3.1 Conidia production of T. asperellum Th-Th4 (3)
3.1.1 First production cycle using fresh rice as the substrate
In cycle 1, T. asperellum Th-Th4 (3) was cultivated in SSC using fresh rice as the substrate (i.e., sterile parboiled rice that had not been previously used). During this cycle, fungal mycelia quickly covered the rice grains and a uniform conidia layer was observed 3 days after inoculation (Fig. 1b). This point was considered the end of cycle 1, and therefore, the incubation was shortly interrupted in order to achieve the harvesting and counting of the conidia.
The conidia production yield was 1.0×109 con/gds, with a productivity of 3.5×108 con/gds⋅d (Table 1). Although T. asperellum Th-Th4 (3) is not a commercial strain, this production was higher than the optimum reported for Trichoderma harzianum growing in rice (5.8×108 con/gds; [6]), and in the same order of magnitude as the one obtained with Trichoderma asperellum TF1 growing in mixtures of agricultural residues (8.6×109 con/gds; [13]). In addition, the conidia of T. asperellum Th-Th4 (3) presented a viability higher than 98 %, which agreed with values previously reported for other Trichoderma strains [14].
Table 1
Production of T. asperellum Th-Th4 (3) conidia in successive production cycles.
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Conidia production cycles
|
|
Cycle 1
(fresh rice)
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Cycle 2
(recycled rice)
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Cycle 3
(recycled rice)
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Cycle length (d)
|
3
|
2
|
3
|
Production yield (con/gds)
|
1.0×109 ± 2.1×108 a
|
2.9×109 ± 4.4×108 b
|
9.9×108 ± 1.7×108 a
|
Productivity (con/gds·d)
|
3.5×108 ± 7.2×107 a
|
1.4×109 ± 2.2×108 b
|
3.2×108 ± 5.9×107 a
|
Conidia viability (%)
|
98.1 ± 1.7 a
|
97.2 ± 1.4 a
|
94.3 ± 4.9 a
|
Initial moisture content (%)
|
34.2 ± 0.74 a
|
46.8 ± 2.96 b
|
58.1 ± 2.74 c
|
Cumulative substrate consumption (%)
|
18.3 ± 6.1
|
53.2 ± 9.8
|
63.8 ± 8.9
|
d: days; con/gds: conidia per gram of dry substrate; con/gds·d: conidia per gram of dry substrate per day. This experiment was performed in two independent times using four replicates at each time. Each individual determination was carried out two times. Means (± SD) followed by the same letter within the same line are not significantly different according to Tukey (p < 0.05).
|
Regarding substrate consumption, at the end of cycle 1, around 80 % of the initial substrate that was added remained unused. The visual inspection of the residual rice revealed that most grains maintained their initial geometry and consistency, and only a slight change in color, from orange to white, was detected. Considering these observations, the residual substrate was reused in a second production cycle.
3.1.2 Second production cycle using recycled rice as the substrate
In the second cycle, the residual rice generated in cycle 1 was used as the substrate. This residual rice was not re-sterilized or re-inoculated; consequently, the conidia that remained attached to the substrate after the first harvest acted as the inoculum to start the second cycle. Also, it is important to highlight that the residual rice was not subjected to a drying process, so the moisture content at the beginning of cycle 2 corresponded to the moisture content recorded after the conidia harvesting in cycle 1.
During the second cycle T. asperellum Th-Th4 (3) grew faster and sporulated earlier than in cycle 1. The rice grains were completely covered with a dense layer of conidia after only 2 days of incubation (Fig. 1c). Therefore, the second cycle was ended at this point and the conidia were harvested and counted. Interestingly, the production yield was 2.9×109 con/gds, which represented a 3-fold increase in comparison with cycle 1. In addition, due to early sporulation, the productivity increased 4-fold, going from 3.5×108 to 1.4×109 con/gds·d. No significant differences were found between the viability of the conidia produced in cycles 1 and 2 (Table 1).
