Wood Degradation by Thermotolerant and Thermophilic Fungi for

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Wood degradation by thermotolerant and thermophilic fungi for sustainable heat production Leire Caizan Juanarena, Annemiek Ter Heijne, Cees J.N. Buisman, and Annemieke van der Wal ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00914 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016

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ACS Sustainable Chemistry & Engineering

Wood degradation by thermotolerant and thermophilic fungi for sustainable heat production Leire Caizán Juanarena a*, Annemiek Ter Heijne a, Cees J. N. Buisman a,b, Annemieke van der Wal c a

Sub-Department of Environmental Technology, Wageningen University, Bornse Weilanden 9, P.O. Box

8129, 6708 WG Wageningen, The Netherlands. b

Wetsus, Centre of Excellence for Sustainable Water Technology, Agora 1, P.O. Box 1113, 8900 CC

Leeuwarden, The Netherlands. c

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg

10, 6708 PB Wageningen, The Netherlands. * Corresponding author. Tel.: +31 617584653; E-mail address: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT The use of renewable biomass for production of heat and electricity plays an important role in the circular economy. Degradation of wood biomass to produce heat is a clean and novel process proposed as an alternative to wood burning, and could be used for various heating applications. So far, wood degradation has mostly been studied at ambient temperatures. However, the process needs to occur at elevated temperatures (40-55 °C) to produce useable heat. Our objective was to study wood degradation at elevated temperatures for its potential application on heat production. Two (a thermotolerant and a thermophilic) fungi with different degradation strategies were chosen: lignindegrading Phanerochaete chrysosporium and cellulose-degrading Chaetomium thermophilum. Each fungus was inoculated on non-sterile and sterile birch woodblocks to respectively study their wood degradation activity with and without natural biota (i.e., microorganisms naturally present in wood). The highest wood decay rates were found with C. thermophilum in presence of natural biota, followed by P. chrysosporium under sterile conditions. The estimated theoretical value of heat production with C. thermophilum under non-sterile conditions was 0.6 W kg-1 wood. In conclusion, C. thermophilum seems a promising fungus to degrade wood together with natural biota, as sterilization of wood is not feasible in practice. Further testing on larger scale is needed to implement the obtained results and validate the potential of biological wood degradation for heat production. Key words: wood, bio-oxidation, heat, high temperature, oxygen, mass loss


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INTRODUCTION The rising world energy consumption, depletion of fossil fuels and the increasing environmental concerns have led to the necessity of alternative energy sources that are clean and renewable 1. Waste management systems that fully use agricultural, forestry and municipal wastes will be essential to enable the circular economy. Biomass (plant material and animal waste) is currently the major global contributor to renewable energy (14% of the total world’s energy consumption 2). It has the potential to convert into three main forms of energy via bio-chemical/biological or thermo-chemical treatments: electricity, transportation fuels and chemicals 3. Special attention has been paid to lignocellulosic biomass, which represents about 50% of the total biomass in the world and have an estimated annual production of 10-50 billion tonnes 4. A big advantage of using lignocellulosic biomass as an energy source is that it does not compete with food production and can be widely found as low cost agricultural feedstock, which makes it a sustainable alternative to fossil fuels 5. Due to the increasing expansion of agro-industrial activity, large quantities of lignocellulosic residues are being accumulated every year in the form of waste streams (e.g., pulp and paper mill and food industry), energy crops (e.g., switchgrass and Miscanthus), agricultural residues (e.g., corn stover and wheat straw) and wood 5. In contrast to the easily degradable sugars and oils extracted from arable crops to produce first generation biofuels, the above mentioned lignocellulosic biomass is more difficult to convert into valuable products due to its complex structure formed by long-chain molecules, referred to as cellulose (40%), hemicellulose (20-30%) and lignin (2030%) 6. Combustion of wood is the most common way, practised in many places around the world, to use lignocellulosic biomass for heating. However, this process creates environmental problems due to the high amounts of fine particles and volatile organic compounds released to the atmosphere 7. Rather



