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Optimization of solid-state fermentation for selective delignification of aspen wood with Phlebia tremellosa
Optimization of solid-state fermentation for selective delignification of aspen wood with Phlebia tremellosa* lan D. Reid Plant Biotechnology Institute, National Research Council o f Canada, Saskatoon, Sask., Canada S 7 N O W 9
The white-rot fungus Phlebia tremellosa can delignify aspen wood and increase the accessibility of its polysaccharides to enzymatic hydrolysis, under solid-state fermentation conditions. Fermentations at the 50 g scale were conducted to provide information for the design of larger ferrnentations. Agitation was not required. Forced aeration was needed for delignification of wood layers more than a few millimeters thick, but air circulation between the particles was not blocked by mycelium. Shavings were the best particles size. Sterilization of the wood was essential, but preadaptation of the inoculum to growth in wood was not. Inoculum levels as low as 2% were adequate.
Keywords: Phlebia tremellosa; delignification; aspen; enzymatic hydrolysis
Introduction Polysaccharides in lignocellulosic materials like wood and straw must be liberated from their association with lignin by a pretreatment before they can be used as fibers in pulp and paper, as feed for ruminant animals, or as sugars after enzymatic hydrolysis. To be commercially viable, any pretreatment method must be effective and inexpensive. This is especially true for the relatively low-price markets of animal feeds and fermentation feedstocks. ~ Biological delignification using white-rot fungi is a potential alternative to chemical and physical methods of delignification, but little quantitative information about its relative costs and benefits is available yet. 2 The ability of the whiterot fungus Phlebia tremellosa (Schrad.:Fr.) Nakas. et Burds. to delignify aspen wood and increase its cellulase digestibility has been demonstrated in small-scale solid-state fermentations. 3 The suitability of aspen wood delignified with P. tremellosa for cellulase hydrolysis and subsequent fermentation has also been established. 4 To produce enough material for further product testing, and to estimate the costs of a biological delignification process, it was necessary to scale-up
Address reprint requests to Dr. Reid at the Biotechnology Research Institute, National Research Council of Canada, 6100 Ave. Royalmount, Montreal, Quebec, Canada H4P 2R2 *Issued as NRCC No. 30069 Received 26 July 1988; revised 28 September 1988
804
the solid-state fermentation. A previous study 3 of aspen wood delignification by P. tremellosa showed PRL 2845 to be the most effective isolate, and 27.5°C, pH 5, a moisture content of 2 g water g-i wood, no added nutrients, 02 levels above 7%, and incubation for 6 weeks to be the optimal culture conditions. Using these conditions, some variations in vessel design, aeration, wood preparation, sterilization, and inoculation have been tested, and the results are reported here.
Materials and methods Fungus
Strain PRL 2845 of Phlebia tremellosa (= Merulius tremellosus) was used, as in previous studies. 3-5 This isolate came from the culture collection of Forintek Canada Corp. (isolate A-350) and is also available from A T C C (#48745). Wood
Wood from Populus tremuloides trees cut in June near Saskatoon was debarked and air-dried. Unless otherwise indicated, the wood was ground in a Wiley mill to pass a 10 mesh screen. Fermentation conditions
Most of the fermentations were carried out in widemouth, 500-ml cylindrical glass bottles. The bottles were closed with rubber stoppers, fitted with two glass
Enzyme Microb. Technol., 1989, vol. 11, December
© 1989 Butterwo~h Publishers
Aspen delignification with Phlebia tremellosa: I. D. Reid tubes. The air inlet tube reached almost to the bottom of the bottle and the air outlet tube ended just below the rubber stopper. Both tubes were plugged with glass wool. Dry preweighed bottles were filled with approximately 50 g of ground wood, dried overnight in a vacuum oven at 65°C, and weighed again. Then the wood was moistened with 100 ml of distilled water, mixed to distribute the water evenly, and sterilized by autoclaving for 30 min. Unless otherwise indicated, the wood was inoculated with 10 ml of a culture of PRL 2845 grown in 10mM glutamate medium3 and homogenized in a sterile Waring blender for 30 s. The wood was either mixed with a sterile spatula to distribute the inoculum or the inoculum was simply spread evenly over the surface of the wood and allowed to percolate down into it. The bottles were placed in an incubator at 27.5°C. The air inlet tube of each bottle was connected to a manifold which supplied air at 15 ml min -I per bottle. The air was humidified and freed of CO2 by passing it through a dilute NaOH solution warmed to 40°C outside the incubator, followed by a condensate trap inside the incubator. The air leaving each bottle was bubbled through l0 ml of 2 M NaOH solution to trap CO:; the CO: traps were replaced at regular intervals.
Analytical methods Carbon dioxide trapped in NaOH solution was determined by carbonate precipitation with BaCI2 and titration of the residual NaOH with 1 M HC1, using thymol blue as indicator. Initial and final moisture contents of the wood were determined by weighing the filled fermentation bottles and subtracting the weights of the bottles, stoppers, and dry wood determined separately. After fermentation, the wood was dried and weighed in the culture bottles. It was then removed from the bottles and thoroughly mixed before taking samples for further analysis. All dry weights were determined after overnight drying in a vacuum oven at 65°C. Cellulase digestibility and Klason lignin content were measured as previously described 5 on duplicate 0.5-g samples of wood from each fermentation bottle. Duplicate samples of a standard timothy hay (50.7% in vivo digestibility in sheep) were included with each digestibility assay. The cellulase digestibility of this hay measured in our laboratory was 52. I - 0.3%.