Regarding substrate consumption, a conspicuous increase in the substrate consumption was detected in cycle 2 compared to cycle 1. At the end of the cycle 2, the fungus had consumed 53.2 % of the initial substrate that was added. Considering that the substrate consumption in cycle 1 was 18.3 %, the fungus consumed 34.9 % of the initial substrate in only the second cycle. This behavior can be clearly observed in Fig. 2, which graphically illustrates the substrate consumption profile.
For T. asperellum Th-Th4 (3), the amount of substrate consumed between days 3 and 5 (cycle 2) was significantly higher than that registered between days 0 and 3 (cycle 1). These results support the proposed recycling strategy, since the substrate consumption, sporulation yield, and productivity were significantly improved by using recycled rice in comparison with fresh rice. Furthermore, it was not necessary to subject the residual substrate to a drying process. Table 1 shows that the initial moisture content in cycle 1 was 34.2 %, while in cycle 2, it was almost 47 %. According to [6], the optimum moisture content for the sporulation of Trichoderma strains growing in rice is around 35 %; however, as shown here, T. asperellum Th-Th4 (3) sporulated more profusely when it was cultivated in recycled rice, even when this substrate presented a moisture content of 47 %.
At the end of the conidia harvesting, the residual substrate was visually inspected again. At this time, the rice grains were turned light brown, some of them appeared broken, and, in general, the substrate began to show a slightly pasty appearance. The moisture content of this rice was 58.1 %.
3.1.3 Third production cycle using recycled rice as substrate
In the third cycle, the residual rice generated after the conidia harvesting in cycle 2 was used as the substrate. As previously mentioned, the moisture content of this residue (58.1 %) was out of the optimal range for the growth and conidia production of Trichoderma strains. However, since the objective of this work was to study a recycling strategy free of extra processing steps (drying, sterilization, etc.), this residual substrate was reused without any further treatment.
Interestingly, T. asperellum Th-Th4 (3) was able to grow and sporulate under these conditions, and after three days of incubation, the conidia production reached 9.9×108 con/gds. Figure 3a shows that the excess humidity caused a decline in the sporulation yield, probably because of the substrate compaction and lack of oxygen diffusion [15, 10]. However, no significant differences were observed between the production yield obtained in cycle 1 (fresh rice) and cycle 3 (two times recycled rice), and the viability of these conidia was as high as in the two previous cycles (Table 1). These results showed that, under the conditions tested, the rice can be reused at least two times, obtaining, at each time, production yields equal or greater than those obtained using fresh rice.
At the end of cycle 3, most rice grains had lost their original structure and presented a pasty appearance (Fig. 1d). Besides that, the moisture content in the residual rice was 63 %. Consequently, the production scheme was stopped at this point, and the residual substrate was not reused in another production cycle.
Considering the 3 cycles, the production scheme took 8 days and produced 4.9×109 con/gds, which was almost 5-fold more conidia than the conventional single-cycle process (only cycle 1). In addition, this production yield was 2-fold higher than that reported by [16] in a single production cycle of 15 days (2.5×109 con/gds), and 8-fold higher than what was reported in other optimized processes (5.8×108 con/gds; [6]).
3.2 Conidia production of M. robertsii Xoch-8.1
In order to evaluate the effectiveness of the proposed recycling strategy with another biological control agent, a similar set of assays was performed using the entomopathogenic fungus M. robertsii Xoch-8.1. Metarhizium strains have been used for the production of several commercial bioinsecticides [5].
3.2.1 First production cycle using fresh rice as the substrate
As previously described for T. asperellum Th-Th4 (3), in the first cycle, M. robertsii Xoch-8.1 was cultivated in SSC using fresh rice as the substrate. This first cycle lasted 10 days and produced 5.7×108 con/gds with a productivity of 5.7×107 con/gds·d (Table 2). The viability of these conidia was 63.2 %.