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than through combustion, wood could also be degraded by microorganisms in the presence of oxygen, an exothermic oxidation reaction in which only carbon dioxide and water vapour are released to the atmosphere (Eq. (1)). CH1.5O0.7+ 1.03 O2 CO2 + 0.734 H2O + Heat

(1)

The heat produced in this reaction, usually seen as a main drawback in large-scale solid-state fermentation (SSF) processes 8, is considered now to be a renewable source of energy. In fact, biological wood degradation has been proposed as a potential new technology for sustainable heat production at temperatures around 40-55 °C 9. Many applications need heat at such temperatures, for instance, warming-up of swimming pools or low temperature heating systems (e.g., floor heating) in houses and other kind of buildings (e.g., schools, offices, stables). The use of local lignocellulosic waste materials (from woods, orchards or tree nurseries, among others) to provide heat on these areas might result in a considerable reduction of natural gas usage. An additional advantage of this technology is that the residue from the process is peat instead of ash. Peat soil is rich in stable organic components and can be used as fertilizer, closing the carbon cycle and restoring soil organic matter. Wood degradation in nature is the result of simultaneous action of several microorganisms

10

.

Saprotrophic fungi are the major contributors to this process, and can be divided in three main groups: white-rot fungi are capable of breaking down lignin, cellulose and hemicellulose; brown-rot fungi do not degrade lignin but only partially modify it to reach and degrade carbohydrate polymers; and soft-rot fungi can only degrade cellulose and hemicellulose 11. Apart from fungi, bacterial activity has also been reported during wood decay. Actinomycetes are bacteria showing hyphal growth, which allows them to degrade insoluble organic polymers like chitin or cellulose. Together with rot-degrading fungi, bacteria can create interactions that might be of high importance for wood decay 12,13. While wood degradation


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has widely been studied at ambient temperatures, little is known about wood degradation and interactions between fungi and bacteria at elevated temperatures (40-55 °C). Our objective, therefore, was to study wood degradation at elevated temperatures and in addition, estimate heat production from this process. We inoculated sterile and non-sterile woodblocks with two thermotolerant and thermophilic fungi (Phanerochaete chrysosporium and Chaetomium thermophilum) to study the effect of their (individual) interaction with natural biota (wood-inhabiting microorganisms) on wood degradation. P. chrysosporium is the model organism among white-rot fungi and an effective lignin degrader, while the soft-rot fungus C. thermophilum is an active cellulose degrader. We analyzed wood degradation based on wood mass loss and oxygen consumption. This latter one was correlated to the amount of heat produced during this oxidation reaction.

MATERIALS AND METHODS Fungal strains and growth conditions Two saprotrophic fungal species were selected to assist wood degradation based on their ecological strategy and resistance to elevated temperatures: Phanerochaete chrysosporium (CBS 316.75), originally isolated from wood material in Indonesia, and Chaetomium thermophilum (CBS 180.67), originally isolated from straw and leaf mold in California. P. chrysosporium has an optimum growth temperature between 36 and 45 °C and a maximum growth temperature of 46-55 °C, while C. thermophilum can optimally grow between 40 and 52.5 °C and has its maximum growth temperature at 54-61 °C, depending on the type of experiment

14

. Based on the definition by Conney and Emerson (1964)

15

,

thermophilic fungi have a maximum growth temperature of 50 °C or higher, while thermotolerant fungi have a maximum growth temperature of about 50 °C. Crisan (1973)

16

defined thermophilic fungi as

those with an optimum temperature of 40 °C or higher. Therefore, while C. thermophilum can definitely



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be characterized as a thermophilic, P. chrysosporium could also be referred as thermotolerant based on its slightly lower optimum and maximum growth temperatures. Fungal strains were incubated on Malt Extract Agar (MEA) and Potato Dextrose Agar (PDA) to check whether they had any preferred medium to grow on. As no differences were found, fungi were incubated in PDA at 41 °C throughout the entire period of study. New agar plates were inoculated every two months in order to ensure continuous fungal growth. Characterization and preparation of wood Branches of a birch tree (hybrid of Betula pendula and Betula pubescens) were collected in October 2013 and January 2014 in Rhenen, the central area of the Netherlands (51°57'33'' N, 5°34'5'' E). Selected branches had a diameter of 1.5 cm approximately and were cut into pieces of 1.5 cm long. Every woodblock was numbered, weighed (fresh and dry weight, when applicable) and associated to a certain experiment and replicate. The chemical composition of birch was not determined, instead the following general composition of wood was assumed: 49-51% carbon (C), 43-44% oxygen (O), 6-7% hydrogen (H) and around 1% of nitrogen (N), sulfur (S) and other inorganic compounds

