics. The most common bacterial species were associated with a citric acid content, whereas the least number of bacterial species correlated with malic acid content. Findings could be helpful for the bioreactor optimization, and thus reaching the level of pilot scale and industrialization.

Solid-state fermentation-based enzyme-assisted extraction of eicosapentaenoic acid-rich oil from Nannochloropsis sp

Enzymatic treatment of microalgal biomass is a promising approach for extraction of microalgal lipid, but high cost of commercially sourcing enzyme is a major drawback in industrial implementation. Present study involves extraction of eicosapentaenoic acid-rich oil from Nannochloropsis sp. biomass using low cost cellulolytic enzymes produced from Trichoderma reesei in a solid-state fermentation bioreactor. Maximum total fatty acid recovery of 369.4 ± 4.6 mg/g dry weight (total fatty acid yield of 77%) was achieved in 12 h from the enzymatically treated microalgal cells, of which the eicosapentaenoic acid content was 11%. Sugar release of 1.70 ± 0.05 g/L was obtained post enzymatic treatment at 50 °C. The enzyme was reused thrice for cell wall disruption without compromising on total fatty acid yield. Additionally, high protein content of 47% in the defatted biomass could be explored as a potential aquafeed, thus enhancing the overall economics and sustainability of the process.

Utilization of waste pine needles for the production of cellulolytic enzymes in a solid state fermentation bioreactor and high calorific value fuel pellets from fermented residue: Towards a biorefinery approach

The present study addresses valorisation of abundantly available, low-cost pine needle (PN) waste into cellulolytic enzymes and high calorific value (CV) fuel pellets (FP). Initial studies in flasks involving hot water steam pre-treatment (HWSP) using Aspergillus niger MTCC 9652 for 5 days resulted in an increase in Cellulase, Endoglucanase and Xylanase activities compared to the untreated pine needles (UPN). Fermentation experiments were scaled-up in a 6-L Solid State fermentation (SSF) packed bed reactor (PBR) at different bed heights (BH) using UPN. The maximum cellulase and xylanase yields were achieved as 9.97 ± 0.27 FPU g-ds−1 and 8276 ± 254 IU g-ds−1, respectively, at 72 h using UPN in the PBR. The fermented residue showed 53.5% consumption of holocellulose, a decrease in crystallinity index to 29.5% from 46%, with an increase in total lignin content of 45.9%. The fermented residue was processed for pellet production, resulting in a higher CV (19.13 ± 0.69 MJ kg-1) than the pellet made from unfermented PN (17.63 ± 0.66 MJ kg−1), along with enhanced compressive strength. Sulphur content (0.14%) was below the permissible limit of SOx emission for fuels with ash content less than 5%. Therefore, the present study represents a novel and sustainable biorefinery approach for PN valorisation.

Rapid and mass production of biopesticide Trichoderma Brev T069 from cassava peels using newly established solid-state fermentation bioreactor system

Converting agricultural waste into value-added biopesticides to replace chemical pesticides for plant protection is a good alternative for environmental sustainability and resource recycling. In this study, five tropical wastes (cassava peels, banana pseudostem, coconut shell, sugarcane bagasse, and pineapple peels) were screened as substrates for the rapid production of biopesticide Trichoderma Brev T069. Five single tests and a Box-Behnken design (BBD) with response surface methodology were used to optimize the culture conditions to improve the spore yield. The results showed that cassava peel was the optimal solid fermentation substrate, and the optimization enabled a spore yield of 9.31 × 109 spores/g at 3rd day, which was equal to 93.19% of spore yield obtained at 5th day (9.99 × 109 spores/g). A newly packed-bed bioreactor with agitation and ventilation system was developed and used to expand the production that 250 kg of biopesticide (2.89 × 109 spores/g) could be available on the 3rd day. A pot experiment indicated that the biopesticide T. Brev T069 obtained under this production system, when applied at 1 × 107 spores/g of soil had a 64.65% biocontrol efficiency on banana fusarium wilt. This study provides a practical solution for turning a tropical waste into an effective biopesticide which can prevent banana wilt disease, thereby helping to reduce disease management cost and overcome environmental hazards caused by synthetic pesticides.