Table 2
Production of M. robertsii Xoch 8.1 conidia in successive production cycles.
|
Conidia production cycles
|
|
Cycle 1
|
Cycle 2
|
Cycle 3
|
Cycle 4
|
Cycle length (d)
|
10
|
6
|
6
|
6
|
Production yield (con/gds)
|
5.7×108 ± 6.7×107 a
|
1.4×109 ± 3×108 b
|
1.2×109 ± 1.3×108 b
|
6.0×108 ± 6.2×107 a
|
Productivity (con/gds·d)
|
5.7×107 ± 6.7×106 a
|
2.4×108 ± 5×107 b
|
2.1×108 ± 2.1×107 b
|
1.0×108 ± 1×107 a
|
Conidia viability (%)
|
62.3 ± 2.6 a
|
71.2 ± 23 ab
|
88.1 ± 1.2 b
|
89.4 ± 3.3 b
|
Initial moisture content (%)
|
32.0 ± 1.2 a
|
43.1 ± 2.8 b
|
50.4 ± 0.8 c
|
57.0 ± 3.1 d
|
Cumulative substrate consumption (%)
|
15.1 ± 6.8
|
37.3 ± 6.7
|
46.4 ± 8.7
|
48.2 ± 5.5
|
d: days; con/gds: conidia per gram of dry substrate; con/gds·d: conidia per gram of dry substrate per day. This experiment was performed in two independent times using four replicates at each time. Each individual determination was carried out two times. Means (± SD) followed by the same letter within the same line are not significantly different according to Tukey (p < 0.05).
|
During this cycle, the fungus consumed 15.1 % of the added substrate, so almost 85 % of the initial rice remained unused at the end of the cycle. The residual substrate practically conserved its original structure, consistency, and color (Fig. 4c), so it was reused in a second production cycle.
3.2.2 Second production cycle using recycled rice.
In a similar way to T. asperellum Th-Th4 (3), M. robertsii Xoch-8.1 grew faster and sporulated earlier in cycle 2 than in cycle 1. The second cycle lasted 6 days and produced 1.4×109 con/gds. This production yield was 2.5-fold higher than that registered in the first cycle (5.7×108 con/gds). Also, the productivity improved from 5.7×107 to 2.3×108 con/gds·d, a 4-fold increase in comparison with cycle 1. In addition, no significant differences were found between the viability of these conidia and that of the conidia produced in the first cycle (Table 2).
At the end of cycle 2, the residual substrate practically maintained its initial structure and consistency (Fig. 4e). Besides, at this point, 60 % of the initial substrate that was added was still unused, and therefore available for setting up another production cycle.
3.2.3 Third production cycle using recycled rice
In the third cycle, conidia production yields were as high as in the second cycle, so once again, the use of recycled rice resulted in an average conidia production that was 2-fold higher than that obtained using fresh rice (Table 2). The conidia productivity was 3.7-fold higher in cycle 3 in comparison with cycle 1, and the conidia viability was unaffected.
At the end of this cycle, the residual substrate showed more significant changes in its macroscopic structure. Some rice grains appeared broken and the whole substrate presented a pasty appearance. Nevertheless, since T. asperellum Th-Th4 (3) was shown to be able to grow and sporulate in a substrate with these characteristics, it was decided to reuse the residual substrate again and evaluate the behavior of M. robertsii Xoch-8.1 under these conditions.
3.2.4 Fourth production cycle using recycled rice
Metarhizium robertsii Xoch-8.1 was able to grow and sporulate using 3-times recycled rice. More interesting was the finding that the conidia yield reached in cycle 4 was statistically equal to that obtained in cycle 1 (Table 2). In addition, the conidia viability was unaffected, so the use of 3-times recycled rice did not reduce the conidia quality or conidia yields in comparison with the use of fresh rice.
At the end of the fourth cycle, 50 % of the initial substrate that was added was still unused (Table 2), but this residual substrate presented a highly deteriorated appearance, so it was decided to end the assay and not to reuse the residual rice in a fifth cycle.