Effect of sterilization A freshly cut aspen tree was debarked in the field and brought back to the laboratory in clean plastic bags. It was chipped, reduced to slivers in a hammer-mill with a 1/8-inch screen, and weighed into sterile fermentation bottles (90 g fresh weight per bottle). Both the chipper and the hammer-mill had been disinfected with 70% ethanol. Two of the filled bottles were autoclaved for 30 min at 120°C. Steam was passed through four other filled bottles (in the air inlet tube and out the air outlet) for 30 min. Two nonsterilized bottles, two steamed bottles, and the two autoclaved bottles were inoculated with 25 ml each of a blended culture of PRL 2845. The duplicate noninoculated bottles received 25 ml of sterile water. All the bottles were incubated for 6 weeks and then autoclaved before analysis.
Effect of inoculum type To prepare wood colonized by P. tremeilosa for use as inoculum, Petri plates containing 5 g of 10-mesh aspen wood and l0 ml of water were autoclaved, inoculated with blended mycelium of PRL 2845, and incubated at 27.5°C for 2 weeks. Triplicate fermentation bottles were inoculated as usual with liquid-grown mycelium. Three other bottles were inoculated with an amount of the precolonized wood calculated to contain the same amount of nitrogen. The bottles were incubated under standard conditions for 30 days.
Effect of inoculum quantity Weighed amounts (ca. 50 g) of 10-mesh aspen wood were mixed with twice their weight of water in autoclavable polypropylene bags and autoclaved. Sufficient precolonized wood was then weighed into the bags to give two bags with 2% inoculum, three bags with 5% inoculum, two bags with 10% inoculum, and one bag with 20% inoculum. The contents of each bag were thoroughly mixed by kneading and then mostly transferred to a sterile fermentation bottle. The amount of wood in each bottle was determined by weighing the filled bottles and determining the moisture content of the left-over wood in each bag. The filled bottles were incubated under standard conditions for 6 weeks.
Results
Aeration and fermenter configuration Effect of particle size Chips were cut with a band saw to 40 mm (axial) by 30 mm (tangential) by 4 mm (radial). Shavings were produced on a planer set for a 3-mm (1/8-inch) cut. Hammer-milled wood was prepared from a portion of the shavings in a machine with a 3-mm mesh screen. "10-mesh" and "40-mesh" wood powders were prepared in a Wiley mill with the appropriate screen.
In an initial experiment, the amount of wood added to the 60-ml bottles used previously3 was varied. Both weight loss and digestibility increase showed strong negative correlations with the degree of fullness of the bottle (Figure 1). Above 4 g of wood per bottle (half full), there was tittle increase in digestibility. There was evidence of inadequate aeration: even though the inoculum had been evenly mixed with the wood, the characteristic reddish-brown discoloration resulting
Enzyme Microb. Technol., 1989, vol. 11, December
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Figure I Effect of bottle fullness on solid-state fermentation of aspen wood by P. tremellosa. Eight grams of w o o d filled a bottle. Each bottle received 1.8 ml of water and 0.2 ml of inoculum per gram of wood. The bottles were incubated at 27.5°C for 8 weeks and were flushed with air weekly. The initial digestibility of the wood was 20%. Points are means of three replicates; the bars represent standard errors
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initial value of 20%, and its lignin content had decreased to 13.1% from an initial 21%. These values are similar to those found in small-scale fermentations. 3 In the column, air could enter the entire bottom surface of the wood bed through the fritted glass plate. Glass bottles with a central air inlet tube required less incubator space. However, the central aeration tube might not effectively supply air to the wood near the outside of the bottles. To check this possibility, fermentations were simultaneously conducted in the column, in a bottle as described in Materials and methods, and in a similar bottle with a 2.5-cm layer of 1-cm-diameter glass beads below the wood. The patterns of CO2 production and the compositions of the fermented wood from the three fermentations were similar (Table 1). Evidently the lateral diffusion of air between the wood particles was rapid enough to supply air to all parts of the bottle, even with a simple central aeration tube. This simple configuration was used in all subsequent experiments. Stirring, mixing, or agitation of the fermentations was not required. When the fermentation bottles were continuously or intermittently rolled, or intermittently agitated by shaking, delignification was less than in undisturbed bottles.