17

. From these values, the

approximate molecular composition of wood was determined to be CH1.5O0.7 (the rest of components were not considered due to their low amount). For experiments with sterile wood, woodblocks were first dried for 72 hours at 70 °C. Hereafter, they were sterilized in a steamer (inside closed glass vessels) with demineralized water for 21 minutes within an interval of 24 hours 18. Pre-inoculation of woodblocks with fungal species Woodblocks were pre-inoculated to have sufficient initial activity for wood degradation. Small agar plugs (around 2 mm2) were taken from the periphery of fungal colonies of P. chrysosporium and C. thermophilum and inoculation of woodblocks was carried out by positioning one agar plug from one of the fungal species on top of each woodblock. Inoculated woodblocks were then placed inside pots of


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100 cm3 (one per pot) containing 2 g of wet vermiculate and incubated at 41 °C for twenty days. The lids of the pots were loosely closed and surrounded with parafilm in order to allow oxygen exchange. To determine the extent of wood degradation during incubation, six woodblocks of each fungus were sacrificed after this period and their average mass loss was calculated. Experimental set-up The experimental set-up is shown in Figure 1. Bottles (500 cm3) were filled with 6 g of sterile vermiculite to completely cover the bottom of the bottles. 15 cm3 of autoclaved water were added to vermiculite (2.5 cm3 g-1) to reach 250% of moisture level, which was considered enough to maintain the moisture content of a bottle during the whole period of experiments. A nitrogen (NH4NO3) solution was added to sterile and non-sterile woodblocks to prevent that nitrogen was limiting microbial growth. The amount and concentration of nitrogen solution added was determined by the dry weight of each woodblock with the aim to reach a nitrogen content of 3% (dry weight basis) combined with a moisture content of 42.6%. All bottles were closed with lids and septa. Temperature was maintained at 41 °C.

Figure 1: Set-up of experiments: sterile and non-sterile woodblocks with (P. c. /C. t.) and without (control) fungal inoculation. Five replicates were prepared for each of the six scenarios.



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Measurement of oxygen In every bottle, oxygen (O2) concentration was measured with gas chromatography. Although wood degradation is normally determined via CO2 production

19–22

, we measured oxygen concentrations to

determine wood degradation activity. Oxygen consumption can be used not only to determine wood degradation itself

23,24

, but it is also the main indicator to measure and control heat production in

degradation processes of organic substances, e.g. composting

25,26

. Therefore, oxygen measurements

can be also used to estimate heat production from wood. Oxygen was measured every two to four days. When the oxygen level was equal to or below 5% (value at which O2 was considered depleted 27), bottles were opened in the flow cabinet to refresh their inside air and make sure the concentration of oxygen returned to atmospheric levels. The gas chromatograph used for oxygen measurements was Thermo ScientificTM FOCUSTM Gas Chromatograph. It had one Instant Connect Split/Splitless (SSL) injector and one Rt-Qplot column (30 m x 0.53 mm). The carrier gas was helium (at 10 kPa pressure and 20 cm3 min-1 of column flow) and it had a thermal conductivity (TCD) detector. 0.05 cm3 of sample were injected with a syringe. Chromeleon software was used to analyze data from the chromatograph. The results were expressed as percentage (v/v) and transformed to oxygen consumption by calculating the difference with the atmospheric value (21% of oxygen) in case the bottle was opened after the previous gas analysis, or with the value of the previous measuring point in case the bottle was kept closed since then. Moisture content, mass loss and decay rate calculations of woodblocks Moisture content (dry weight basis) of each individual sterile woodblock was calculated before and after the experiment according to Eq. (2). % = ℎ !"ℎ " − $% !"ℎ"⁄$% !"ℎ " ∙ 100%

