Incubation temperature affects growth and efficacy of white-rot fungi to improve the nutritive value of rice straw

Preparation of fungal strains and spawn

Three WRF species (C. subvermispora (MES 13094), L. edodes (MES 02121), and P. eryngii (MES 13049)) maintained in the liquid nitrogen collection of Plant Breeding, Wageningen, The Netherlands, were used. Fungal species were propagated on malt extract agar (MEA, malt extract, 20.0 g/L; KH₂PO₄, 0.5 g/L; MgSO₄·7H₂O, 0.5 g/L; Ca(NO₃)₂·4H₂O, 0.5 g/L) with a pH of 5.4 at 24°C, until mycelia colonised most of the agar surface. Spawn was prepared by adding a piece (1.5 × 2.0 cm) of colonised agar to sterilised sorghum grains, and incubated in a container (O95/60 + OD95, Sac O₂, Deinze, Belgium) at 24°C until all grains were colonised by the mycelium. The spawn was kept at 4°C until further use.

Substrate preparation, fungal inoculation and sampling

To determine the impact of temperature on the colonisation speed of the WRF, rice straw variety ‘Ha Phat 3’ from Vietnam was collected, transported to Wageningen University and ground to pass a 1 mm screen by using a cross-beater mill (Peppink, Deventer, the Netherlands). Then, the ground straw was soaked in tap water for 1 h at room temperature before the water was drained by applying manual pressure to remove excess water. The final dry-matter (DM) content was ~19%. The hydrated straw was subsequently used to manually fill glass tubes (Ø 22 × L 150 mm) open at one side, which were autoclaved at 121°C for 1 h before being cooled to room temperature. Tubes were positioned vertically in six racks, with each rack containing nine tubes. A piece (1 × 1 mm) of malt extract agar from each WRF was placed on top of the rice straw of three tubes per rack before a lose-fitting cap was fitted. A permanent marker was used to mark the top edge of the substrate where the spawn was located, before two vertical lines opposite each other were drawn from the top to the bottom of the tube for measurement of mycelium growth. The tubes in six racks were incubated in four temperature-controlled water baths (water level was below the rack) at the following six different temperature regimes: continuous at 24°C, 30°C, 35°C, and 40°C, 35°C for 3 days, and subsequently 24°C and 40°C for 3 days, and subsequently 24°C. Every Tuesday and Friday of each week, the colony front was marked between the two lines and the distance to the top was measured using each line. At Day 26, the mycelium of one of the strains reached the bottom of the tube, whereafter the experiment was terminated.

In a follow-up experiment, rice straw (Khang Dan 18 cultivar) was obtained from farmers located in central Vietnam, transported to Wageningen University and chopped into particles of approximately 3–5 cm by using a cutting machine at Unifarm (Wageningen, the Netherlands). Thereafter, the rice straw was soaked in tap water for 3 days at room temperature. The water was drained from the wet straw by using a perforated strainer for 5 h, which resulted in a final dry-matter content of 26.2% of the wetted rice straw. The moistened straw was used to fill micropropagation containers (TP1200 + TPD1200, Sac O₂, Deinze, Belgium) before all filled containers were autoclaved at 121°C for 1 h and allowed to cool overnight in an aseptic room at room temperature. The weights of empty container, container with wetted straw before and after autoclaving and after spawn inoculation were recorded to determine the weight change at each step. The autoclaved rice straw was aseptically inoculated with spawn at 5% fresh spawn weight to autoclaved rice straw fresh weight. Containers (triplicates for each time point) with inoculated rice straw were incubated at either 24°C or 30°C for 8 weeks in two dedicated climate-controlled chambers. Three containers inoculated with each fungus from each climate-controlled room were collected at Weeks 0, 4, 6, 7, and 8 of incubation. The contents of each container were weighed, freeze-dried, and milled to pass a 1 mm sieve by sing a Wiley mill (100AN, Peppink, Olst, The Netherlands), before being stored at room temperature for chemical analysis and in vitro gas production.

