Application of wood sawdust microbial transformation for the utilization of long-term stored bark-wood waste

doi:10.21203/rs.3.rs-7628066/v1

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Application of wood sawdust microbial transformation for the utilization of long-term stored bark-wood waste

https://doi.org/10.21203/rs.3.rs-7628066/v1

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The issue of bark-wood waste (BWW) utilization is challenging for the regions adjacent to pulp and paper mills. Bark is difficult to recycle, and the additives used in paper production further complicate this process. This work focuses on the potential application of wood sawdust microbial transformation, the technology of which was developed at the Irkutsk Institute of Chemistry SB RAS, for the processing of bark-wood waste into environmentally safe soil. Laboratory experiments revealed that long-term stored BWW can undergo microbial degradation. The best results were obtained with the BWW/sawdust mixture. The substrate neutralization conditions were selected to simultaneously lower the pH and introduce the necessary mineral additives for microbial function. A semi-industrial experiment with a BWW/sawdust mixture of 20 m³ (3:1 ratio) was performed. The experiment demonstrated that pH stabilization occurred more efficiently than in the laboratory experiment. A temperature increase, which is characteristic of aerobic processing of lignocellulosic waste was also observed. The product obtained was non-toxic to both plants and Chlorella vulgaris (hazard class 5). The processing improved of the agrochemical parameters of the substrate, such as hydrolytic acidity, cation exchange capacity, and the sum of absorbed bases. The increase in humic acid content indicates a deep microbial degradation of the lignin component of BWW. Consequently, the microbial transformation of BWW was successfully implemented to produce a non-toxic soil. This technology can be scaled up for large volumes of BWW at storage sites.

bark-wood waste

microbial processing

soil

composting parameters

The volume of wood harvested worldwide is large (3.91 billion m³ in 2022) and is increasing annually by an average of 0.8% (State report 2023). Owing to the long-term activity of large wood processing enterprises, environmental pollution has become a biosphere-wide process. In Russia, no more than half of the biomass of harvested wood is used sustainably. Consequently, wood processing enterprises generate approximately 68–74 million m³ of wood waste annually, of which only 48–58% is processed. One of the main types of sawmill waste is bark, which accounts for 8–15% of the waste structure. Due to its complex chemical and mechanical structure, bark is not widely used in secondary processing (Petrunina et al. 2022). The only industrial application of conifer bark is in the landscape, agrotechnical and design works to mulch the soil and improve the aesthetic appearance of parks (Hallman et al. 2023). The microbial decomposition of bark is a slow process due to its high antibacterial and disinfectant contents (Janah et al. 2025; Dessalegn et al. 2025; Hazizah et al. 2025; Hossain et al. 2024). Bark-wood waste (BWW) accumulates in large quantities in pulp and paper mill dumps (Kulikova et al. 2022). This waste is almost not involved in further utilization. This leads to the formation of environmental disaster zones in the areas surrounding pulp and paper mills due to the toxic effects of BWW components on soil and groundwater. The inconsistent composition and the presence of hard-to-degrade polymers in BWW make it extremely unfavorable substrate for chemical and thermal utilization. Moreover, long-term stored BWW can undergo microbial processing to produce fungal enzymes (Martynov et al. 2024) and even composting (Margina et al. 2023). A method for the microbial processing of sawmill waste into organic-mineral fertilizer was developed at the Irkutsk Institute of Chemistry (Belovezhets 2019). This work aimed to investigate the potential application of this technology to process bark-wood waste into environmentally safe soil.

Long-term stored BWW from Bratsk Pulp and Paper Mill (elemental composition: C 18.01, H 2.29, P 0.45, N 0.0, S 0.0, ash 45.51, lignin 37.0, cellulose 28.0, pH 9.4) and fresh softwood sawdust (elemental composition: C 17.84, H 6.17, P 0.3, N 0.03, S 0.61, ash 3.68, lignin 31.3, cellulose 47.3, pH 4.5) were used as substrates. Elemental analysis was performed automatically on a Flash EA 1112 CHN-O/MAS 200 microanalyzer. The moisture content was determined on a moisture meter AND MX-50 (Japan), and the pH was determined on a pH-meter Expert-pH (“Econix” Russia) according to GOST 26423-85 (Interstate standard of the USSR, 1986). The substrates were neutralized with 0.8% NH₄NO₃ or a mixture of 4% HNO₃ and H₃PO₄.