Substrate preparation 6
0
E 3 E
~
1'4
2'1
2'a
3'5
4'2
Time (days)
Figure 2 Kinetics of CO2 release from a solid-state fermentation in a column continuously sparged with air at 25 ml min -1. The cylindrical glass column (designed for chromatography) was 6.5 cm i.d. by 30 cm tall, and had a coarse fritted glass plate across its bottom. It received 50 g of wood, 75 ml of water, and 25 ml of inoculum
from the growth of P. tremellosa was only visible in the top 1 cm of the wood layers. Solid-state fermentation with P. tremellosa could be accomplished in deep layers of wood by placing the cultures in continuously aerated columns. Figure 2 shows the kinetics of respiration of such a culture with a wood layer 30 cm deep. There was a 6-day lag period, a period of accelerating activity, and then a period of approximately constant respiration rate. The reddish-brown discoloration developed evenly throughout the wood bed. After 6 weeks, the digestibility of the wood had increased to 56.6% from an
806
Particle size. The Wiley-milled and hammer-milled wood contained particles with diameters between 150 ~m and 2 mm. The chips and the shavings were too large to pass through a screen with 2-mm openings and contained negligible amounts of fines. The patterns of CO2 release from fermentations of the shavings, the hammer-milled wood, and the Wileymilled wood were very similar (Figure 3). The curve for 10-mesh Wiley-milled wood was almost identical to that for 40-mesh wood, and the curve for hammermilled wood was indistinguishable from that for shavings. Development of the fermentation on the chips was slower and more variable than on the smaller particles. There were large air spaces between the chips; the inoculum tended to run off their surfaces, and they were actually colonized by mycelium growing up from the bottoms of the bottles. Lignin loss and digestibility increase were compa-
Table 1 Composition of wood after solid-state fermentation in three fermenter types Fermenter type Column Bottle + beads Plain bottle Original w o o d
Weight loss (%)
Lignin a loss (%)
Digestibility b (% of original dw)
10.8 11.0 10.7 -
43 46 45 -
48.2 50.3 49.2 18.6
a Std. deviation of duplicate determinations was 0.5 b Std. deviation of duplicate determinations was 0.1
Enzyme Microb. Technol., 1989, vol. 11, December
Aspen delignification with Phlebia tremellosa: I. D. Reid
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;
~
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it
2'8
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4'2
Time (days)
Figure 3 Kinetics of CO2 production from solid-state fermentations with different particle sizes. Points are the means of two replicates; bars show standard errors
200
~
rn Raw,Non-inoculated • Raw,Inoculated ~.,~ O Steamed,Non-inoculated O / ~
~o150
Steaming the wood for 30 min greatly decreased the initial rate of respiration (Figure 4). There was a delayed burst of respiratory activity that peaked on day 3 in the noninoculated bottles and then gradually declined. There was no visible growth or sporulation of molds on the steamed wood. The steamed bottles inoculated with P. tremellosa showed higher rates of respiration, which persisted throughout the fermentation. However, the maximum rate of respiration reached in the steamed, inoculated wood was lower than the maximum rate in the autoclaved, inoculated wood. During steaming, considerable amounts of water condensed in the wood; the steamed wood contained 2.8 g of water per gram & d r y weight, compared to 0.65 g of water per gram of dry weight in the unsteamed wood. The steamed, noninoculated wood showed very little weight loss, no lignin loss, and no change in digestibility (Table 3). The steamed, inoculated wood showed relatively high weight loss, moderate lignin loss, and a moderate increase in digestibility. The autoclaved, inoculated wood showed the usual lag period before respiration accelerated (Figure 4) but reached higher rates of respiration than the steamed wood. Weight loss in the autoclaved wood was comparable to that in the steamed wood, but lignin loss and digestibility increase were much higher in the autoclaved wood (Table 3).
Inoculation Inoculum type. The pattern of respiration, including
-~100 '5 E
the lag period, was very similar in fermentations inoculated with P. tremellosa grown on wood to that in fermentations inoculated with liquid-grown mycelium (Figure 5). There were also no significant differences in weight loss, lignin loss, or digestibility between fermentations with the two inoculum types.
C so c)
Time (days)
Rgure 4 Effect of sterilization and inoculation with P. tremelIosa on CO2 evolution from early stages of solid-state fermentations of aspen wood. Points are the means of two replicates; bars show standard errors
Inoculum quantity. Increasing the amount of inoculum added to the wood in the range from 2% to 20% slightly decreased the lag period before rapid respiration began (Figure 6). Thereafter, the acceleration of respiration and the maximum rates of respiration were similar at all inoculum levels.
rable in the shavings and the smaller particles, but significantly less in the chips (Table 2).
Table 2 Effect of particle size on delignification of aspen wood
Sterilization. Wood from a freshly-cut aspen tree was prepared for fermentation under conditions that minimized contamination. The nonsterilized wood, even without inoculation, had a very high initial rate of respiration. After day 4, this respiration gradually declined. Inoculation with P. tremellosus had no effect on CO2 production (Figure 4). Mold growth and sporulation was profuse on the nonsterilized wood. After 6 weeks of incubation, it had lost 7% of its initial dry weight and about 3% of its lignin, its cellulase digestibility was close to that of the original wood
Particle type
(Table 3).
by P. tremellosus
Chips Shavings Hammer-milled 10 mesh 40 mesh Original wood Standard error
Weight loss (%)
Lignin loss (%)
Digestibility (% of original dw)
3.4° 6.4b 6.5 b 4.8ab 4.9°b -+0.5
13 31 34 30 35 -+1
30.5 44.08 42.58 42.38 46.1 18.7 -+0.5
a,bValues in each column followed by the same letter are not significantly different at the 95% probability level, according to the Student-Neuman-Kuels test
Enzyme Microb. Technol., 1989, vol. 11, December
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Papers Table 3
Effect of sterilization on delignification of aspen w o o d by P. tremellosa
Sterilization
Inoculation
None
Weight loss (%)
Lignin loss (%)
Digestibility (% of original dw)
7.1 ab 7.0 a~ 2.2" 16.0 b 0.0 16.26 -+2.2
1.6" 4.7" -4.5 a 26.5 0.0 47,6 -+2.8
16.1" 19.1ab 18.7 ab 29.36 18.0 ab 48.3 -+2.2
+ + +
Steaming Autoclaving Standard error
.,b Values in each column followed by the same letter are not significantly different at the 95% probability level, according to the S t u d e n t - N e u m a n - K u e l s test
There was a small statistically significant increase in delignification and digestibility with increasing amounts of inoculum (Figure 7). The amount of inoculum did not affect the lignin content or the digestibility of the initial wood-inoculum mix.