(2)


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In the case of non-sterile woodblocks, moisture content was directly calculated once the experiment was finished. However, prior to the experiment woodblocks could not be dried as the natural biota had to be kept alive. Therefore, only few of them were selected and separately dried in order to deduce their initial dry weight. From there, moisture content of each woodblock was calculated and the average of all those values was used to determine the approximated initial dry weight of each woodblock that could not be dried, also with Eq. (2). Mass loss of all woodblocks was determined based on their initial and final dry weight (at the end of the experiment) as indicated in Eq. (3). Fungal and bacterial biomass was not calculated nor subtracted from the dry weights used for this equation. Because this biomass is considered to only represent a few percentage (around 5%) of the total wood mass 28,29, its contribution can be considered negligible. ( ) % = *() +% !"ℎ " − () +% !"ℎ"⁄*() +% !"ℎ " ∙ 100%

(3)

From the final mass loss of woodblocks and with a first order differential equation, wood decay rates (k) were calculated (see Eq. (4)): (4)

+, ⁄+ = −- ∗ ;

where x is the amount of wood in gram and t is time in days. Heat production As the small scale of the experiment did not allow actual measurements of heat production, we related oxygen consumption to heat production as commonly done in biomass combustion also in microbial respiratory metabolisms

30,31

processes and

32,33

. Theoretical heat production of the reaction was

calculated from the cumulative oxygen consumption reached during the whole experiment. A linear equation was associated to the oxygen consumption and, from the slope (mmol g-1 d-1), heat was calculated (in W kg-1) by using the heat released in aerobic respiration of glucose (686 kcal mole-1) 32, as



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this is the more abundant carbohydrate present in lignocellulosic materials 34. Because in the reaction (see Eq. 5) 1 mole of glucose reacts with 6 moles of oxygen, the value we use to calculate heat production based on oxygen is 467.5 kJ mol-1 O2. C6H12O6+ 6 O2 6 CO2 + 6 H2O

(5)

Statistical analysis All data were analyzed per scenario with one-Way ANOVA at p< 0.05 using IBM SPSS Statistics 20, using the following factors: type of fungal inoculation (P. chrysosporium or C. thermophilum), and presence of natural biota (sterile or non-sterile woodblocks). The assumption of normality was tested with ShapiroWilk statistics and homogeneity of variance was assessed with Levene’s test. In case the variance between groups was high or the number of samples between groups was unequal, Welch’s t test was performed.

RESULTS AND DISCUSSION Wood degradation by P. chrysosporium and C. thermophilum under sterile conditions Under sterile conditions, so in absence of natural biota, P. chrysosporium consumed 4 mmol g-1 of oxygen after 36 days of incubation, which was higher than the 2 mmol g-1 of oxygen consumed by C. thermophilum (Figure 2, left) after the same time period (P< 0.01). Mass loss of woodblocks was also higher in the case of P. chrysosporium (Table 1), as the value reached was 12.2% compared to the 6.5% of C. thermophilum (P< 0.01).


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Figure 2: Cumulative oxygen consumption (mmol g-1 DM) for sterile (left) and non-sterile (right) woodblocks with fungal inoculation of P. chrysosporium (P. c.) (□) or C. thermophilum (C. t.) (o) and without fungal inoculation (controls) (-, x).

A closer look at the oxygen consumption pattern of both fungal species revealed similar oxygen consumption during the first 17 days. During this time period, both fungi colonized the rest of woodblocks within the bottles. It is expected that soluble and easily degradable carbon sources, such as monosaccharides, starch and lipids, are first degraded

14

. After day 17, however, P. chrysosporium

showed a clear increase in wood decay activity over C. thermophilum, since its oxygen consumption became higher over time. This might be due to the ability of P. chrysosporium to degrade lignin and, therefore, give access to carbohydrates within the lignocellulosic matrix. The enzymatic hydrolysis of cellulose is also known to increase in rate and extent when lignin is removed

35

. In contrast, C.

