Fungal Solid-state Fermentation Research Articles

Effect of feeding of 10 % prefermented feed on fatty acid profile and oxidation changes in chicken breast meat

Abstract The aim of this work was to evaluate the effect of addition of 10 % fermented feed (FF) into commercial broiler feed on fatty acid profile, chemical composition and oxidative stability of chicken breast meat fat. FF was prepared by the process of fungal solid-state fermentation. Application of FF into the commercial feed mixtures resulted in increased content of gamma-linolenic and oleic acid in broiler breast meat (P < 0.05). Oxidative stability of meat fat was significantly higher in experimental broilers group fed diet with the addition of 10% FF (P < 0,05) after storage period of samples for seven days at temperature of 4 °C.

Closing the loop of cereal waste and residues with sustainable technologies: An overview of enzyme production via fungal solid-state fermentation

Abstract The circular economy is one of the leading sustainable development strategies that can help utilize waste to produce new products. Cereal food waste and residues represent a significant resource of million tonnes globally and a feasible feedstock for enzyme production via environmentally-friendly solid-state fermentation (SSF). This study presents a targeted review of 487 examples of quantified enzyme production from 209 studies reported on Science Direct. The collection of data enabled the identification of the key substrates and enzymes for potential production pathways. The main substrates were wheat (53 experiments), rice (22 experiments), and corn (22 experiments), while the most common enzymes reported were xylanase, CMCase, FPase with 92, 83, and 61 examples, respectively. Their activities showed a wide range with xylanase activities 7-10 000 U/gds, CMCase activities 1.4-950 U/gds, and FPase activities 1.7-438 U/gds. Other enzymes reported were β-glucosidase, Laccase, β-xylosidase. Both single substrates and mixed substrates were included, and several mixtures showed a synergistic effect for higher enzyme activities. Xylanase from wheat bran, rice straw, and their combination showed great potential for reaching high activity and promising revenues. Scale-up experiments were reported and demonstrated increased production of enzymes as a crucial step towards industrial application. Identified improvements in transparency and knowledge sharing for cross-disciplinary research are: i) harmonized enzyme names ii) expression of enzyme activity per mass unit specifying moisture content as found in 52% of studies. Only the results using the same unit (or enabling unit conversion) can be compared and used to identify the most promising production.

Valorisation of fungal hydrolysates of exhausted sugar beet pulp for lactic acid production.

Exhausted sugar beet pulp pellets (ESBPP) were used as raw material for lactic acid (LA) fermentation. The enzymatic hydrolysis of ESBPP was performed with the solid obtained after the fungal solid-state fermentation of ESBPP as a source of hydrolytic enzymes. Subsequently, a medium rich in glucose and arabinose was obtained, which was used to produce LA by fermentation. For LA production, two Lactobacillus strains were assayed and the effects of the supplementation of the hydrolysate with a nitrogen source and the mode of pH regulation of the fermentation were investigated. Moreover, a kinetic model for LA fermentation by Lactobacillus plantarum of ESBPP hydrolysates was developed. L. plantarum produced a LA concentration 34% higher than that produced by L. casei. The highest LA concentration (30g L-1 ) was obtained with L. plantarum when the hydrolysate was supplemented with 5g L-1 yeast extract and the pH was controlled with CaCO3 . The concentration of acetic acid differed depending on the concentration of CaCO3 added, producing its maximum value with 27g L-1 CaCO3 . The proposed kinetic model was able to predict the evolution of substrates and products depending on the variation of the pH in the hydrolysate, according to the amount of CaCO3 added. ESBPP can be revalorised to produce LA. A pure LA stream or a mixture of LA and acetic acid, depending on the pH control method of the fermentation, can be produced. Thus, this control is of great interest depending on the destination of the effluent. © 2020 Society of Chemical Industry.

Animal Fat as a Substrate for Production of n-6 Fatty Acids by Fungal Solid-State Fermentation.

