Fungal Spores Production by solid-state fermentation under hydric stress condition

De la Cruz-Quiroz, R.1, Roussos, S.2,Tranier, M.T.1,2, Rodríguez-Herrera, R.1,Ramírez-Guzmán, N.1 and Aguilar, C.N.1*

1Research Group of Bioprocesses and Bioproducts (DIA-UAdeC). School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, México.
2Equipe Ecotechnologies et Bioremédiation, IMBE-UMR CNRS-7263/IRD-237, Case 421, Aix Marseille Université; Campus Etoile, Faculté St Jérôme; 13397 Marseille cedex 20; France
*Autor de correspondencia: Tel: (844)416 12 38

JBCT Vol. 11, No. 21
Enero – Junio 2019
Artículo PDF

El control biológico se aplica en más de 30 millones hectáreas en todo el mundo para reducir las plagas que causan importantes pérdidas en la agricultura. Norteamérica tiene las mayores ventas de agentes de control biológico (ABC). Un fuerte crecimiento en el uso de ABC, particularmente de agentes microbianos, está teniendo lugar en América Latina, seguido por Asia. Trichoderma harzianum es un hongo importante como biopesticida, particularmente contra microorganismos patógenos de plantas. En el presente estudio, se evaluó la influencia de la tensión hídrica como estrategia para aumentar la producción de esporas y la viabilidad celular de T. harzianum por fermentación de estado sólido (SSF). El bagazo de caña de azúcar, el salvado de trigo y la pulpa de olivo fueron evaluados como apoyos del crecimiento fúngico y monitoreados por respirometría. La producción de esporas, la viabilidad y la actividad de la celulasa se monitorearon cinéticamente. Los resultados demostraron que la tensión hídrica aplicada en SSF no funcionó para aumentar la producción de esporas, pero aumentó la viabilidad de las esporas (1,3 x109 espora/g de fuente de carbono, con 24,7% de viabilidad). Una buena funcionalidad de bagazo de caña de azúcar y del salvado de trigo fue encontrada como sustratos de SSF para producir las esporas y las celulasas de T. harzianum.

Palabras clave: estrés hídrico, fermentación de estado sólido, celulasas, esporas, Trichoderma harzianum.

Biological control is applied in over 30 million hectares worldwide to reduce pests that cause significant losses in agriculture. North America has the largest sales of biological control agents (ABC). Strong growth in the use of ABC, particularly microbial agents, is taking place in Latin America, followed by Asia. Trichoderma harzianum is an important fungus as a biopesticide, particularly against pathogenic microorganisms of plants. In the present study, the influence of water tension was evaluated as a strategy to increase the production of spores and the cellular viability of T. harzianum by solid state fermentation (SSF). Sugar cane bagasse, wheat bran and olive pulp were evaluated as support for fungal growth and monitored by manometric. Spore production, viability and cellulase activity were monitored kinetically. The results showed that the water stress applied in SSF did not work to increase the production of spores but increased the viability of the spores (1.3 x109 spore/g of carbon source, with 24.7% viability). A good functionality of sugar cane bagasse and wheat bran was found as SSF substrates to produce spores and cellulase of T. harzianum.

Keywords: Hydric stress, solid-state fermentation, cellulases, spores, Trichoderma harzianum.

Biological control is an environmentally friendly strategy and effective tool to reduce, or mitigate the pests and pest effects using natural enemies, including the excessive use and the effect of chemical pesticides, since these generate resistance of pathogenic microorganisms, high environmental damages and human health among other negative effects1. Some fungi can be considered as biological control agents (BCA), because they act as natural enemies of plant pathogens2; particularly, the filamentous fungus Trichoderma harzianum has physiological, enzymatic and biochemical properties, which give it the capacity to grow on low water activity substrates3,4. Also, it is industrially used to produce cellulase, antibiotics, flour protein enrichment, flavored compounds and biopesticides5,6. T. harzianum inhibits the species of Fusarium oxysporum, Botrytis cinerea, Crinipellis perniciosa, Rhizoctonia solani, etc.7-10.

BCA can be produced by SSF, which is a microbial process carried out on solid materials surface in the absence of free water. The materials should have the property to absorb the water with, or without soluble nutrients, since the substrate must have enough water for supporting the microbial growth and metabolism11,12. A fundamental step of SSF is the selection of substrate with low cost and good availability. All the substrates have a common feature, a basic macromolecular structure of starch, cellulose, lignocellulose, pectin, or other polysaccharides. Generally, the substrates for SSF are agricultural products, or agroindustrial wastes, which have heterogeneous composition, and offer attractive advantages for applications in fermentation processes13.