Discussion As Zadrazil and Brunnert 6 have remarked, solid-state fermentations do not present many opportunities for dynamic control. Conditions must be arranged at the start to guide the fermentation along the desired path. Any deviations from that path while the fermentation is underway are difficult to correct. Consequently much of this report concerns substrate preparation and inoculation. Solid-state fermentations of aspen wood with P. tremellosa require active aeration; passive gas diffusion is only adequate in very shallow layers. I have not determined the minimum or optimum aeration rates, but 0.3 ml air min -~ g-~ wood dry weight seems sufficient. Air circulated freely between the wood
particles, and a single central air inlet effectively aerated the whole fermenter. P. tremellosa does not form much mycelium outside the wood particles. Some other species, including Pleurotus ostreatus, can produce enough external mycelium to block the channels between substrate particles and restrict air circulation. Kamra and Zadrazil 7 found that continuous forced aeration did not improve fermentation of straw with Pleurotus sajor-caju or Pl. eryngii. The optimum particle size is determined by a balance between air diffusion into the particles and circulation between them. Small particle size should facilitate aeration by shortening the distances that gases must diffuse through the internal pores, probably liquid-filled, of the wood to the sites where the hyphae are degrading lignin. However, reducing the particle size increases the packing density of the particles and could restrict air circulation between them. Size reduction is expensive, so it is desirable to use the largest particles consistent with adequate gas exchange. Shavings I/8-inch thick seemed to be the optimum size, although the results with chips were not
200 150 • Liquid inoculum O
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2% 5% 10% 20%
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100 ¢0
~ 100
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o
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1'0
2'0
3'0
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Time (days)
Figure 5
Effect of inoculum type on kinetics of CO2 production. Points are the means of three replicates; bars show standard errors
808
Figure 6 Effect of inoculum quantity on kinetics of C02 production from solid-state fermentations of aspen w o o d with P. tremellosa. Points are the means of three (5%),~t w o (2%, 10%), or one (20%) replicate; bars s h o w standard errors
Enzyme Microb. Technol., 1989, vol. 11, December
Aspen delignification with Phlebia tremellosa: L D. Reid better insulating the vessel, steaming might be an acceptable sterilization method for these fermentations. Pleurotus spp. are similarly unable to overcome competition from the indigenous microflora of straw) Pasteurization did not completely suppress the competition, but anaerobic incubation of the straw for 48 h at 50°C did allow good colonization by Pl. ostreatus and
60 Final
~
Digestibility Lignin loss
50
.,~ 40
Pl. sajor-caju. 8
-o 30 "6 O 20
o
O
Initial ¢3 Digestibility
| Weight loss 10
10 20 Amount of inoculum (%) Figure 7 Effect of inoculum quantity on c o m p o s i t i o n of aspen w o o d delignified by solid-state fermentation with P. tremelIosa. Points are the means of three (5%), t w o (2%, 10%), or one (20%) replicate; bars s h o w pooled standard errors. The standard error for digestibility measurements was 0.4%
definitive; a better inoculation method for the chips might have given better results. The shavings had the lowest packing density of the particle types tested. Sterilization is another expensive part of substrate preparation and should be just intense enough to prevent the growth of harmful contaminating microbes and permit the desired fungus to establish itself. P. tremellosa was unable to effectively colonize clean, but nonsterile, wood. Sterilization by moist heat seems the most suitable method for wood: dry heat would dry the wood, and gas or radiation sterilization requires special equipment and is more expensive. Autoclaving gave better results than treatment with steam at atmospheric pressure. However, steaming did effectively eliminate competing fungi from the wood. Condensation during steaming led to water-logging of the wood, probably resulting in poor gas exchange. If the condensation could be reduced by
The fermentations showed a variable lag period before rapid respiration began. Clearly this lag period should be minimized in a practical fermentation. The lag does not seem to be a period of adaptation to growth on wood or of recovery from damage during homogenization; fermentations inoculated with precoIonized wood showed lags equal to those inoculated with homogenized liquid-grown mycelium. The lag showed a small inverse dependence on the amount of inoculum; this could mean that an inhibitor present in the wood must be destroyed, or a fungal metabolite must accumulate, before rapid growth can proceed. Although weight loss was unaffected by the quantity of inoculum, delignification and the cellulase digestibility of wood fermented for 6 weeks were positively correlated with the amount of inoculum. The implications of these results for design of solid-state fermentation processes for biological delignification are discussed in a companion article. 2
Acknowledgements I thank Mr. Cliff Mallard for expert technical assistance and Dr. R.K. Chaplin for providing a standard hay sample of known in vivo digestibility.
References 1 2 3 4 5 6 7 8
Dale, B. E. and Linden, J. C. Annu. Rept. Ferm. Proc. 1984, 7, 107-134 Reid, I. D. Enzyme Microb. Technol. 1989, U, 786-803 Reid, I. D. Appl. Environm. Microbiol. 1985, 50, 133-139 Mes-Hartree, M., Yu, E. K. C., Reid, I. D. and Saddler, J. N. Appl. Microbiol. Biotechnol. 1987, 26, 120-125 Reid, I. D. and Seifert, K. A. Can. J. Bot. 1982, 60, 252-260 Zadrazil, F. and Brunnert, H. Eur. J. Appl. Microbiol. Biotechnol. 1981, 11, 183-188 Kamra, D. N. and Zadrazil, F. Agric. Wastes 1986, 18, 1-17 Zadrazil, F. and Peerally, A. Biotechnol. Lett. 1986, 8, 663-666
Enzyme Microb. Technol., 1989, vol. 11, December
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The white-rot fungus Phlebia tremellosa can delignify aspen wood and increase the accessibility of its polysaccharides to enzymatic hydrolysis, under solid-state fermentation conditions. Fermentations at the 50 g scale were conducted to provide information for the design of larger ferrnentations. Agitation was not required. Forced aeration was needed for delignification of wood layers more than a few millimeters thick, but air circulation between the particles was not blocked by mycelium. Shavings were the best particles size. Sterilization of the wood was essential, but preadaptation of the inoculum to growth in wood was not. Inoculum levels as low as 2% were adequate.