The method of solid-state fermentation (SSF) represents a powerful technology for the fortification of animal-based by-products. Oleaginous Zygomycetes fungi are efficient microbial cell factories used in SSF to valorize a wide range of waste and rest cereal materials. The application of this fermentation technique for utilization and biotransformation of animal-based materials represents a distinguished step in their treatment. In this study, for the first time, the strain Umbelopsis isabellina CCF2412 was used for the bioconversion of animal fat by-products to the fermented bioproducts enriched with n-6 polyunsaturated fatty acids, mainly γ-linolenic acid (GLA). Bioconversion of both cereals and the animal fat by-product resulted in the production of fermented bioproducts enriched with not just GLA (maximal yield was 6.4 mg GLA/g of fermented bioproduct), but also with high yields of glucosamine. Moreover, the fermentation on the cornmeal matrix led to obtaining bioproduct enriched with β-carotene. An increased amount of β-carotene content improved the antioxidant stability of obtained fermented bioproducts. Furthermore, the application of Fourier-transform infrared spectroscopy for rapid analysis and characterization of the biochemical profile of obtained SSF bioproducts was also studied.

Conversion of Exhausted Sugar Beet Pulp into Fermentable Sugars from a Biorefinery Approach

In this study, the production of a hydrolysate rich in fermentable sugars, which could be used as a generic microbial culture medium, was carried out by using exhausted sugar beet pulp pellets (ESBPPs) as raw material. For this purpose, the hydrolysis was performed through the direct addition of the fermented ESBPPs obtained by fungal solid-state fermentation (SSF) as an enzyme source. By directly using this fermented solid, the stages for enzyme extraction and purification were avoided. The effects of temperature, fermented to fresh solid ratio, supplementation of fermented ESBPP with commercial cellulase, and the use of high-solid fed-batch enzymatic hydrolysis were studied to obtain the maximum reducing sugar (RS) concentration and productivity. The highest RS concentration and productivity, 127.3 g·L−1 and 24.3 g·L−1·h−1 respectively, were obtained at 50 °C and with an initial supplementation of 2.17 U of Celluclast® per gram of dried solid in fed-batch mode. This process was carried out with a liquid to solid ratio of 4.3 mL·g−1 solid, by adding 15 g of fermented solid and 13.75 g of fresh solid at the beginning of the hydrolysis, and then the same amount of fresh solid 3 times every 2.5 h. By this procedure, ESBPP can be used to produce a generic microbial feedstock, which contains a high concentration of monosaccharides.

Enhanced antioxidant activity of okara through solid state fermentation of GRAS Fungi

Abstract The enhancement of antioxidant activity of fermented okara was investigated using fungal solid-state fermentation. The initial fungal concentration and water activity were found to influence the enhanced antioxidant capacity of fermented okara. Extracellular enzymes, especially proteases and carbohydrases might facilitate the hydrolysis of okara protein that yielded antioxidative amino acids and peptides, as well as the release of isoflavones-derived components. Fermentation of okara with R. oligosporus with 1.04×10-3 gDCW/gokara as initial concentration, water activity of 0.931 was found to yield the highest antioxidant strength. Whereas for A. Oryzae, 1.6×10-3 gDCW/gokara was optimum as the initial concentration with water activity of 0.913. With these optimum conditions, solid-state fermentation of okara could enhance the antioxidant activity up to 2.6- and 5.5-fold, respectively for R. oligosporus and A. oryzae. Besides having an enhanced antioxidant activity, the fermented okara also had higher protein content, amino nitrogen content, and protein digestibility.

Discoloration of rhodamine b dye by white-rot fungi in solid bleached sulfate paperboard coated with polyethylene terephthalate: scale-up into non-sterile packed-bed bioreactor

Treatment of dye-contaminated effluents and proper disposal of solid waste from the processing industry has been intensely studied. The use of basidiomycetes fungi has been highlighted over conventional treatments using green technologies. This work presents the first scale-up of a rhodamine B discoloration under non-sterile conditions using solid bleached sulfate (SBS) paperboard coated with polyethylene terephthalate (PET) by filamentous fungi. The dye discoloration was conducted first in agar plates and then in flasks and packed-bed bioreactor with SBS/PET. The fungus Agaricus brasiliensis (EF 03) presented the best dye discoloration results (42.94 ± 0.64) in agar plates. Optimization studies (flasks) of the culture medium showed a higher percentage of discoloration (93.65 ± 4.60) by A. brasiliensis (EF 03) when cultivated in solid-state fermentation with SBS/PET waste and Socarean medium and sodium nitrate variables. The pilot packed-bed bioreactor scaled-up and optimized medium were successful in discoloring of rhodamine B. The fungal population, later identified as the genera Agaricus and Phlebia by rRNA gene sequencing approach, was predominant up to 30 days and the highest laccase peaks were observed in 20 days (198.44 IU L−1) and 45 days (203.47 IU L−1). This packed-bed bioreactor operated with filamentous fungi in solid-state fermentation proved suitable for the discoloration of rhodamine B dye for 51 days, for repeated fed-batch under non-sterile conditions.