Biotechnological potential of Zymotis-2 bioreactor for the cultivation of filamentous fungi.

A new prototype of Solid-State Fermentation Bioreactor, namely "Zymotis-2 ", was developed to produce fungal spores. A fermentation process for fungal spores, and hydrolase enzymes (endo and exoglucanases, amylases) production by Trichoderma asperellum DWG3, Aspergillus niger G131 and Beauveria bassiana was scaled-up from flasks and glass Raimbault column packed with 20g of solid substrates (dry weight) to 5kg of solid substrate by using the new Zymotis-2 bioreactor. Fungi strains growth using a mix of vine shoots, wheat bran, and olive pomace was tested under similar experimental conditions in Zymotis-2 bioreactor, column bioreactor and flasks in a parallel fermentation system. Overall, significant spore production on Zymotis-2 bioreactor was obtained, achieving 22.01 ±0.01×109 spores/g DM 16.30 ±0.07 × 109 spores/g DM, and 3.30 ±0.07 × 109 spores/g DM for B. bassiana, T. asperellum DWG3, and A. niger G131, respectively. Forced aeration increased the endoglucanases, exoglucanases and amylases activities for T. asperellum DWG3 but B. bassiana and A. niger G131 were affected negatively by the aerated process, showing the lowest enzyme activities. In conclusion, a high yield of spores was obtained at 137 h of cultivation time, confirming the validity of the new Zymotis-2 bioreactor to produce virulent spores at low cost by T. asperellum, B. bassiana and A. niger G131.

Bacterial Whole Cells Synthesis of Whisky Lactones in a Solid-State Fermentation Bioreactor Prototype

Agro-industrial side streams such as oilseed cakes were used as a medium in solid-state fermentation (SSF) for microbial oxidation of anti- and syn-3-methyl-octane-1,4-diols to obtain corresponding trans- and cis-whisky lactones. In preliminary screening transformations, a wide range of whole bacterial cells were tested on the basis of oxidation activity, which is rarely described in the literature, in contrast to the widely studied lipolytic activity on SSF. Among the different oil cakes tested, biotransformations carried out on linseed cake were characterized by the highest conversion and stereoselectivity. Several preparative-scale oxidations performed in a self-constructed SSF bioreactor catalyzed by Rhodococcus erythropolis DSM44534, Rhodococcus erythropolis PCM2150 and Gordonia rubripertincta PCM2144 afforded optically active trans-(+)-(4S,5R), cis-(+)-(4R,5R) and cis-(-)-(4S,5S) isomers of whisky lactones, respectively. Bacteria of the Rhodococcus, Gordonia, Dietzia and Streptomyces genera carried out transformations with complete conversion after three days. Various extraction methods were applied for the isolation of the products, and among them, the combination of steam distillation with simple extraction were the most efficient. Biotransformations were conducted under precise control of conditions in a bioreactor based on a Raspberry Pi Zero W. The proposed low-cost (ca. USD 100) bioreactor is a standalone system that is fully autoclavable and easy to use.

A model-based strategy for scaling-up traditional packed-bed bioreactors for solid-state fermentation based on measurement of O2 uptake rates