In vitro gas production

Gas production of the WRF-treated rice straw samples was determined for 72 h during incubation in buffered rumen fluid, using fully automated gas-recording equipment (Cone et al. 1996). Samples of each substrate were freeze-dried and ground over a 1 mm sieve by using a Wiley mill (100AN, Peppink, Olst, The Netherlands). An accurately weighed amount (~0.5 g DM) of each sample was transferred into 250 mL fermentation bottles (Schott, Mainz, Germany), with each substrate and blanks (rumen fluid without sample) run in duplicate. Approximately 250 mL rumen fluid was collected from the front ventral, middle ventral and cranial dorsal sac of each of three non-lactating, rumen-cannulated donor cows provided only hay ad libitum. Rumen fluid from each cow was collected before the morning feeding in pre-warmed insulated flasks flushed with CO₂. Then, rumen fluid was filtered through two layers of cheese cloth and subsequently mixed (1:2, v/v) with an anaerobic buffer-mineral solution (Cone et al. 1996) under continuous flushing with CO₂. Prior to inoculation, fermentation bottles were placed in a shaking water bath and kept at 39°C and pre-flushed with CO₂. The fermentation bottles containing fungal-treated rice straw samples were then filled with 60 mL of buffered rumen fluid and connected to the fully automated gas-recording equipment (Cone et al. 1996).

Chemical analysis

Dry matter was determined by drying at 103°C to a constant weight (ISO6496 1999) and ash content after combustion at 550°C for 3 h in a muffle furnace (ISO5984 2002). Nitrogen was determined by means of the Kjeldahl method (ISO5983 2005) and crude protein (CP) was calculated as N × 6.25. Neutral detergent fibre (NDF) was determined using heat-stable amylase according to the standard procedures of Van Soest et al. (1991), with acid detergent fibre (ADF) and acid detergent lignin (ADL) being determined as described by Van Soest et al. (1991), by using an automatic Ankom fibre analyser (A2000I; ANKOM Technology, Macedon, NY, USA). The NDF and ADF contents are expressed exclusive of residual ash. Cellulose was calculated as the difference between ADF and ADL and hemicellulose as the difference between NDF and ADF. Loss of DM was calculated on the basis of the weight change of the content of a container between the beginning and each time point (4, 6, 7 and 8 weeks) of the incubation. The pH of the dried and ground samples was measured using a pH meter (Metrohm, Herisau, Switzerland) after adding 30 mL demineralised water to 1 g of sample and mixing for 30 min at 24 radians per second in a shaking water bath.

Statistical analyses

Linear regression analysis with zero intercept was conducted on the growth data of each individual tube, with growth (mm) as the dependent variable and time (days) as the independent variable. The slopes of the regression lines were subjected to ANOVA by using the general linear model procedure in SPSS, as follows:

where Y_ij = response variable, μ = overall mean, T_i = incubation temperature (i = 6; 24, 30, 35, 40, 35/24 or 40/24°C), S_j = strains (j = 3; C. subvermispora, L. edodes, or P. eryngii), (T × S)_ij = interaction term between incubation temperature and strains, and e_ij = residual error.

All data from the follow-up experiment were subjected to ANOVA by using the same general linear model where Y_ij = response variable, μ = overall mean, Ti = incubation temperature (i = 2; 24 or 30°C), S_j = W_j = week (j = 5; 0, 4, 6, 7, or 8) and (T × W)_ij = interaction term between incubation temperature and time. Differences among treatment means were compared using Tukey’s statement for multiple comparisons. The level of statistical significance was declared at P < 0.05. Prior to statistical analysis, duplicate in vitro values were averaged.

Growth of WRF incubated at different temperatures

At 40°C, no growth of any of the fungal species was observed. Except for C. subvermispora at 35°C, all growth rates showed a linear pattern. In general, C. subvermispora grew faster than did L. edodes and P. eryngii (P < 0.001), with L. edodes growing, on average, 11.4% slower than P. eryngii (P = 0.009). Ceriporiopsis subvermispora grew 3.3 ± 0.21 and 4.0 ± 0.16 mm/day at 24°C and 30°C respectively, whereas the growth rate was 2.3 ± 0.27 mm/day during the first 8 days at 35°C, with the subsequent 16 days yielding 1.5 mm growth. Pleurotus eryngii mycelium reached a growth rate of 2.1 ± 0.06 and 2.5 ± 0.07 mm/day at 24°C and 30°C respectively, and grew 1.1 ± 0.16 mm/day at 35°C (Fig. 1). After three incubation days at 35°C and the subsequent 23 days at 24°C, P. eryngii and L. edodes had a growth rate of 1.8 ± 0.26 and 1.4 ± 0.10 mm/day respectively. Lentinula edodes, in contrast, did not show any difference in growth rate when incubated between 24°C and 35°C.