Beyond Enzyme Production: Solid State Fermentation (SSF) as an Alternative Approach to Produce Antioxidant Polysaccharides

Solid state fermentation (SSF) is a sustainable process that uses low amounts of water and transforms plant-based agro-industrial residues into valuable products such as enzymes, biofuels, nanoparticles and other bioactive compounds. Many fungal species can be used in SSF because of their low requirements of water, O2 and light. During SSF, plant-based wastes rich in soluble and insoluble fiber are utilized by lignocellulolytic fungi that have enzymes such as lignases, celullases or hemicelullases that break fiber hard structure. During the hydrolysis of lignin, some phenolic compounds are released but fungi also synthetize bioactive compounds such as mycophenolic acid, dicerandrol C, phenylacetates, anthraquinones, benzofurans and alkenyl phenols that have health beneficial effects such as antitumoral, antimicrobial, antioxidant and antiviral activities. Another important group of compounds synthetized by fungi during SSF are polysaccharides that also have important health promoting properties. Polysaccharides have antioxidant, antiproliferative and immunomodulatory activities as well as prebiotic effects. Fungal SSF has also proved to be a process which can release high contents of phenolics and it also increases the bioactivity of these compounds.

From stale bread and brewers spent grain to a new food source using edible filamentous fungi

ABSTRACT By-products from the food sector with a high load of organic matter present both a waste-handling problem related to expenses and to the environment, yet also an opportunity. This study aims to increase the value of stale bread and brewers spent grain (BSG) by re-introducing these residues to the food production chain by converting them to new protein-enriched products using the edible filamentous fungi Neurospora intermedia and Rhizopusoryzae. After 6 days of solid state fermentation (at 35°C, with a95% relative humidity and moisture content of 40% in the substrate) on stale bread, a nutrient-rich fungal-fermented product was produced. The total protein content, as analyzed by total amino acids, increased from 16.5% in stale sourdough bread to 21.1% (on dry weight basis) in the final product with an improved relative ratio of essential amino acids. An increase in dietary fiber, minerals (Cu, Fe, Zn) and vitamin E, as well as an addition of vitamin D2 (0.89 µg/g dry weight sample) was obtained compared with untreated stale bread. Furthermore, addition of BSG to the sourdough bread with the aim to improve textural changes after fermentation showed promising outcomes. Cultivation of N. intermedia or R. oryzae on stale sourdough bread mixed with 6.5% or 11.8% BSG, respectively, resulted in fungal-fermented products with similar textural properties to a commercial soybean burger. Bioconversion of stale bread and BSG by fungal solid state fermentation to produce a nutrient-enriched food product was confirmed to be a successful way to minimize food waste and protein shortage.

Assessment of fungal and thermo-alkaline post-treatments of solid digestate in a recirculation scheme to increase flexibility in feedstocks supply management of biogas plants

Agricultural biogas plants can suffer occasional feedstock shortages (poor harvest, storage …) and recirculation of solid digestate (SD) into digester has been identified as a simple way to offset methane production loss from these situations. Calculations show that recirculation of SD could offset for losses in plant methane production by up to 2.4%. In that context, two post-treatments were evaluated to enhance residual potential methane of agricultural SD. Effect of fungal solid state fermentation (SSF) of SD on subsequent methane production has never been explored before. It was hypothesized that: (i) ligninolytic fungi would be able to specifically use the complex fraction of SD for their growth and (ii) energy generation from the subsequent anaerobic digestion of the colonized SD will be enhanced. However, experiments showed that thermo-alkaline treatment of SD (used as alkalinization and sterilization process) and a high spawn level (20% w/w) were necessary to perform fungal SSF. Besides, the observed fungal activities on SD did not target specifically the most complex fractions. This led to uncontrolled organic matter losses and subsequent decreases of biodegradability and methane yield of SD (up to 50%). Therefore, fungal SSF of SD before its recirculation into biogas plants appeared not to be a viable option. Only thermo-alkaline treatment (CaO 2% w/w and 121 °C 30 min) enhanced methane yield of SD by 13% and decreased its complex fraction by 25%. Further studies on optimization of this post-treatment may enhance efficiency of SD recirculation strategy to offset plant methane production losses.