• O 2 uptake profiles are used to obtain the kinetics of metabolic heat generation. • The kinetics are incorporated in a two-phase heat and mass transfer model. • The model is validated with temperature profiles in a pilot-scale bioreactor. • The model is then used to explore packed-bed design and operation at large scale. • The approach is superior to previously published scale-up strategies for packed-beds. Model-based strategies for scaling up traditional packed-bed bioreactors used in solid-state fermentation have been available for over two decades. However, these strategies are theoretical, not being based on experimental data. A recently proposed strategy did use experimental data; however, it was based on data from a slow-growing fungus and also considered heat transfer in both the axial and radial directions, ignoring the fact that radial heat transfer is negligible in large-scale packed-bed bioreactors. The current work demonstrates a scale-up strategy based on a model describing heat transfer only in the axial direction. The strategy is demonstrated for the cultivation of Aspergillus niger , a fast-growing filamentous fungus. In the model, the kinetics of production of metabolic heat are based on an experimental time-course profile for the O 2 uptake rate. The model is validated by comparing its predictions for temperature profiles in the bed with temperatures measured experimentally in a pilot-scale bioreactor. Finally, the validated model is used to explore strategies for optimizing the performance of large-scale traditional packed-bed bioreactors. We conclude that a key strategy is to decrease the temperature of the inlet air during periods of peak heat generation.

Estimation of heat and mass transfer coefficients in a pilot packed-bed solid-state fermentation bioreactor

Although mathematical models of heat and water transfer in packed-bed bioreactors for solid-state fermentation have been available for several decades, there have been few efforts to determine the heat and mass transfer coefficients between the solids and air phases experimentally. In the current work, we undertook drying, cooling and heating experiments in a pilot packed-bed bioreactor, using a solid substrate prepared as for a fermentation, but uninoculated. We then used the solids-air heat and mass transfer coefficients as fitting parameters to adjust a heat and mass transfer model to temperature data obtained at different bed heights. The model fit the data well, but only if the air flow rate was allowed to vary as a function of position, indicating that the air flow in the bed was not uniform. In a sensitivity analysis, the air flow rate had a greater effect on the predicted temperature profiles than did the solids-air heat and mass transfer coefficients themselves. However, despite the lesser effect of these coefficients, it is not possible to treat the solids and air phases as being in thermal and hydric equilibrium. Rather, it is essential to use models that recognize the solids and air as separate phases and use expressions based on driving forces to describe evaporation and convective heat removal. The experimentally determined heat and mass transfer coefficients can be substituted into a mathematical model that also describes microbial growth and metabolic heat production and this model can be used to guide the scale-up from our pilot bioreactor to larger scales.

CFD simulation of a packed-bed solid-state fermentation bioreactor

In recent years, Solid-State Fermentation (SSF) has shown much promise for the development of bioprocesses and products. SSF involves the growth of microorganisms within a bed of moist solid particles permeated by a continuous gas phase and a minimum of visible water. SSF offers potential advantages over submerged culture: since the concentrations of products are often higher, smaller bioreactors can be used, reducing operational costs. However, there is a major challenge in obtaining adequate heat and mass transfer when this fermentation method is used at large scales. Mathematical models and computer simulations are useful tools for designing strategies to overcome this challenge; use of these tools can reduce costs of experimental development programs at pilot-scale and production scale, by reducing the number of fermentation experiments required. In the current work, we used the commercial CFD software ANSYS FLUENT® 16.0 to develop a mathematical model for heat and mass transfer in a pilot-scale packed-bed bioreactor. The model was used to simulate two different experiments that had been carried out previously in the bioreactor: first, the cooling of a bed of soybeans and, second, the heating of a bed composed of a mixture of wheat bran and sugarcane bagasse. The simulations considered the dynamics of airflow in the porous substrate bed and the non-equilibrium transfer of heat and moisture between the solid and gas phases. The second simulation considered a heterogeneous distribution of porosity within the substrate bed. Even though the two experiments were quite different, the same mathematical model was able to represent the temperature profiles observed experimentally. In the second simulation, the average temperature difference between the experimental and predicted values was 0.07°C. A third simulation was done for the growth of the filamentous fungus Aspergillus niger in SSF, with the predictions being compared to the results of a traditional mathematical model based on differential equations. Our work provides the basis for the development of a suitable and reliable mathematical model for testing operating conditions and control strategies for large-scale cultivation of microorganisms in SSF bioreactors.

