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Mycelia Growth and Spore Yield of Trichoderma harzianum in Batch and Fed-Batch Cultures: Influence of pH and Temperature

Trichoderma species are the most common saprophytic fungi in the rhizosphere and are found in almost any soil. They have mycoparasitic ability against economically important aerial and soil borne plant pathogens (Dubey et al., 2007; Kuhls et al., 1996; Papavizas and Lumsden, 1980). However, Trichoderma spp. have been of great interest to many researchers who have been contributing to biological control pursuit through the use of fungi (Ortiz and Orduz, 2001; Heraux et al., 2005). Trichoderma strains have been successfully used as biological control agents (BCAs) due to their high adaptability and reproducibility in diverse conditions, efficiency in the utilization of nutrients, ability to modify the rhizosphere, antagonistic activity against phytopathogenic fungi which promotes plant growth and defense mechanisms (Chet et al., 1997). T. harzianum is a common BCA, used as against phytopathogenic and viral vector fungi (Grondona et al., 1997).


Introduction
Trichoderma species are the most common saprophytic fungi in the rhizosphere and are found in almost any soil. They have mycoparasitic ability against economically important aerial and soil borne plant pathogens (Dubey et al., 2007;Kuhls et al., 1996;Papavizas and Lumsden, 1980). However, Trichoderma spp. have been of great interest to many researchers who have been contributing to biological control pursuit through the use of fungi (Ortiz and Orduz, 2001;Heraux et al., 2005). Trichoderma strains have been successfully used as biological control agents (BCAs) due to their high adaptability and reproducibility in diverse conditions, efficiency in the utilization of nutrients, ability to modify the rhizosphere, antagonistic activity against phytopathogenic fungi which promotes plant growth and defense mechanisms (Chet et al., 1997). T. harzianum is a common BCA, used as against phytopathogenic and viral vector fungi (Grondona et al., 1997).
during product formulation and field use, in contrast to their mycelia and chlamysdospore forms as microbial propagules (Amsellem et al., 1999). However, the presence of mycelia along with conidia would insure presence of various essential metabolites (e.g., antibiotics) for BCA activity (Roberts et al., 2005).
When planning the application of biocontrol strains, it is very important to consider the environmental stresses affecting microbial activities. As in all microorganisms even in Trichoderma, the external factors modify their morphological characteristics as well as physiological functions. Among these factors, pH and temperature are probably the most important environmental parameter affecting the mycoparasitic activities of Trichoderma strains (Kredics et al., 2004). Biocontrol strains should have better stress tolerance levels than the plant pathogens against which they are going to be used during biological control. Therefore, it is also of great importance to collect information about the effects of pH and temperature on mycelia growth and spore yield of T. harzianum in order to know conditions that will favour high production yield T. harzianum propagules for BCAs.
Liquid fermentation has been pursued by researchers than solid state fermentation due to its compatibility with pre-existing large scale facilities. Of the different types of liquid fermentation, fed-batch systems appear to have potential for commercial spore production by fungi (Casino et al., 1990;Molla et al., 2004). Unfortunately accurate information regarding the factors affecting the production of spores in liquid culture still remains a challenging task and warrants considerable research inputs. This study was designed to examine the influence of pH and temperature on mycelial growth and sporulation of T. harzianum in batch and fedbatch cultures.

Fungal culture
The fungal culture of T. harzianum was obtained from The Culture Collection Centre of The Department of Microbiology University of Ibadan, Nigeria. The strain was maintained on Potato dextrose agar (PDA).
Batch culture fermentation was done as described by Cascino et al., (1990). 250 mL Erlenmeyer flask containing 50 mL of liquid medium was inoculated with 1 mL of the spore suspension prepared according to the method of Nahar et al., (2008) and was later incubated at specified temperature under static condition in the dark for 7 days.
Fed-batch culture fermentation was conducted using the modified method of Cascino et al., (1990). A fed-batch vessel containing 50 mL of liquid medium was inoculated with 1 mL of the spore suspension and incubation at specified temperature under static condition for 7 days.
At every 12 h intervals, 4 mL of the limiting nutrient (yeast extract 0.05 mg/mL) was added.