There are several uses of the SSF such as biomass production, enzyme production and secondary metabolites production, like mycotoxins, or fragrances, etc. The SSF could be used for BCA production, including spores, biocatalysts, inoculum and biomasses, facilitating the biopesticide application on the field crops. However, it has been proposed that the desirable quality of a BCA can be significantly improved through the application of hydric stress, because it could promote the virulence and viability for a stock long time1,14.Nevertheless, it is necessary to demonstrate such effectively of hydric stress. Hence, this study was carried out to evaluate the influence of hydric stress to increase the spore production by Trichoderma harzianum in SSF using sugarcane bagasse, wheat bran, olive pulp and potatoes flour in column bioreactors.


Microorganims and culture conditions

The fungal strain of T. harzianum IRDT22C was provided by the Institute Mediterranean of Biodiversity and Marine Ecology and Continental (IMBE), Aix-Marseille University, France. The strain was inoculated and cultured on potato dextrose agar (PDA)and incubated at 29ºC for five days.Spores suspension was prepared by adding 20 mL of Tween 80 (0.1 g/L) in the flask; the spores were counted in a cell counter chamber (Malassez).

Spore germination

A kinetic study of spore germination was carried out using asterile mix (70:30, w/w)of sugarcane bagasse and wheat branas substrate of SSF at 75% of humidity, 29°C and2x107 spore/g inoculum3. The spore germination kinetics was done through direct observation at microscope, each hour during two days until germination of all the spores. The results are shown asthe percentage of spores germination. A germinated spore iswhen the germinated tube is equal, or higher at the diameter of spore15.

Solid-state fermentation (SSF)

The fermentations were done in column bioreactors16.Several treatments were conducted to evaluate the fungal spores production comparing several solid substrates as supports of SSF. The fermentations were coded as SSF-1, SSF-2, SSF-3 and SSF-4. Each treatment was done in triplicates and monitored kinetically to evaluate the physiology of T. harzianum. The material used was sugarcane bagasse (SCB), wheat bran (WB), olive pulp (OP) and potatoes flour (PF). The material was sterilized at 120ºC for 30 min. After inoculation (2×107 spore/g of support), bioreactors were incubated at 29ºC. Culture conditions are showed on table 1. The Czapeck Dox medium containing (g/L) NaNO3 3.0, KH2PO4 1.0, MgSO4 0.5 and KCl 0.5 g/L were the mineral solution used in SSF-1.

Table 1- Culture conditions of the solid-state fermentations.

Sampling, treatment and analytical determinations

After the fermentation, 5.0 g of sample was withdrawn from each column and placed in conical tubes (50 mL),and then were frozen for post-analysis. The remainder of the sample was used to check the moisture and, pH and to make morphological observations on fungal growth in function of the time. The values of air-flow, temperature, humidity percentage and CO2 of SSF were monitored through respirometry using the PNEO equipment17.

Kinetic sporulation of T. harzianum on SSF

One gram of each sample from the fermented material was placed in an Erlenmeyer flask with 100 mL of Tween 80 at 0.01 % and stirred for 10 min. The spore counting was done every 12 h, using the cell counter chamber (Malassez). The results were expressed as the spores number per gram of carbon source initially present in the culture media (spore/g CS). A culture of T. harzianum on PDA medium was used as control.

Spores viability of T. harzianum under different stock conditions

Four warehouse conditions were evaluated to observe the spore viability. The lyophilized sample (LSS), frozen sample(FSS), dry sample (DSS) and PDA sample (PSS).The samples DSS (0.5 g), LSS (0.5 g) and FSS (1.0 g) were placed in a conical tube with 40 mL of Tween 80 at 0.1% and stirred. 10 mL of PSS were added in the flask; after this,the samples were diluted and each one was inoculated (0.2 mL) on Petri plates with PDA/ rose Bengal. Colonies were counted and calculated to know the viable spores number per gram of the sample3.

Cellulase determination of T. harzianum

Defrosted samples (5.0 g wet weight) were mixed with 50 mL of distilled water, the suspension was homogenized using an Ultra-turrax for 1 min and then pH was measured. Several dilutions were done (1/2, 1/5, 1/10) for other analysis. Reducing sugars were spectrophotometrically determined by the Miller method18. Samples (2.0 mL) and DNS reagent (3.0 mL) were taken in a test tube, vortexed and placed in a bath at 100 ºC for 5 min. Reaction was stopped by placing the tube in anice frozen bath. Calibration curve was made from 0 to 1 g/L of glucose and the absorbance was measured at 575 nm. The carboxymethyl cellulase (CMCA, endo 1,4-β-D-glucanase, EC and filter paper (FPA, exo 1,4-β-D-glucanase, EC were determined by the methodology of Mandels19 and expressed as international unit IU). One IU of cellulase activity was the amount of enzyme to release a micromole of glucose per minute of the reaction.