Design and Operation of a Pilot-Scale Packed-Bed Bioreactor for the Production of Enzymes by Solid-State Fermentation.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

Strategies for scaling-up packed-bed bioreactors for solid-state fermentation: The case of cellulolytic enzymes production by a thermophilic fungus

Mathematical models can be useful to predict the behavior of particulate systems, as it is the case of packed-bed bioreactors (PBBs) used for solid-state fermentation (SSF). The models also simplify the processes scale-up, and depending on the accuracy of the model, critical parameters can be predicted, as temperature, moisture content and maximum bed height. In the current paper, mathematical models and simulations were used to predict the optimal conditions for enzymes production by means of SSF in PBBs. Two models available in literature were applied: a recently proposed two-phase and two-dimensional (2-D) model and a modified Damköhler number (Dam) approach, considering maximum temperatures of the bed and maximum bed height to allow good production of enzymatic activities by the fungus Myceliophtora thermophila I-1D3b. Based on simulation results, experiments in bench and pilot-scale PBBs were performed and analyzed. By comparing experimental and simulated results of temperatures, the 2-D model showed to be more accurate than Dam approach. Despite of the temperature increase in pilot-scale PBB, the bioreactor scale-up with the fungus M. thermophila in substrate composed of sugarcane bagasse (SCB) and wheat bran (WB) (weight proportion 7:3) could be considered as feasible for cellulolytic and xylanolytic enzymes provision, for instance, for second generation ethanol production chain, although issues related to airflow distribution and substrate shrinkage in pilot-scale PBB must still be further overcome.

A review of bioreactor technology used for enzymatic hydrolysis of cellulosic materials

Cellulases are costly, a principal challenge of enzymatic hydrolysis of cellulosic materials for bioethanol production. For an efficient cellulase production, fungi are preferred over bacteria due to their permeation capability and versatile substrate consumption. Some limitations in the enzymatic hydrolysis step prevent the process to be economically feasible. Different strategies have been investigated to overcome these limitations, including genetic engineering, enzyme recycling, high solid loadings, pretreatment technologies, supplementation of cellulases with additives and application of nanomaterials for improving the thermal and pH stability of cellulases. Several studies have been performed in various bioreactors with the target to procure higher yields of glucose in the enzymatic hydrolysis step. The key factors for designing a bioreactor include efficient mixing, sufficient mass transfer, low shear stress, low foaming problems and low consumption of water and energy. In this scenario, different bioreactor configurations, including stirred tank bioreactor, horizontal rotating tubular bioreactor, airlift bioreactor, membrane bioreactor, reciprocating plate bioreactor, solid-state fermentation bioreactors have been reviewed for cellulase production with the aim to investigate main factors for designing a bioreactor.

Aroma compounds production by solid state fermentation, importance of in situ gas-phase recovery systems.

Flavour and fragrance compounds are extremely important for food, feed, cosmetic and pharmaceutical industries. In the last decades, due to the consumer's increased trend towards natural products, a great interest in natural aroma compounds has arisen to the detriment of chemically synthesised ones. Recently, solid state fermentation (SSF) has been applied in the production of many metabolites. Aroma compounds can be produced by SSF with a higher yield compared to submerged fermentation (SmF). In SSF processes, aroma compounds can be produced in the solid matrix or in the headspace, but they can be lost or stripped when aeration is required. This review focuses on the production of aroma compounds by SSF processes with a special highlight on in situ systems to recover the volatiles released in the gaseous phase and stripped due to aeration. Following a brief presentation of specificities of SSF processes concerning the choice of microorganisms and the solid matrix used for the production of aroma compounds, bioreactor aspects, factors affecting production of aroma compounds and in situ gas phase aroma recovery systems in aerated SSF bioreactors are discussed.