Keywords: Phlebia tremellosa; delignification; aspen; enzymatic hydrolysis
Introduction Polysaccharides in lignocellulosic materials like wood and straw must be liberated from their association with lignin by a pretreatment before they can be used as fibers in pulp and paper, as feed for ruminant animals, or as sugars after enzymatic hydrolysis. To be commercially viable, any pretreatment method must be effective and inexpensive. This is especially true for the relatively low-price markets of animal feeds and fermentation feedstocks. ~ Biological delignification using white-rot fungi is a potential alternative to chemical and physical methods of delignification, but little quantitative information about its relative costs and benefits is available yet. 2 The ability of the whiterot fungus Phlebia tremellosa (Schrad.:Fr.) Nakas. et Burds. to delignify aspen wood and increase its cellulase digestibility has been demonstrated in small-scale solid-state fermentations. 3 The suitability of aspen wood delignified with P. tremellosa for cellulase hydrolysis and subsequent fermentation has also been established. 4 To produce enough material for further product testing, and to estimate the costs of a biological delignification process, it was necessary to scale-up
Address reprint requests to Dr. Reid at the Biotechnology Research Institute, National Research Council of Canada, 6100 Ave. Royalmount, Montreal, Quebec, Canada H4P 2R2 *Issued as NRCC No. 30069 Received 26 July 1988; revised 28 September 1988
804
the solid-state fermentation. A previous study 3 of aspen wood delignification by P. tremellosa showed PRL 2845 to be the most effective isolate, and 27.5°C, pH 5, a moisture content of 2 g water g-i wood, no added nutrients, 02 levels above 7%, and incubation for 6 weeks to be the optimal culture conditions. Using these conditions, some variations in vessel design, aeration, wood preparation, sterilization, and inoculation have been tested, and the results are reported here.
Materials and methods Fungus
Strain PRL 2845 of Phlebia tremellosa (= Merulius tremellosus) was used, as in previous studies. 3-5 This isolate came from the culture collection of Forintek Canada Corp. (isolate A-350) and is also available from A T C C (#48745). Wood
Wood from Populus tremuloides trees cut in June near Saskatoon was debarked and air-dried. Unless otherwise indicated, the wood was ground in a Wiley mill to pass a 10 mesh screen. Fermentation conditions
Most of the fermentations were carried out in widemouth, 500-ml cylindrical glass bottles. The bottles were closed with rubber stoppers, fitted with two glass
Enzyme Microb. Technol., 1989, vol. 11, December
© 1989 Butterwo~h Publishers
Aspen delignification with Phlebia tremellosa: I. D. Reid tubes. The air inlet tube reached almost to the bottom of the bottle and the air outlet tube ended just below the rubber stopper. Both tubes were plugged with glass wool. Dry preweighed bottles were filled with approximately 50 g of ground wood, dried overnight in a vacuum oven at 65°C, and weighed again. Then the wood was moistened with 100 ml of distilled water, mixed to distribute the water evenly, and sterilized by autoclaving for 30 min. Unless otherwise indicated, the wood was inoculated with 10 ml of a culture of PRL 2845 grown in 10mM glutamate medium3 and homogenized in a sterile Waring blender for 30 s. The wood was either mixed with a sterile spatula to distribute the inoculum or the inoculum was simply spread evenly over the surface of the wood and allowed to percolate down into it. The bottles were placed in an incubator at 27.5°C. The air inlet tube of each bottle was connected to a manifold which supplied air at 15 ml min -I per bottle. The air was humidified and freed of CO2 by passing it through a dilute NaOH solution warmed to 40°C outside the incubator, followed by a condensate trap inside the incubator. The air leaving each bottle was bubbled through l0 ml of 2 M NaOH solution to trap CO:; the CO: traps were replaced at regular intervals.
Analytical methods Carbon dioxide trapped in NaOH solution was determined by carbonate precipitation with BaCI2 and titration of the residual NaOH with 1 M HC1, using thymol blue as indicator. Initial and final moisture contents of the wood were determined by weighing the filled fermentation bottles and subtracting the weights of the bottles, stoppers, and dry wood determined separately. After fermentation, the wood was dried and weighed in the culture bottles. It was then removed from the bottles and thoroughly mixed before taking samples for further analysis. All dry weights were determined after overnight drying in a vacuum oven at 65°C. Cellulase digestibility and Klason lignin content were measured as previously described 5 on duplicate 0.5-g samples of wood from each fermentation bottle. Duplicate samples of a standard timothy hay (50.7% in vivo digestibility in sheep) were included with each digestibility assay. The cellulase digestibility of this hay measured in our laboratory was 52. I - 0.3%.