Experimental design and data analysis

For each fermentation process, an experimental design with mono-factorial arrangement was used to evaluate each response as the function of time. The ANOVA was done by the software UANL version 2.5 and the mean comparison tests by the Tukey test.


The aim of this study was the production of spores of T. harzianumin SSF and to compare this conventional fermentation conditions under the hydric stress conditions. The sporulation kinetic showed a highest value (8.6×109 spores/ g CS) at 147 h. After this time, the sporulation level was stable with a slowly decrease (Table 2). The behavior of the kinetic spore production on SSF-1 and SSF-2 was similar. The moisture and pH were stable in both the cases and after 48 h, the pH increased until neutral levels. The maximum concentration of CO2 released was 2% at 48 h, and then it was decreased at 0 % at the end of SSF. The spores concentration obtained on SSF-1 reached a maximum value of 4.33×109 spore/ g CS at 161 h, with a productivity of 2.7×107 spore/ g * h. In the case of SSF-2,the sporulation was 8.90×109 spore/ g CS at 120 h, with a productivity of 6.9×107 spore/ g * h (Table 2).

Table 2. Spore production by T. harzianum on SSF.

The pH was stable in SSF-3 and SSF-4 but the same was not with the moisture, which decreased to 59 and 8 %, respectively. This was due to the application of dry air throughout the columns in SSF-4. SSF-3 resulted 1.03×1010 spores/ g CS at 168 h, with a productivity of 5.8×107 spore/ g * h. The maximum concentration of CO2 released was 1.29% at 72 h, and then it was decreased to 0% at the end of fermentation. SSF-4 resulted 1.3×109 spore /g CS at 115 h, with a productivity of 1.1×107 spore/ g * h (Table 2).SSF-3 and SSF-2 showed higher spores production. The conditions used in both the experiments were the same, except for the flow of the dry air, which was 35 and 40 mL/min, respectively. Comparing the conditions of all the fermentation process, the SSF-3 was the best for the production of spores, although the productivity was better in SSF-2.The hydric stress applied on SSF-4 did not show spores production comparable with SSF-2 or SSF-3. The spore production was ten times less than SSF-3. But it was important to note that after the process, the product was already dry, so it was easy to store.

Four treatments were evaluated to store the spores produced by SSF: lyophilization (LSS), drying (DSS), freezing (FSS) and refrigeration on PDA (PSS).The spores stored on PDA medium at 4ºC were the control (25% of viability). The spores obtained from the dry samples from SSF-4 reached a 24.7% of viability (Table 3), showing that the SSF with hydric stress applied allowed an easier way to produce the spores with viability similar to PDA without additional energy expenditure.

Table 3. Spore viability percentage of T. harzianum under several storage treatments.

The cellulase activity obtained during the growth of T. harzianum on a mix of sugarcane bagasse and wheat bran increased over the time and was maximum at 48 h. These enzymes are synthesized during the active growth of mycelium and they are excreted to outside of the cell3.The filter paper activity (FPA) indicates the action of cellulases against the insoluble cellulose. In this work, higher FPA was observed in SSF-1 (86.7 IU/L at 96 h). The carboxymethyl-cellulose activity showed the effect of cellulases on soluble cellulose and was maximum in SSF-1 (1666 IU/L at 89 h) (Fig. 1). The CMCA values in SSF-1 remained stable during 91 h, from the maximum production of cellulase until the end of fermentation. This showed high stability of CMCA over the time.

Figure 1 – Cellulase production by T. harzianum on SSF, A (FPA) and B (ACMC).

There are some studies on the production of several enzymes and spores under SSF (20-22). SSF is a preferred system because the raw materials used in it as the substrates are cheaper such as sugarcane bagasse, wheat bran, among others23. Efforts have been made on the production of biological control agents using several systems. For example, Shahzadi et al.24 reported the use of corn cob under SSF by T. asperellum obtaining a yield of 3.13×109 spores/g. Motta and Santana25 used empty fruit bunch and a Trichoderma sp in SSF and found. 4.4×109 spores/ g in columns bioreactors.Roussos3 evaluated a mix of sugarcane bagasse and wheat bran as substrate in SSF and obtained 1570 and 21052 IU/L/100 g CS to FPA and CMCA, respectively. The corresponding values obtained in liquid media with wheat straw /bran substrate mixture were 1770 and 17280 IU/L / 100 g CS to FPA and CMCA, respectively. The CMCA values were at least 10 fold higher over the FPA under the conditions used, which was similar to the present findings.