thermophilum was probably not able to reach all carbohydrates within this time period, since we found a low oxygen consumption rate in the last 19 days as well as a low final mass loss. Wood degradation by P. chrysosporium and C. thermophilum under non-sterile conditions A large difference in oxygen consumption for C. thermophilum was observed when comparing sterile (Figure 2, left) and non-sterile conditions (Figure 2, right). Together with the natural biota, C. thermophilum achieved a cumulative oxygen consumption (4.4 mmol g-1) that doubled the value 11 ACS Paragon Plus Environment


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reached with sterilized woodblocks after 36 days of incubation (P< 0.01). In addition, after 36 days, a higher but not significant mass loss was observed in non-sterile woodblocks (11%) compared with sterile ones (6.5%) (P= 0.06, Table 1). This increase in oxygen consumption in non-sterile woodblocks may be attributed to synergistic interactions between C. thermophilum and sugar fungi that were naturally present in wood. Sugar fungi are not able to degrade cellulose, but they can uptake inhibitory sugars leading to an increased enzymatic activity and thereby an increased wood degrading ability of the cellulolytic fungi

35–37

. In contrast, P. chrysosporium achieved around 1 mmol g-1 higher cumulative

oxygen consumption after 36 days of incubation under sterile conditions (Figure 2, left) than when acting together with natural biota in wood (Figure 2, right) (P< 0.01). Mass loss, however, was not higher with sterile woodblocks (12%) than with non-sterile ones (8.3%) (P= 0.32, Table 1). The presence of natural biota, therefore, had a negative effect on wood degradation activity of P. chrysosporium, in contrast to C. thermophilum. This is in line with studies performed at ambient temperatures, where it was found that competitive interactions are common among wood-rot fungi and may increase or decrease wood decay rates, depending on the combination of the species 38. This lower degradation activity in presence of natural biota can be due to other fungi or bacteria unable to overcome the lignin barrier and that compete for low molecular weight intermediates released by the white-rot fungus 8. In addition, some bacteria can secrete harmful substances, such as antibiotics 39 or volatile compounds 40, that slow down growth and wood decay activity of fungi. More specifically for P. chrysosporium, Radtke and co-workers 37 showed the antagonistic effect of several soil bacteria on the growth of this fungus by the secretion of phenazines. Nevertheless, the presence of these or other harmful substances as well as the competition mechanisms between inoculated fungi and other organisms, cannot be assured with complete certainty as no chemical nor genetic analyses were performed for non-sterile woodblocks. Oxygen consumption in the control treatment under non-sterile conditions (i.e., natural biota alone) was lower than together with C. thermophilum (P= 0.01). Also the mass loss achieved in the control was 12 ACS Paragon Plus Environment

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lower than with the cellulolytic fungus (P= 0.04). However, when P. chrysosporium was added to the natural biota, no improvement in oxygen consumption (P= 0.09) or mass loss (P= 0.36) was found compared to the non-sterile control. The addition of P. chrysosporium to non-sterile woodblocks, therefore, does not contribute to increasing wood degradation rates. For the sterile control, cumulative oxygen consumption (< 0.5 mmol g-1) and mass loss (2.4%) remained lower than in the other scenarios, as expected. Visual inspection of the woodblocks at the end of the experiment showed diversity of fungal species in non-sterile woodblocks both with and without inoculation of thermotolerant and thermophilic fungi, while only one fungal species could be seen in sterile woodblocks (see Figure 3). This observation reflects the higher variability of oxygen consumption and mass loss values found among replicates with non-sterile woodblocks. Microbial community, however, was not further analyzed and characterized.



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Figure 3: Visible fungal growth under different conditions: P. c. sterile (A), C. t. sterile (B), P. c. nonsterile (C), C. t. non-sterile (D), control non-sterile (E), and control sterile (F).