Influence of pH
For the influence of various pH levels on mycelial growth and sporulation, the growth medium was prepared using citrate buffer regulated to varying pH, ranging from 3.0 to 6.0. 1 mL spore suspension of T. harzianum was inoculated into Erlenmeyer flask (250 mL) containing 50 mL of liquid medium and incubated at 30 0 C for both batch and fed-batch cultures. The setup was replicated thrice after which the mycelial dry weight and spore yield was recorded after 7 days of incubation (Steyaert et al., 2010a).

Influence of temperature
The influence of various temperatures on mycelial growth and sporulation was done according to the modified method of Schemaeza, et al., (2013). Fifty milliliters of the liquid medium adjusted to pH 5.5 using citrate buffer was dispensed into 250 mL Erlenmeyer flasks. The liquid medium was later inoculated with 1mL spore suspension of T. harzianum and incubated at four different temperatures, viz, 25, 30, 37 and 45 0 C for 7 days in both batch culture and fed-batch cultures. The setup was replicated thrice. Mycelial dry weight and spore yield was determined.

Spore yield determination
The spore yield was determined using the modified method of Waghunde et al., (2010). Each flask was harvested by filtering the spore suspension through a sterilized double layered muslin cloth. The stock suspension was kept on Rotary Flask Shaker (MAC, MSW-301) for 2 minutes, after which 3mL of the suspension was added into a cuvette. The equipment was calibrated with 3 mL of blank solution (liquid medium). The spore yield was determined at a wavelength of 550 nm using Perkin Elmer Lambda 25 UV Spectrophotometer.

Assessment of dry cell mass
The fungus biomass yield was assessed by collecting fungal biomass on pre-weighed filter paper after incubation. The mycelium dry weight was determined after drying at 80 °C to constant mass. The actual weight of fungal mycelium was calculated by difference (Al-Taweil et al., 2009).

Influence of pH
The effect of pH on the mycelia growth and spore yield by T. harzianum after 7 days in batch and fed-batch culture are shown in Figure 1 and 2. Analysis revealed that pH 4.0 supported the maximum mycelial growth of 14.29±0.56 g/L in batch culture while the lowest mycelial growth of 9.97±0.38 g/L was recorded at pH 6.0 (Figure 1a). Mycelial growth of T. harzianum was favourable within the optimum pH range of 3.5-5.0 in batch culture. Similarly, the maximum spore yield with optical density (OD) of 1.05±0.02 was supported at pH 4.0 while the lowest spore yield with OD of 0.17±0.03 was produced at pH 3.0 (Figure 1b).
Results of effect of initial pH in fed-batch culture are shown in Figure 2 reveals that pH 4.0 supported the highest mycelial growth of 11.92±0.43 g/L while the lowest mycelial growth of 3.28±0.61 g/L was recorded at pH 6.0 (Figure 2a).
The result reveals that optimum pH range of 3.5-5.0 favours the mycelial growth of T. harzianum in fed-batch culture. Similarly, the highest spore yield with OD of 1.24±0.05 was recorded at pH 4.0 while the lowest spore yield with OD of 0.21±0.12 was recorded at pH 3.0 (Figure 2b). The optimum pH range that favoured high spore yield of T. harzianum in fed-batch culture is pH 4.0-5.5.