Fig. 1.

Average growth rate of Ceriporiopsis subvermispora (□), Lentinula edodes (), and Pleurotus eryngii (■) incubated on rice straw for 26 days at six temperature regimes (24°C, 30°C, 35°C, 40°C, 35°C/24°C, and 40°C/24°C). No growth was observed for 40°C and 40°C/24°C. Error bars indicate the standard deviation. Means with different letters differ significantly (P < 0.05).

pH changes of WRF-treated rice straw over 8 weeks

There was an effect of strain, temperature, time and their interaction on pH of treated rice straw (P < 0.001). Ceriporiopsis subvermispora and L. edodes decreased the pH over time, whereas P. eryngii increased the pH of the substrate (P < 0.001). The pH was also affected by the incubation temperature, with incubation at 24°C decreasing the pH more than that at 30°C (P < 0.001). Ceriporiopsis subvermispora significantly decreased the pH of rice straw from 4.9 ± 0.08 to 4.0 ± 0.01 when incubated at 24°C and to 4.4 ± 0.03 when incubated at 30°C, with the major decrease occurring during the first 4 weeks of incubation. At both temperatures, no differences were detected from Week 4 to Week 8 of the incubation. A similar trend was observed for L. edodes, with the pH of the treated straw decreasing when incubated at both temperatures. At 24°C, the lowest pH was detected at Week 4 (4.5 ± 0.28), whereafter it significantly increased from Week 6 to Week 8. There were no significant (P = 0.065) differences in the pH of treated rice straw when incubating L. edodes from Week 6 to Week 8 at both temperatures. Pleurotus eryngii significantly increased the pH of treated rice straw for both temperatures.

IVGP and compositional changes of fungal treated rice straw

Tables 1, 2, and 3 show the change in the IVGP and chemical composition of the rice straw during the fungal treatment at 24°C, and 30°C for C. subvermispora, L. edodes and P. eryngii. There was a significant (P = 0.976) effect of incubation time but not the incubation temperature on the IVGP of rice straw treated with WRF. Ceriporiopsis subvermispora significantly (P = 0.015) increased the IVGP of treated rice straw after 7 weeks of incubation. Gas production also increased at Week 6 (281 and 272 mL/g organic matter (OM)) and Week 8 (270 and 280 mL/g OM) of incubation compared with Week 0 (254 mL/g OM), but this difference was not significant (P > 0.05). For L. edodes, a similar trend was observed, with a significant effect of incubation time but not of temperature. The IVGP significantly decreased at Week 4 before being increased from Week 6 to Week 8. The average improvement in IVGP was 12.8% after 6 weeks of incubation at both temperatures (P = 0.059). Pleurotus eryngii also showed a significant (P = 0.011) change in IVGP, but unlike for the other fungi, this was a decrease.

In rice straw treated with C. subvermispora, only the organic matter (OM) content and the absolute amount of hemicellulose (Table 1) were significantly affected by an interaction between incubation time and temperature (P = 0.033 and P = 0.02 respectively). The chemical composition of rice straw changed significantly, with a continuous decrease in OM, ADL and hemicellulose concentration. The absolute amounts of lignin and hemicellulose were decreased by 80% and 55% at 24°C compared with 90% and 69% at 30°C respectively. In contrast, the cellulose amount did not change when incubated at different temperatures. The absolute amount of CP did not change after 8 weeks at either temperature.

When rice straw was treated with L. edodes, there was an interaction only between incubation temperature and time on CP content (P = 0.008), but not in terms of the absolute amount. There was a significant impact of temperature on OM and cellulose loss. Organic matter and cellulose loss of 21% and 10%, compared with 27% and 22% when incubating at 24°C and 30°C respectively, were observed. On average, 58% and 78% of hemicellulose and lignin contents were degraded by L. edodes after 8 weeks of incubation at both temperatures respectively, with no difference being detected from Week 6 to Week 8.

Pleurotus eryngii showed a significant (P = 0.007) effect of incubation temperature on lignin degradation, with 42% and 45% of the lignin amount being degraded at 24°C and 30°C respectively. In general, P. eryngii degraded 13%, 6%, 3% and 20% in terms of absolute amount of OM, CP, cellulose and hemicellulose respectively, during incubation time.