Fungal sporulation starts when environmental and nutritional factors become critical to the life-cycle of the fungus. The spores are the units of reproduction and conservation to filamentous fungi, capable to support the extreme conditions and resist to physical and chemical attacks1. The sporulation occurs on the surface and in all the cavities that offer free spaces. Hence, solid media represent the best place for the production spores by the filamentous fungi. If a liquid medium is stirred, the spores cannot be formed on the surface, rather are dispersed in the medium with a very low yield. The liquid media have high water content, therefore, the biomass obtained in this process have a risk that the spores could germinate3.

To understand about the hydric stress, it is necessary to know about the water and its influence on the life. Water always has an impact on biological systems due at interactions mechanisms with several organic molecules, and has two mainly functions, as solvent and structural at molecular and cellular level26. Therefore, the water plays an important role in all culture systems such as solid-state fermentation (SSF) and submerged cultures.  In the SSF, the moisture requirement is very low, which results on a limiting factor to growth and metabolism of microorganisms. Oriol et al.27 reported that water content on SSF should be on a range between 30 to 80 % depending on the substrate used.  The maintenance of functional features of some enzymes and the mechanic structure of plasmatic membrane could be denatured due to lack of proper water and could affect the permeability properties and transport through the membrane, causing cell perturbation28.

The water activity (ɑW) has been used as predictive criteria of the physiology functions of the microorganisms. It influences the cellular mechanism, metabolites production, aroma, fungal growth (radial and apical) and sporulation26. The increase of ɑW on a substrate could cause a reduction on the spore germination time and increase the specific growth rate of microorganism27. It is well known that low levels of ɑW could inhibit the growth of filamentous fungi, causing low levels of mass transfer and low water availability, therefore, the conversion of substrate to biomass is not carried out completely27.

The spore formation is induced when a limiting factor is decreasing in a culture media, therefore, the lack of nutrients cause the start of the sporogenesis (physiological stage that correspond at formation and release of the resistance form and reproduction)3. The nature of limit factor is very variable and generally is the water and carbon and nitrogen sources. However, there are physicochemical factors that have the ability to start the sporogenesis, such as pH, CO2, O2, temperature and secondary metabolites3.

In SSF, the hydric stress is a condition applied with dry air, which could favor the sporulation and minimize the time of production. This offers an early induction to sporulation and the excess of water is evaporated, resulting in the release of the space for the sporulation, which contributes in increase of sporulation index. However, to make a hydric stress process more productive, it is necessary to optimize the processes to allow maximum production of the spores.

A successful biological control product needs a long shelf-life. Commercially, the most recommendable is a dry product, because it is easy to store, formulate and apply29. The high temperatures and moisture content are important environmental conditions that affect the deterioration rate of biological systems. This deterioration can be reduced by a slowly drying with air, without high temperatures, keeping high levels of germinal propagulates. This work achieved to conserve 25% of spores on the dry materialwith the possibility to germinate, which reduced the cost of storage because it did not require refrigeration, lyophilization, or other subsequent process. The obtained biomass could be used for several applications such as starter inoculum for fermentations, biopesticides, bioherbicides, valorization of agricultural byproducts, production of primary and secondary metabolites, etc.

The respirometry system was a useful tool due to the monitoring of physiological stages of microorganism. The present results showed that at 48 h SSF, the maximum metabolic activity of T. harzianum was maximum under specific conditions, when the sporulation began. The mixed substrate SSF with sugarcane bagasse and wheat bran showedhigh influence on cellulase production, specifically on the activity levels (CMCA and FPA) and stability over thetime and was effective for the production of spores and cellulase.

These results suggested the good functionality of sugar cane bagasse and wheat bran as substrates for the SSF to produce the spores and cellulases by T. harzianum and the spores concentration was comparable with that obtained on PDA. Therefore, this added value to these wastes and also it could reduce the cost of biomass production (mycelium, or spores).The application of hydric stress did not increase the spores production, but it increased the viability of the spores.


The authors thank National Council of Science and Technology of Mexico (CONACYT) for the financial support during the stage on France (Scholarship No. 305744). The authors thank Department of Food Research (Mexico), to Institut de Recherché pour le Development (IRD) and the Institut Méditerranén de Biodiversité et d’Ecologie Marine et Continentale (IMBE) for the technical facilities.


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