Fungal Laccase Production from Lignocellulosic Agricultural Wastes by Solid-State Fermentation: A Review.

Laccases are copper-containing oxidase enzymes found in many fungi. They have received increasing research attention because of their broad substrate specificity and applicability in industrial processes, such as pulp delignification, textile bleaching, phenolic removal, and biosensors. In comparison with traditional submerged fermentation (SF), solid-state fermentation (SSF) is a simpler technique for laccase production and has many advantages, including higher productivity, efficiency, and enzyme stability as well as reduced production costs and environmental pollution. Here, we review recent advances in laccase production technology, with focus on the following areas: (i) Characteristics and advantages of lignocellulosic agricultural wastes used as SSF substrates of laccase production, including detailed suggestions for the selection of lignocellulosic agricultural wastes; (ii) Comparison of fungal laccase production from lignocellulosic substrates by either SSF or SF; (iii) Fungal performance and strain screening in laccase production from lignocellulosic agricultural wastes by SSF; (iv) Applications of laccase production under SSF; and (v) Suggestions and avenues for future studies of laccase production by fungal SSF with lignocellulosic materials and its applications.

Assessment of fermented broken rice extracts for their potential as functional ingredients in cosmeceutical products

Rice-derived bioactive compounds are now widely used as active ingredients in health foods, pharmaceutical and cosmeceutical products and as food additives. Polyphenolic compounds, gamma oryzanol, and vitamin E are among the bioactive constituents in rice that have important antioxidant, anti-aging, and anti-pigmentation properties among others. In this present study, broken rice was subjected to solid-state fermentation for 20 days by using two types of fungi, Aspergillus niger and Rhizopus oligosporus. Cosmeceutical-related functionalities of the fermented broken rice extracts were tested by evaluating antioxidant activities as well as tyrosinase and elastase inhibition activities. Total phenolic content and total flavonoid content of fermented broken rice extracts were also measured. Fermentation caused a substantial increase in the total phenolic and flavonoid content of broken rice. Ferric-reducing antioxidant activity was the highest (18.47 mM FE(II)/g sample) in broken rice fermented with R. oligosporus on day 20. Fermentation of broken rice with the same fungus resulted in higher DPPH radical-scavenging antioxidant activity than A. niger, with the highest activity recorded was 94.17%. Broken rice fermented with R. oligosporus for 20 days had the highest tyrosinase and elastase inhibition activities with the value of 46% and 51.9%, respectively. The results demonstrate that the fermentation process increased the biological activities and the numbers of bioactive compounds for potential use in the cosmeceutical industry. This undervalued by-product of the rice milling industry can effectively be used for the production of high value products through fungal solid-state fermentation.

Production of Isomalto-Oligosaccharides from Available Economic Starchy Materials

ABSTRACT:Isomalto-oligosaccharides have a great potential to improve the physiochemical quality of many foods as anti-fading agent for food pigments, as prebiotic, food antioxidant and as a sweetener. Solid-state fermentation (SSF) has been applied in the production of many fermented foods (Couto and Sanroman, 2006). Various fungi have been used in order to produce amylolytic enzymes for starch degradation. Many fungal amylolytic enzymes are used to advantage in prebiotic oligosaccharide production. Isomaltooligosaccharides which are known as prebiotic branched-oligosaccharides have been synthesized from starchy grains (Kuriki et al, 1993; Pan and Lee, 2005). The effects of agricultural substrate and fungal strain were studied to maximize the isomalto- oligosaccharides yield. The production of Isomalto-oligosaccharides by fungal fermentation using available economic starchy materials as wheat, white corn, and starch was investigated. This study was designed to investigate the potential use of economic crops of Egypt to produce prebiotic isomalto-oligosaccharides using the process of solid state fungal fermentation. Solid state fermentation with Aspergillus Oryzae EMCC 126 was studied to increase the isomalto-oligosaccharides yield. Results show that the fermentation of wheat with A. Oryzae produced the highest concentrations of total reducing sugar (460.8 mg/g) and free amino nitrogen (20.3 mg/g) with the highest levels of amylolytic activity (87.0 U/g), α-amylase (32.5 U/g) and α-glucosidase (1.2 U/g) compared with that produced from fermentation of white corn or starch. The appropriate fermentation time for 5 days, the fermented wheat slurry was experimented on further in mashing for syrup production. The obtained wheat syrup contained the highest amounts of isomaltose, panose, and isomaltotriose, which were detected by very small amounts in the syrup of corn and starch.