Effect of stirring on growth and cellulolytic enzymes production by Trichoderma harzianum in a novel bench-scale solid-state fermentation bioreactor

A novel bench-scale stirred bioreactor for solid-state fermentation was used to determine the effect of the stirring rate on growth and enzymes production by Trichoderma harzianum PBLA. Lab-scale static tubular bioreactors were first used to assess the effect of bioreactor diameter on heat accumulation, growth, and production of cellulases and xylanases. The increased diameters (1.8–4.2 cm) led to increases in temperature up to 36 °C (at a rate of 1.08 °C/cm), which negatively affected the growth and enzyme production. Afterward, in the bench-scale bioreactor operated at rates up to 3.0 rpm, maximum xylanases production (107 ± 0.3 U/g dm) was attained at rates of 0.5 and 1.0 rpm, reaching a maximum of 34 ± 0.3 °C. Cellulases production was reduced (up to 79%) due to stirring. Therefore, the production of xylanases by T. harzianum can be performed in this cross-flow stirred SSF bioreactor at rates up to 1.0 rpm, avoiding heat accumulation and damage on metabolic activity.

Pectinolytic enzymes-solid state fermentation, assay methods and applications in fruit juice industries: a review.

A plethora of solid substrates, cultivation conditions and enzyme assay methods have been used for efficient production and estimation of polygalacturonase and pectin methylesterase enzymes. Recent developments in industrial biotechnology offer several opportunities for the utilization of low cost agro-industrial waste in Solid State Fermentation (SSF) for the pectinolytic enzyme production using fungi. Fruit waste mainly citrus fruit waste alone and along with other agro-industrial waste has been explored in SSF for enzyme production. Agro-industrial waste, due to the economic advantage of low procuring cost has been employed in SSF bioreactors for pectinolytic enzyme production. Acidic pectinases produced by fungi are utilized especially in food industries for clarification of fruit juices. This review focuses on the recent developments in SSF processes utilizing agro-industrial residues for polygalacturonase and pectin methylesterase production, their various assay methods and applications in fruit juice industries.

Oxygen uptake rate as a tool for on-line estimation of cell biomass and bed temperature in a novel solid-state fermentation bioreactor.

Direct measurement of cell biomass is difficult in a solid-state fermentation (SSF) process involving filamentous fungi since the mycelium and the solid substrate are often inseparable. However, respiratory data are rich in information for real-time monitoring of microbial biomass production. In this regard, a correlation was obtained between oxygen uptake rate (OUR) and biomass concentration (X) of Rhizopus oryzae MTCC 1987, during phytase production, in an intermittently mixed novel SSF bioreactor. To obtain the correlation, various models describing sigmoidal growth were tested, namely the logistic, Gompertz, Stannard, and Schnute models. Regression analysis of experimental results, at different operating conditions of inlet air flow rate and relative humidity suggested that OUR and X were correlated well by the logistic model (R2 > 0.90). To corroborate the use of respiratory data for on-line measurement of metabolic activity, OUR was related to metabolic heat generation rate (Rq), and the logistic model was found to satisfactorily correlate Rq and X as well. The model parameter, YQ/X, when substituted into a heat transfer design equation, along with the values of other parameters and operating variables, gave reliable estimates of bed temperature. The correlations developed in the present study, between respiratory activity and biomass concentration may be extended on to other SSF processes for further validation and real-time monitoring of cell biomass and bed temperature.

Effect of mixing events on the production of a thermo-tolerant and acid-stable phytase in a novel solid-state fermentation bioreactor

Abstract Mixing constitutes a critical design parameter in solid-state fermentation (SSF) bioreactor and its effect on heat and water transport, and microbial growth in substrate bed can significantly influence overall productivity. Effect of mixing events on the production of a thermo-tolerant and acid stable phytase, by Rhizopus oryzae, was studied in a novel SSF bioreactor, using optimized growth medium containing wheat bran and linseed oil cake (1:1) as main substrate. A critical mixing phase was identified, in the absence of which fungal growth led to the onset of heat accumulation and subsequent bed drying. The tensile strength of hyphal bonds between two substrate particles, at this critical phase, was estimated and related to the mixing intensity in the bioreactor that resulted in an optimum working value of 15 rpm. Effect of mixing time on bioreactor performance was also investigated where a 3 min mixing duration, at every 6 h, increased biomass and phytase productivity to 2.2- and 4.5-fold, respectively, in comparison to packed bed bioreactor (PBR). The proposed bioreactor system with intermittent mixing gave superior performance than PBR and tray bioreactor, in terms of maximum bed temperature, axial bed temperature and biomass gradient, average bed moisture content, biomass and phytase productivity.