Effect of sterilization A freshly cut aspen tree was debarked in the field and brought back to the laboratory in clean plastic bags. It was chipped, reduced to slivers in a hammer-mill with a 1/8-inch screen, and weighed into sterile fermentation bottles (90 g fresh weight per bottle). Both the chipper and the hammer-mill had been disinfected with 70% ethanol. Two of the filled bottles were autoclaved for 30 min at 120°C. Steam was passed through four other filled bottles (in the air inlet tube and out the air outlet) for 30 min. Two nonsterilized bottles, two steamed bottles, and the two autoclaved bottles were inoculated with 25 ml each of a blended culture of PRL 2845. The duplicate noninoculated bottles received 25 ml of sterile water. All the bottles were incubated for 6 weeks and then autoclaved before analysis.
Effect of inoculum type To prepare wood colonized by P. tremeilosa for use as inoculum, Petri plates containing 5 g of 10-mesh aspen wood and l0 ml of water were autoclaved, inoculated with blended mycelium of PRL 2845, and incubated at 27.5°C for 2 weeks. Triplicate fermentation bottles were inoculated as usual with liquid-grown mycelium. Three other bottles were inoculated with an amount of the precolonized wood calculated to contain the same amount of nitrogen. The bottles were incubated under standard conditions for 30 days.
Effect of inoculum quantity Weighed amounts (ca. 50 g) of 10-mesh aspen wood were mixed with twice their weight of water in autoclavable polypropylene bags and autoclaved. Sufficient precolonized wood was then weighed into the bags to give two bags with 2% inoculum, three bags with 5% inoculum, two bags with 10% inoculum, and one bag with 20% inoculum. The contents of each bag were thoroughly mixed by kneading and then mostly transferred to a sterile fermentation bottle. The amount of wood in each bottle was determined by weighing the filled bottles and determining the moisture content of the left-over wood in each bag. The filled bottles were incubated under standard conditions for 6 weeks.
Results
Aeration and fermenter configuration Effect of particle size Chips were cut with a band saw to 40 mm (axial) by 30 mm (tangential) by 4 mm (radial). Shavings were produced on a planer set for a 3-mm (1/8-inch) cut. Hammer-milled wood was prepared from a portion of the shavings in a machine with a 3-mm mesh screen. "10-mesh" and "40-mesh" wood powders were prepared in a Wiley mill with the appropriate screen.
In an initial experiment, the amount of wood added to the 60-ml bottles used previously3 was varied. Both weight loss and digestibility increase showed strong negative correlations with the degree of fullness of the bottle (Figure 1). Above 4 g of wood per bottle (half full), there was tittle increase in digestibility. There was evidence of inadequate aeration: even though the inoculum had been evenly mixed with the wood, the characteristic reddish-brown discoloration resulting
Enzyme Microb. Technol., 1989, vol. 11, December
805
Papers 10
~=i~i(
55
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Figure I Effect of bottle fullness on solid-state fermentation of aspen wood by P. tremellosa. Eight grams of w o o d filled a bottle. Each bottle received 1.8 ml of water and 0.2 ml of inoculum per gram of wood. The bottles were incubated at 27.5°C for 8 weeks and were flushed with air weekly. The initial digestibility of the wood was 20%. Points are means of three replicates; the bars represent standard errors
[][]
initial value of 20%, and its lignin content had decreased to 13.1% from an initial 21%. These values are similar to those found in small-scale fermentations. 3 In the column, air could enter the entire bottom surface of the wood bed through the fritted glass plate. Glass bottles with a central air inlet tube required less incubator space. However, the central aeration tube might not effectively supply air to the wood near the outside of the bottles. To check this possibility, fermentations were simultaneously conducted in the column, in a bottle as described in Materials and methods, and in a similar bottle with a 2.5-cm layer of 1-cm-diameter glass beads below the wood. The patterns of CO2 production and the compositions of the fermented wood from the three fermentations were similar (Table 1). Evidently the lateral diffusion of air between the wood particles was rapid enough to supply air to all parts of the bottle, even with a simple central aeration tube. This simple configuration was used in all subsequent experiments. Stirring, mixing, or agitation of the fermentations was not required. When the fermentation bottles were continuously or intermittently rolled, or intermittently agitated by shaking, delignification was less than in undisturbed bottles.