Heat production Biomass is a renewable energy source, and can be used to produce heat and electricity. Although biomass can originate from many different sources, wood is by far the most common. Biological wood degradation by fungi and/or natural biota has been proposed as a novel technology to recover sustainable heat 9, as it is an exothermic reaction driven by microorganisms that produces heat. Since no direct measurements were done for heat production (but rather temperature was controlled), heat production was estimated from oxygen consumption values achieved during the study (see Table 1). The highest heat production (0.63 W kg-1) was obtained by C. thermophilum under non-sterile conditions, which achieved much higher values than the same fungus alone under sterile conditions (0.28 W kg-1). In the second place, P. chrysosporium alone achieved a theoretical heat production of 0.60 W kg-1, which 14 ACS Paragon Plus Environment

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was higher than the same fungus together with the natural biota (0.40 W kg-1). Between these treatments, natural biota (non-sterile woodblocks without inoculation of a fungus) was placed with a value of 0.47 W kg-1. The pattern of mass loss was similar to the pattern of heat production (or oxygen consumption) in all scenarios except for the non-sterile control. Table 1: Theoretical heat production (W kg-1 DM) and wood mass loss (%) with P. chrysosporium (P. c.) and C. thermophilum (C. t.) on sterile and non-sterile woodblocks. Ordered from highest to lowest values of heat production.

Type of treatment

Heat production (W kg-1 DM)

Mass loss (%)

C. t. Non-Sterile

0.63

11.0

P. c. Sterile

0.60

12.2

Control Non-Sterile

0.47

4.7

P. c. Non-Sterile

0.40

8.3

C. t. Sterile

0.28

6.5

Control Sterile

0.04

2.4

In conclusion, P. chrysosporium without natural biota (sterile) and C. thermophilum together with natural biota (non-sterile) achieved the highest wood degradation rates in this study. The natural biota alone, without addition of a fungus, also produced considerable amount of heat, however, wood mass loss was very low.

OUTLOOK P. chrysosporium alone achieved the highest degradation of wood, higher than the naturally occurring biota. From a practical point of view, however, wood sterilization is a laborious and energy consuming process that is not suitable for use at larger scale, i.e., in a bioreactor system. Because performance of P. chrysosporium in combination with the natural biota was not optimal, this fungus does not seem suitable for wood decay processes on larger scale. In contrast, C. thermophilum achieved higher 15 ACS Paragon Plus Environment


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degradation rates when acting together with the natural biota than on sterile woodblocks. Therefore, the inoculation or presence of this latter fungus is a potential strategy to increase wood degradation rate. When no fungus was inoculated, degradation of wood by natural biota happened at a lower extent, but it could also lead to relevant wood decay rates and heat production. Nevertheless, wood decay rates with other tree species that might have different structure, chemical composition or natural biota, remain to be tested. In the present study using only birch wood, we found much more variation among replicates of inoculated non-sterile woodblocks than in sterile woodblocks. This is consistent to the finding that a large variety of fungal communities can be found in individual tree stumps present in the same forest plot, and that these different fungal communities can explain variation in wood decay rates even under similar environmental conditions 41. Therefore it is likely that, as a consequence of the diverse fungal and bacterial communities present in the woodblocks, as well as their interactions, larger differences in wood decay activities were found under non-sterile conditions; these differences being less pronounced under sterile conditions. This variability is an issue to take into consideration when scaling up the system, as even different individual trees of the same species can contain different microbial communities. In addition, the use of different tree species will add an extra variability factor on wood decay since different wood properties will affect composition and activity of degrader communities as well

13

. All of these elements will lead to changes in wood degradation

efficiency and process stability and are important to study in more detail. Another important parameter to take into account is aeration. Additional experiments performed with longer exposition time periods of woodblocks to atmospheric oxygen (data not shown) reflected that in the current experimental design (batch system) oxygen was a limiting factor in the biological wood degradation. Its continuous and homogeneous supply is, therefore, crucial. Richard and co-workers

42

developed mathematical models that related oxygen concentration with biodegradation kinetics in aerobic solid-state systems. They found that degradation rates at low temperatures (below 50 °C) were 16 ACS Paragon Plus Environment