It is clear from Fig. 3a-b that for both fungi (M. robertsii Xoch-8.1 and T. asperellum Th-Th4 (3)), the excess moisture content negatively affected the conidia yields. The relation between these two variables was described by 3-parameter quadratic models defining curves where y (conidia production yield) rises to a maximum, but declines with further increases in x (substrate moisture content). According to this, it is likely that a reduction in the substrate moisture at the beginning of cycles 3 and 4, for T. asperellum Th-Th4 (3) and M. robertsii Xoch-8.1, respectively, could have resulted in higher conidia yields in these particular cycles. However, as is shown in Tables 1 and 2, the cumulative conidia production obtained without drying processes or humidity adjustments were highly competitive in comparison with a single-cycle process using fresh rice, and also in comparison with the results reported in previous works. Therefore, the employment of drying processes or humidity adjustments is not recommended.
On the other hand, the use of cheap texturizers mixed with the initial dry-substrate in cycle 1 could be an interesting alternative. The use of water hyacinth or sugar cane bagasse improve the porosity of rice, which in turn, increase oxygen and heat transfer across the substrate bed [17, 8]. Therefore, it is probable that the use of these kinds of materials from the beginning of the first cycle could allow the substrate to be reused for at least one more cycle without needing extra drying or sterilization steps. However, this should be studied in future works.
3.3 Kinetic analysis of substrate consumption
The substrate consumption data obtained for each fungus were adjusted to the modified Gompertz model in order to estimate the kinetic parameters: P (maximum dry substrate consumed at the end of the whole production scheme) and Rs (maximum dry substrate consumption rate). As can be observed in Table 3, the P value for both fungi corresponded to a final substrate consumption close to 50 %. This showed that the proposed recycling strategy significantly increased the substrate usage in comparison with a single-cycle scheme where the substrate consumption is 20 % or less [7].
Table 3
Parameters of substrate consumption estimated by the modified Gompertz model.
|
P (g)
|
Rs (g/d)
|
R2
|
T. asperellum Th-Th4 (3)
|
1.57 ± 0.20 a
|
0.56 ± 0.07 a
|
0.944
|
M. robertsii Xoch-8.1
|
1.12 ± 0.13 b
|
0.08 ± 0.02 b
|
0.927
|
P: Maximum dry substrate consumed at the end of the whole production scheme (2.5 g of substrate was added at the beginning of the production scheme), Rs: Maximum dry substrate consumption rate. Means with different lowercase letters within the same column are significantly different (p < 0.05).
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Regarding the consumption rates, T. asperellum Th-Th4 (3) showed the higher Rs value; however, it was interesting to observe that both fungi reached the maximum substrate consumption rate in the second cycle (i.e., when they grew on recycled rice) (Table 3 and Fig. 2). This observation and the high conidia production yields obtained for both fungi in their corresponding cycle 2 were clear indications that the use of recycled rice did not negatively affect the growth and sporulation physiology of these fungi, on the contrary, it increased it. Previous works have shown that the geometry, shape and size of the substrates used in SSC significantly affect the production of extracellular enzymes and the fungal growth rates [18]. Therefore, it is likely that the changes in rice geometry during the course of each of the production cycles have favored the growth and sporulation of the tested fungi. On the other hand, it has been reported that sporulating fungal colonies produce volatile organic compounds (VOCs) (e.g. 1-octen-3-ol, 3-octanol and 3-octanone) that can act as chemical elicitors of conidiation [19]. According to this, the accumulation of VOCs in the residual rice, could have caused some induction of the conidiation process, which could explain the increase in the conidia yields observed when recycled rice was used as the substrate. The analysis of this hypothesis will be the subject of further study.
Finally, since T. asperellum Th-Th4 (3) and M. robertsii Xoch-8.1 belong to different taxonomic families (Hypocreaceae and Clavicipitaceae, respectively), it is likely that the proposed recycling strategy will be useful for other commercially important fungi (e.g., Beauveria bassiana, Isaria fumosorosea, Trichoderma harzianum, etc.).