Influence of temperature
The effect of temperature on the mycelial growth and spore yield by T. harzianum after 7 days in batch and fed-batch culture are shown in Figure 3 (A and B) and 4 (A and B). Analysis revealed that temperature 30 o C is the optimum temperature for mycelial growth with a mycelial weight of 12.13±0.29 g/L in batch culture while the lowest mycelial growth of 8.72±0.56 g/L was recorded at 45 o C (Fig. 3A). Mycelial growth of T. harzianum was favourable within the optimum temperature range of 25-30 o C in batch culture. The highest spore yield with OD of 1.0±0.04 was supported at 25 o C while the lowest spore yield with OD of 0.34±0.11 was produced at 37 o C (Fig. 3B).
Results of effect of temperature in fed-batch culture are shown in Figure 4 (A and B) and it reveals that the optimum temperature that supported the highest mycelial growth of 10.43±0.38 g/L was 30 o C while the lowest mycelial growth of 6.40±0.25 g/L was obtained at 25 o C (Fig. 4A). The result reveals that optimum temperature of 25-37 o C favours the mycelial growth of T. harzianum in fedbatch culture. The spore yield with OD of 1.58±0.02 was highest spore yield produced and it was obtained at 45 o C while the lowest spore yield with OD of 0.42±0.03 was recorded at 25 o C (Fig. 4B). The optimum temperature range that favoured high spore yield of T. harzianum in fed-batch culture was 30-45 o C.
The growth and conidiation of Trichoderma is influenced by some known environmental factors include the ambient pH of the medium, temperature extracellular calcium, physical injury to the mycelium and the presence of fungal-derived volatile organic compounds (Steyaert et al., 2010a). In our study, two fermentation techniques; batch and fed-batch culture, and different pH and temperature conditions affected the mycelial growth and conidiation of T. harzianum. The result of this study showed that in batch and fed-batch culture the optimum pH for mycelial growth and spore yield was pH4. The pH range that favoured the mycelial growth and spore yield was pH 3.5-5 and pH 4-5.5 respectively. It has been demonstrated that Trichoderma strains are active under a wider range of pH (Kredics et al., 2003) and from our analysis it can de deduced that T. harzianum grow and sporulate maximally at a low ambient pH. This result is in line with the work of Papavizas et al., (1982) and Steyaert et al., (2010b). A similar response was observed in another study, it is found that T. harzianum grow in wide range of pH 2.0 to 6.0, with maximum growth at 4.0 (Kredics, 2004). The initial pH of the medium has also been demonstrated to have an effect on mycelial growth and conidiation, and unlike the C: N ratio, pH levels which favour conidiation have been shown to favour mycelia growth as well (Aube and Gagnon, 1969;Bastos, 2001;Brian and Hemming, 1950;Lewis and Papavizas, 1983;Steyaert et al., 2010b). Benitez (2004) reported that growth of Trichoderma is more efficient in acidic than alkaline soils and they modify the rhizosphere soil by acidifying the soil. Low ambient pH of the growth medium has been demonstrated to result in intracellular acidification in Aspergillus niger and Saccharomyces cerevisiae Caspani et al., 1985;Gradisnik-Grapulin and Legisa, 1997). This explains the reason why the T. harzianum strain prefered acidic pH.
Investigating the effect of temperature on T. harzianum in batch and fed batch culture showed that the optimum temperature for mycelial growth is 30 o C while the optimum temperature for spore yield in batch and fedbatch culture is 25 o C and 45 o C respectively. It can be inferred that T. harzianum has a broad range of tolerance as regards its growth and sporulation and that higher temperatures favours sporulation in fed-batch cultures.
Maximum mycelial growth at the optimum temperatures in both batch and fed-batch cultures could be because it also affects their metabolic activity especially the production of volatile antibiotics and enzymes (Tronsmo and Dennis, 1978). Though temperature plays an important role in the growth of organisms, at elevated level, it damages the organisms by denaturing enzymes, transport carriers, integrity of cell membrane (Prescott et al., 2002).
In general, commercial preparations of Trichoderma spp. for biological control consist of bulk-produced conidia (asexual spores), whereas good biocontrol activity in the environment relies upon the fungus remaining vegetative, and thus antagonistically active. The result from this present study suggests that there are considerable and unexpected differences between the mycelia growth and spore yield by T. harzianum in batch and fed-batch cultures.
However, fed-batch culture produced more spore yield than batch culture, this is in line with the work of Onilude et al., (2013). Although more mycelial was produced in batch culture, unfortunately mycelia cannot survive downstream processing steps such as drying and hence are not useful as compared to conidia (Papavizas et al., 1982). The high spore yield obtained in fed-batch culture was at the expense of greatly reduced mycelia, resulting in a high spore yield. The substrate-limited, fed-batch culture increased the amount of spore production by favouring the allocation of nutrients to spore production rather than mycelia production (Cascino et al., 1990).
In conclusion, this work has shown that fedbatch system at acidic pH and temperature range of 30-45 o C will be the best production strategy for maximum spore production of T. harzianum for BCA application than batch culture system.