Surface imaging of the filamentous fungus Penicillium simplicissimum growing in a solid-state fermentation system

A comparative study on the lipase-producing fungus Penicillium simplissicimum, grown on a tray type solid-state fermentation (SSF) bioreactor, was performed using stereoscopy, focus Z-stacking stereoscopic images, field emission scanning electron microscopy (FE-SEM) and environmental scanning electron microscopy (ESEM) to better characterize the morphology of filamentous fungi in SSF and their distribution over the solid matrix. The imaging of live fungal samples using a stereomicroscope with focus Z-stacking showed differences in colonization between a static SSF and an intermittent agitated SSF. A comparison of the stereomicroscopy, hi-vacuum and environmental scanning electron microscopy results obtained using different protocols for sample processing showed that fixation with osmium tetroxide vapor and subsequent hydrated imaging is the best combination of sample preparation and imaging conditions for keeping the arrangement of the aerial hyphae and conidia morphology closer to the natural state. These combined methodologies can be applied in the SSF of fungal growth to characterize the formation of conidiophores over time, the conidia morphology and the spatial organization after their release from conidiophores. Mycelium colonization over the matrix, which is an important characteristic related to the production of different biotechnological products, could be observed and provide more knowledge about fungal physiology behavior during SSF.

Fluid Dynamics Simulation of a Pilot-Scale Solid-State Fermentation Bioreactor

Solid-state fermentation (SSF) has potential advantages for the production of certain biotechnological products, due to its large volumetric productivity and low operating costs. However, there are major challenges in obtaining adequate heat and mass transfer when this fermentation method is used at large scales. Mathematical models and computer simulations are useful tools for designing strategies to overcome these challenges, given the cost of large-scale fermentation experiments. In the current work, we used the commercial CFD software ANSYS FLUENT® 16.0 to develop a mathematical model for heat and mass transfer in a pilot-scale packed-bed bioreactor. The model takes into account the dynamics of airflow in the porous substrate bed and the lack of thermal and moisture equilibrium between the solid and gas phases. The permeability parameters were obtained from pressure drop measurements made in the pilot-scale bioreactor during a fermentation in which Aspergillus niger was grown on a mixture of wheat bran and sugar cane bagasse. The CFD simulations were able to represent the temperature and moisture profiles observed experimentally during the initial heating of the substrate bed by the process air. A parametric analysis was also performed, in order to understand better the physical dynamics of the system. Our work provides the basis for the development of a robust and reliable model for testing operating conditions and control strategies for large-scale cultivation in SSF bioreactors.

Hygroscopic properties of solid agro-industrial by-products used in solid-state fermentation

One of the main drawbacks in operating solid-state fermentation (SSF) bioreactors is the reduction in moisture content (MC) of the solid phase due to microbiological and physicochemical issues. Therefore, heat and mass transfer models require hygroscopic information concerning the solid substrate. This contribution addresses the study of the sorption isotherms of sugarcane bagasse (SCB) and wheat bran (WB) obtained using the static gravimetric method, with temperature and water activity as the controlled variables. The Oswin model was selected to construct the sorption isotherm for SCB, and the Halsey model, modified in the present study, for WB. The Bates and Watts curvature measurements and the Box parameter bias were used to analyze the nonlinear behavior and assess the goodness of the fitting process. Fermentative assays were carried out in glass flasks for the production of endoglucanase employing the fungus Myceliophthora thermophila I-1D3b, controlling the environmental water activity, in order to assess the influence of the loss of moisture from the solid phase on fungal metabolic activity.