Valorization of agro-industrial wastes to produce hydrolytic enzymes by fungal solid-state fermentation.

Nowadays, significant amounts of agro-industrial wastes are discarded by industries; however, they represent interesting raw materials for the production of high-added value products. In this regard, orange peels (ORA) and exhausted sugar beet cossettes (ESBC) have turned out to be promising raw materials for hydrolytic enzymes production by solid state fermentation (SSF) and also a source of sugars which could be fermented to different high-added value products. The maximum activities of xylanase and exo-polygalacturonase (exo-PG) measured in the enzymatic extracts obtained after the SSF of ORA were 31,000 U·kg-1 and 17,600 U·kg-1, respectively; while for ESBC the maximum values reached were 35,000 U·kg-1 and 28,000 U·kg-1, respectively. The enzymatic extracts obtained in the SSF experiments were also employed for the hydrolysis of ORA and ESBC. Furthermore, it was found that extracts obtained from SSF of ORA, supplemented with commercial cellulase, were more efficient for the hydrolysis of ORA and ESBC than a commercial enzyme cocktail typically used for this purpose. In this case, maximum reducing sugars concentrations of 57 and 47 g·L-1 were measured after the enzymatic hydrolysis of ESBC and ORA, respectively.

Solid-state fermentation of oil palm frond petiole for lignin peroxidase and xylanase-rich cocktail production.

In current practice, oil palm frond leaflets and stems are re-used for soil nutrient recycling, while the petioles are typically burned. Frond petioles have high commercialization value, attributed to high lignocellulose fiber content and abundant of juice containing free reducing sugars. Pressed petiole fiber is the subject of interest in this study for the production of lignocellulolytic enzyme. The initial characterization showed the combination of 0.125mm frond particle size and 60% moisture content provided a surface area of 42.3m2/g, porosity of 12.8%, and density of 1.2g/cm3, which facilitated fungal solid-state fermentation. Among the several species of Aspergillus and Trichoderma tested, Aspergillus awamori MMS4 yielded the highest xylanase (109IU/g) and cellulase (12IU/g), while Trichoderma virens UKM1 yielded the highest lignin peroxidase (222IU/g). Crude enzyme cocktail also contained various sugar residues, mainly glucose and xylose (0.1-0.4g/L), from the hydrolysis of cellulose and hemicellulose. FT-IR analysis of the fermented petioles observed reduction in cellulose crystallinity (I900/1098), cellulose-lignin (I900/1511), and lignin-hemicellulose (I1511/1738) linkages. The study demonstrated successful bioconversion of chemically untreated frond petioles into lignin peroxidase and xylanase-rich enzyme cocktail under SSF condition.

Biovalorization of brewers’ spent grain for the production of laccase and polyphenols

Brewers’ spent grain (BSG) is the main solid by-product of the brewing process and is typically disposed of as cattle feed. In this study, BSG was evaluated as a substrate for the production of polyphenols and the lignin-degrading enzyme laccase using fungal solid-state fermentation by Trametes versicolor. Laccases are finding increasing applications in the food industry and polyphenols have benefits for human health. After 14 days of fermentation with T. versicolor, there was a 3.4-fold increase in the extraction of total polyphenols compared with untreated BSG. Using BSG as the sole source of carbon and nitrogen, maximum laccase activity was achieved after seven days of treatment with an activity of 560 U/L. Based on these results, BSG is suggested to be a good lignocellulose waste material to produce value-added products such as the enzyme laccase and polyphenols.