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highest at atmospheric oxygen level (21%) than at low oxygen concentrations (1-4%), while at higher temperature (55-65 °C) degradation rates were increased at lower oxygen levels. Moisture content is also known to affect oxygen diffusion and solubility as well as microbial activity. In fact, too high levels can limit oxygen transport, decrease degradation rates and stimulate anaerobic processes 43. Regarding the optimal temperature for wood degradation, we found that P. chrysosporium at 46 °C degraded only 10% of the wood mass loss achieved at 41 °C (data not shown), which means this temperature was too high for this fungus to grow and degrade the substrate. Tuomela and co-workers 14 stated that white-rot fungi, which are the most efficient lignin degraders, are not able to survive the thermophilic phase (40-60 °C) of composting and, therefore, their role on lignin degradation will be limited under these conditions. Oxygen uptake rate is known to increase at thermophilic conditions over mesophilic ones 44, so if low temperatures (40-50 °C) are applied, degradation would result slow and heat production would not be feasible. Wood degradation by C. thermophilum, however, was not affected at 46 °C (data not shown) as it can handle temperatures up to 52.5 °C 14. Besides, even though cellulolytic fungi are not capable of degrading lignin, they can degrade cellulose and hemicellulose very efficiently. These facts make them better candidates for wood degradation processes with heat generation purposes. Further research on biological degradation of wood will lead to a larger insight on the optimum conditions of the process, and the way it can be best controlled with the purpose to reach high decay rates and heat production levels. Only when degradation is fast and efficient, it will be possible to scaleup the process and make it applicable to close waste cycles and enable the circular economy. With that purpose, parameters like availability of nutrients (N, P), wood structure and composition, surface-areato-volume ratio of woodblocks, moisture content and process temperature, among others, should be further studied in order to investigate their effect on microbial growth and degradation mechanisms of wood. At large scale level, special attention must be paid to commonly occurring problems such as 17 ACS Paragon Plus Environment


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heterogeneous distribution of the inoculum, unsuitable or uneven oxygen and carbon dioxide amounts, unfavorable and irregular wood moisture content or temperature increase beyond 50 °C 45.

ACKNOWLEDGMENTS We thank Vinnie de Wilde (Wageningen University) for his advice in the experimental set-up and his technical assistance during the experimental work. We are also grateful to Hans Zweers (NIOO-KNAW) for the installation of the oxygen gas chromatograph and his technical assistance in sampling and analyzing GC data output.

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Table of Content Wood degradation by thermotolerant and thermophilic fungi for sustainable heat production Leire Caizán Juanarena a*, Annemiek Ter Heijne a, Cees J. N. Buisman a,b, Annemieke van der Wal c a

Sub-Department of Environmental Technology, Wageningen University, Bornse Weilanden 9, P.O. Box

8129, 6708 WG Wageningen, The Netherlands. b

Wetsus, Centre of Excellence for Sustainable Water Technology, Agora 1, P.O. Box 1113, 8900 CC

Leeuwarden, The Netherlands. c

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg

10, 6708 PB Wageningen, The Netherlands. * Corresponding author. Tel.: +31 617584653; E-mail address: [email protected] Synopsis: Heat production was estimated from oxygen uptake under the activity of several microbial wood degraders: the natural biota (a), a thermotolerant fungus (b), a thermophilic fungi (c), and the one-to-one combination of fungus and natural biota. GRAPHICAL TOC:



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Figure 1: Set-up of experiments: sterile and non-sterile woodblocks with (P. c. /C. t.) and without (control) fungal inoculation. Five replicates were prepared for each of the six scenarios. Figure 1 113x72mm (300 x 300 DPI)

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Figure 2: Cumulative oxygen consumption (mmol g-1 DM) for sterile (left) and non-sterile (right) woodblocks with fungal inoculation of P. chrysosporium (P. c.) (□) or C. thermophilum (C. t.) (o) and without fungal inoculation (controls) (-, x). Figure 2 80x30mm (300 x 300 DPI)



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Figure 3: Visible fungal growth under different conditions: P. c. sterile (A), C. t. sterile (B), P. c. non-sterile (C), C. t. non-sterile (D), control non-sterile (E), and control sterile (F). Figure 3 72x93mm (300 x 300 DPI)


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Heat production was estimated from oxygen uptake under the activity of several microbial wood degraders: the natural biota (a), a thermotolerant fungus (b), a thermophilic fungi (c), and the one-to-one combination of fungus and natural biota. Graphical TOC 190x142mm (300 x 300 DPI)


Wood Degradation by Thermotolerant and Thermophilic Fungi for

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