Substrate preparation 6
0
E 3 E
~
1'4
2'1
2'a
3'5
4'2
Time (days)
Figure 2 Kinetics of CO2 release from a solid-state fermentation in a column continuously sparged with air at 25 ml min -1. The cylindrical glass column (designed for chromatography) was 6.5 cm i.d. by 30 cm tall, and had a coarse fritted glass plate across its bottom. It received 50 g of wood, 75 ml of water, and 25 ml of inoculum
from the growth of P. tremellosa was only visible in the top 1 cm of the wood layers. Solid-state fermentation with P. tremellosa could be accomplished in deep layers of wood by placing the cultures in continuously aerated columns. Figure 2 shows the kinetics of respiration of such a culture with a wood layer 30 cm deep. There was a 6-day lag period, a period of accelerating activity, and then a period of approximately constant respiration rate. The reddish-brown discoloration developed evenly throughout the wood bed. After 6 weeks, the digestibility of the wood had increased to 56.6% from an
806
Particle size. The Wiley-milled and hammer-milled wood contained particles with diameters between 150 ~m and 2 mm. The chips and the shavings were too large to pass through a screen with 2-mm openings and contained negligible amounts of fines. The patterns of CO2 release from fermentations of the shavings, the hammer-milled wood, and the Wileymilled wood were very similar (Figure 3). The curve for 10-mesh Wiley-milled wood was almost identical to that for 40-mesh wood, and the curve for hammermilled wood was indistinguishable from that for shavings. Development of the fermentation on the chips was slower and more variable than on the smaller particles. There were large air spaces between the chips; the inoculum tended to run off their surfaces, and they were actually colonized by mycelium growing up from the bottoms of the bottles. Lignin loss and digestibility increase were compa-
Table 1 Composition of wood after solid-state fermentation in three fermenter types Fermenter type Column Bottle + beads Plain bottle Original w o o d
Weight loss (%)
Lignin a loss (%)
Digestibility b (% of original dw)
10.8 11.0 10.7 -
43 46 45 -
48.2 50.3 49.2 18.6
a Std. deviation of duplicate determinations was 0.5 b Std. deviation of duplicate determinations was 0.1
Enzyme Microb. Technol., 1989, vol. 11, December
Aspen delignification with Phlebia tremellosa: I. D. Reid
t
15O
[ [] Chips 1201./ • Shavings ~ / o 4omesh
9°t -~ 6o
;
~
1'4
it
2'8
g
4'2
Time (days)
Figure 3 Kinetics of CO2 production from solid-state fermentations with different particle sizes. Points are the means of two replicates; bars show standard errors
200
~
rn Raw,Non-inoculated • Raw,Inoculated ~.,~ O Steamed,Non-inoculated O / ~
~o150
Steaming the wood for 30 min greatly decreased the initial rate of respiration (Figure 4). There was a delayed burst of respiratory activity that peaked on day 3 in the noninoculated bottles and then gradually declined. There was no visible growth or sporulation of molds on the steamed wood. The steamed bottles inoculated with P. tremellosa showed higher rates of respiration, which persisted throughout the fermentation. However, the maximum rate of respiration reached in the steamed, inoculated wood was lower than the maximum rate in the autoclaved, inoculated wood. During steaming, considerable amounts of water condensed in the wood; the steamed wood contained 2.8 g of water per gram & d r y weight, compared to 0.65 g of water per gram of dry weight in the unsteamed wood. The steamed, noninoculated wood showed very little weight loss, no lignin loss, and no change in digestibility (Table 3). The steamed, inoculated wood showed relatively high weight loss, moderate lignin loss, and a moderate increase in digestibility. The autoclaved, inoculated wood showed the usual lag period before respiration accelerated (Figure 4) but reached higher rates of respiration than the steamed wood. Weight loss in the autoclaved wood was comparable to that in the steamed wood, but lignin loss and digestibility increase were much higher in the autoclaved wood (Table 3).
Inoculation Inoculum type. The pattern of respiration, including
-~100 '5 E
the lag period, was very similar in fermentations inoculated with P. tremellosa grown on wood to that in fermentations inoculated with liquid-grown mycelium (Figure 5). There were also no significant differences in weight loss, lignin loss, or digestibility between fermentations with the two inoculum types.
C so c)
Time (days)
Rgure 4 Effect of sterilization and inoculation with P. tremelIosa on CO2 evolution from early stages of solid-state fermentations of aspen wood. Points are the means of two replicates; bars show standard errors
Inoculum quantity. Increasing the amount of inoculum added to the wood in the range from 2% to 20% slightly decreased the lag period before rapid respiration began (Figure 6). Thereafter, the acceleration of respiration and the maximum rates of respiration were similar at all inoculum levels.
rable in the shavings and the smaller particles, but significantly less in the chips (Table 2).
Table 2 Effect of particle size on delignification of aspen wood
Sterilization. Wood from a freshly-cut aspen tree was prepared for fermentation under conditions that minimized contamination. The nonsterilized wood, even without inoculation, had a very high initial rate of respiration. After day 4, this respiration gradually declined. Inoculation with P. tremellosus had no effect on CO2 production (Figure 4). Mold growth and sporulation was profuse on the nonsterilized wood. After 6 weeks of incubation, it had lost 7% of its initial dry weight and about 3% of its lignin, its cellulase digestibility was close to that of the original wood
Particle type
(Table 3).
by P. tremellosus
Chips Shavings Hammer-milled 10 mesh 40 mesh Original wood Standard error
Weight loss (%)
Lignin loss (%)
Digestibility (% of original dw)
3.4° 6.4b 6.5 b 4.8ab 4.9°b -+0.5
13 31 34 30 35 -+1
30.5 44.08 42.58 42.38 46.1 18.7 -+0.5
a,bValues in each column followed by the same letter are not significantly different at the 95% probability level, according to the Student-Neuman-Kuels test
Enzyme Microb. Technol., 1989, vol. 11, December
807
Papers Table 3
Effect of sterilization on delignification of aspen w o o d by P. tremellosa
Sterilization
Inoculation
None
Weight loss (%)
Lignin loss (%)
Digestibility (% of original dw)
7.1 ab 7.0 a~ 2.2" 16.0 b 0.0 16.26 -+2.2
1.6" 4.7" -4.5 a 26.5 0.0 47,6 -+2.8
16.1" 19.1ab 18.7 ab 29.36 18.0 ab 48.3 -+2.2
+ + +
Steaming Autoclaving Standard error
.,b Values in each column followed by the same letter are not significantly different at the 95% probability level, according to the S t u d e n t - N e u m a n - K u e l s test
There was a small statistically significant increase in delignification and digestibility with increasing amounts of inoculum (Figure 7). The amount of inoculum did not affect the lignin content or the digestibility of the initial wood-inoculum mix.