Mobilization of phenolic antioxidants from grape, apple and pitahaya residues via solid state fungal fermentation and carbohydrase treatment

This paper reports two strategies to mobilize phenolic antioxidants from lyophilized and oven-dried black grape (Vitis vinifera x (Vitis labrusca x Vitis riparia)) pomace, and apple (Malus domestica cv. Jonagold) and yellow pitahaya (Hylocereus megalanthus) peel, core, peduncle and seed mixtures: a solid-state Rhizomucor miehei NRRL 5282 fermentation-based process and a carbohydrate-cleaving enzyme treatment using R. miehei NRRL 5282 cellulase and Aspergillus niger pectinase cocktails. Both methods proved to be suitable to increase the extractable phenolic content and improve the antioxidant properties of the phenolics as determined by 1,1-diphenyl-2-picrylhydrazyl radical inhibition or ferric reducing antioxidant power analyses. During solid-state fermentation, maximal phenolic yields obtained in lyophilized grape, apple and pitahaya residues were 1956 ± 31, 477 ± 37 and 495 ± 27 mg gallic acid equivalents (GAE)/100 g dry matter (DM), respectively, while they were 1385 ± 71, 362 ± 27 and 615 ± 26 mg GAE/100 g DM in oven-dried samples, respectively. The major individual phenolics produced enzymatically from the substrates were identified by HPLC as gallic acid, 4-hydroxybenzoic acid, vanillic acid, (+)-catechin and (−)-epicatechin, with yields ranging from 0.58 ± 0.06 to 215.81 ± 17.17 mg/100 g DM depending on the substrate and the pretreatment. The obtained phenolic-enriched extracts could potentially be applicable as sources of natural antioxidants.

Production of Lipases by a Newly Isolate of Aspergillus niger Using Agroindustrial Wastes by Solid State Fermentation

Background: Lipases are enzymes widely used in many industrial applications. However, its use can be increased by reducing costs of production, being important to this effort the screening of new microorganisms producing and the use of low cost culture mediums, such as agricultural residuals. Objective: To produce lipases using two new isolates of filamentous fungi in solid-state fermentation. Methods: The selection of significant variables on the lipase production was studied through a 27-3 Fractional Factorial Design. The studied variables were: the type of bran used as support in solid state fermentation (wheat or soy), nitrogen source (urea or nitrate), inducer (soy or olive oil), nitrogen source concentration (1 or 3%), inducer concentration (1 or 3%), pH (5 or 7) and filamentous fungal strain (Aspergillus O-4 or Aspergillus E-6). Also, a 24 Full Factorial Design was used in the sequence of optimization. Results: The first experimental design showed that the variables type of bran, nitrogen source, inducer, and pH were the most significant (p<0.05) on lipolytic activity, being the higher activity obtained of 45.95 U.g-1. These variables were studied in the second experimental design, in which high lipolytic activities were obtained (57 U) with soy bran, sodium nitrate as nitrogen source, olive oil as inducer and pH 5. Conclusion: The statistical methods of optimization enabled an increased lipase production in solidstate fermentation from Aspergillus niger (O-4). Keywords: Inducer, nitrogen source, pH, response surface methodology, valuation of residuals, fermentation.

Valorisation of textile waste by fungal solid state fermentation: An example of circular waste-based biorefinery

This study investigated the feasibility of using textile waste as feedstock for cellulase production through solid state fermentation. Aspergillus niger CKB was selected with the highest cellulase activity (0.43±0.01 FPUg−1) after 7days of cultivation on pure cotton. Material modification techniques including autoclaving, alkali pretreatment and milling were applied on six types of textiles with various cotton/polyester blending ratios. The results indicated that using autoclaved textile blending cotton/polyester of 80/20 led to the highest cellulase activity (1.18±0.05FPUg−1) with CMCase, β-glucosidase and avicelase activities of 12.19±0.56Ug−1, 1731±4.98Ug−1 and 2.58±0.07Ug−1, respectively. The fungal cellulase was then extracted and applied to textile waste hydrolysis, in which a sugar recovery yield of 70.2% was obtained. The present study demonstrates a novel circular textile waste-based biorefinery strategy with recovery of glucose and polyester as value-added products.

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.