Discussion As Zadrazil and Brunnert 6 have remarked, solid-state fermentations do not present many opportunities for dynamic control. Conditions must be arranged at the start to guide the fermentation along the desired path. Any deviations from that path while the fermentation is underway are difficult to correct. Consequently much of this report concerns substrate preparation and inoculation. Solid-state fermentations of aspen wood with P. tremellosa require active aeration; passive gas diffusion is only adequate in very shallow layers. I have not determined the minimum or optimum aeration rates, but 0.3 ml air min -~ g-~ wood dry weight seems sufficient. Air circulated freely between the wood
particles, and a single central air inlet effectively aerated the whole fermenter. P. tremellosa does not form much mycelium outside the wood particles. Some other species, including Pleurotus ostreatus, can produce enough external mycelium to block the channels between substrate particles and restrict air circulation. Kamra and Zadrazil 7 found that continuous forced aeration did not improve fermentation of straw with Pleurotus sajor-caju or Pl. eryngii. The optimum particle size is determined by a balance between air diffusion into the particles and circulation between them. Small particle size should facilitate aeration by shortening the distances that gases must diffuse through the internal pores, probably liquid-filled, of the wood to the sites where the hyphae are degrading lignin. However, reducing the particle size increases the packing density of the particles and could restrict air circulation between them. Size reduction is expensive, so it is desirable to use the largest particles consistent with adequate gas exchange. Shavings I/8-inch thick seemed to be the optimum size, although the results with chips were not
200 150 • Liquid inoculum O
T~1~14~ A.~150
o O o •
2% 5% 10% 20%
o
100 ¢0
~ 100
"o
o
E 5O
C 50
o" o
/ 0
1'0
2'0
3'0
Time (days)
Time (days)
Figure 5
Effect of inoculum type on kinetics of CO2 production. Points are the means of three replicates; bars show standard errors
808
Figure 6 Effect of inoculum quantity on kinetics of C02 production from solid-state fermentations of aspen w o o d with P. tremellosa. Points are the means of three (5%),~t w o (2%, 10%), or one (20%) replicate; bars s h o w standard errors
Enzyme Microb. Technol., 1989, vol. 11, December
Aspen delignification with Phlebia tremellosa: L D. Reid better insulating the vessel, steaming might be an acceptable sterilization method for these fermentations. Pleurotus spp. are similarly unable to overcome competition from the indigenous microflora of straw) Pasteurization did not completely suppress the competition, but anaerobic incubation of the straw for 48 h at 50°C did allow good colonization by Pl. ostreatus and
60 Final
~
Digestibility Lignin loss
50
.,~ 40
Pl. sajor-caju. 8
-o 30 "6 O 20
o
O
Initial ¢3 Digestibility
| Weight loss 10
10 20 Amount of inoculum (%) Figure 7 Effect of inoculum quantity on c o m p o s i t i o n of aspen w o o d delignified by solid-state fermentation with P. tremelIosa. Points are the means of three (5%), t w o (2%, 10%), or one (20%) replicate; bars s h o w pooled standard errors. The standard error for digestibility measurements was 0.4%
definitive; a better inoculation method for the chips might have given better results. The shavings had the lowest packing density of the particle types tested. Sterilization is another expensive part of substrate preparation and should be just intense enough to prevent the growth of harmful contaminating microbes and permit the desired fungus to establish itself. P. tremellosa was unable to effectively colonize clean, but nonsterile, wood. Sterilization by moist heat seems the most suitable method for wood: dry heat would dry the wood, and gas or radiation sterilization requires special equipment and is more expensive. Autoclaving gave better results than treatment with steam at atmospheric pressure. However, steaming did effectively eliminate competing fungi from the wood. Condensation during steaming led to water-logging of the wood, probably resulting in poor gas exchange. If the condensation could be reduced by
The fermentations showed a variable lag period before rapid respiration began. Clearly this lag period should be minimized in a practical fermentation. The lag does not seem to be a period of adaptation to growth on wood or of recovery from damage during homogenization; fermentations inoculated with precoIonized wood showed lags equal to those inoculated with homogenized liquid-grown mycelium. The lag showed a small inverse dependence on the amount of inoculum; this could mean that an inhibitor present in the wood must be destroyed, or a fungal metabolite must accumulate, before rapid growth can proceed. Although weight loss was unaffected by the quantity of inoculum, delignification and the cellulase digestibility of wood fermented for 6 weeks were positively correlated with the amount of inoculum. The implications of these results for design of solid-state fermentation processes for biological delignification are discussed in a companion article. 2
Acknowledgements I thank Mr. Cliff Mallard for expert technical assistance and Dr. R.K. Chaplin for providing a standard hay sample of known in vivo digestibility.
References 1 2 3 4 5 6 7 8
Dale, B. E. and Linden, J. C. Annu. Rept. Ferm. Proc. 1984, 7, 107-134 Reid, I. D. Enzyme Microb. Technol. 1989, U, 786-803 Reid, I. D. Appl. Environm. Microbiol. 1985, 50, 133-139 Mes-Hartree, M., Yu, E. K. C., Reid, I. D. and Saddler, J. N. Appl. Microbiol. Biotechnol. 1987, 26, 120-125 Reid, I. D. and Seifert, K. A. Can. J. Bot. 1982, 60, 252-260 Zadrazil, F. and Brunnert, H. Eur. J. Appl. Microbiol. Biotechnol. 1981, 11, 183-188 Kamra, D. N. and Zadrazil, F. Agric. Wastes 1986, 18, 1-17 Zadrazil, F. and Peerally, A. Biotechnol. Lett. 1986, 8, 663-666
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