For the laboratory experiments, the following compositions were used:

1. BWW + association of microorganisms

2. BWW + association of microorganisms + sawdust

Neutralized BWW without microorganisms served as the control.

In all the cases, 4 liters of substrate, pre-neutralized and humidified to the optimum moisture content, were employed. The ratio of BWW/sawdust was 3:1.

Microorganism cultures of Paecilomyces variotii sp., Phanerochaete chrysosporium, Trametes versicolor, Sporotrichum pulverulentum (14B), and Sporotrichum pulverulentum (14G), which were isolated from infected wood in Eastern Siberia and identified on the basis of 26S-RNA in the All-Russian Collection of Industrial Microorganisms, were used in this study. One hour after acidification, a mixture of five-day-old fungal cultures grown individually (on modified Sabouraud's nutrient medium, under stationary conditions at 26°C) was added to the substrates. As neutralization was carried out using a mixture of nitric and phosphoric acids, mineral additives were not employed (Belovezhets 2019). Dilute acid solutions were prepared from the culture filtrate. The substrates were neutralized to a pH of 5.6–6.2. Modeling of real exothermic composting processes, which is impossible with a small substrate volume, was performed by incubation of the mixture in a thermostat at 37°C. The moisture content was maintained at 60–70%. The duration of the experiment was 5 months. Lignin loss was estimated gravimetrically after hydrolysis with 72% Н₂SO₄, and cellulose loss was evaluated indirectly by determining the amount of reducing substances in terms of glucose content via the phenol-sulfuric acid method (Yue et al. 2022). The mobile forms of nitrogen were determined according to GOST 27894.3–88 (State standard of the USSR 1988), phosphorus was determined according to GOST 27894.5–88 (State standard of the USSR 1988), and the cation exchange capacity (mEq/100 g) was determined according to GOST 17.4.4. 01–84 (Interstate standard of the USSR 1985), hydrolytic acidity (mEq/100 g) according to GOST 26212-91 (State standard of the USSR 1993), the sum of absorbed bases (mEq/100 g) according to GOST 27821-88 (State standard of the USSR 1990), the mass fraction of humic acids in terms of dry matter (%) according to GOST 9517-76 (State standard of the USSR 1977), and ash content according to GOST 11306 − 2013 (Interstate standard of the Russian Federation 2013). The phytotoxicity of the samples was established using the biotesting method on higher plant cress (Lepidium sativum) (Kubrina and Supinichenko 2021), and the toxicity of the aqueous extracts was determined via the use of green freshwater unicellular algae (Chlorella vulgaris beijer) according to GOST R 54496 − 2011 (National standard of the Russian Federation 2011).

The semi-industrial experiment was carried out with the following substrates: BWW, 15 m³; sawdust 5, m³ (pH of the mixture was 8.5). The experiment was performed on 26.05.2024 at the site belonging to the A.E. Favorsky Irkutsk Institute of Chemistry of the Siberian Branch of the Russian Academy of Sciences, Russia, Irkutsk (geographic coordinates: 52°24' N 104°26' E). The site was preliminarily cleared from vegetation and leveled. The substrate was spread in an even 0.5 m-thick layer and subjected to neutralization with a 3:1 mixture of nitric and phosphoric acids. For neutralization, the acid was diluted with tap water to a concentration of 4%. The substrate was watered using a submersible pump (Vikhr VN-10N, Russia). Neutralization was carried out once. Microorganisms were grown individually (Belousov et al. 2024).

The starting BWWs are highly toxic (hazard class 3.5), which makes them unsuitable for use as soil. This problem can be solved using the directed microbial transformation. It was hypothesized that the association of non-pathogenic basidial fungi employed for processing wood sawdust could also be effective for BWW utilization. To optimize the conditions for these fungi, one version of the laboratory experiment included a mixture of sawdust and BWW.

Preliminary experiments revealed that BWW and sawdust have highly imbalanced elemental compositions. They are also characterized by high ash content and high pH values (BWW: 9.4; BWW/sawdust mixture: 8.5). Consequently, it was necessary to select an adequate neutralization option in the first stage of the laboratory experiment. As the optimum pH for fungal performance is 5.5–6.9, it was set to between 6.0 and 6.8 before the cultures were applied. Initially, 0.8% NH₄NO₃ was used for neutralization. However, even when 25.7% of the total substrate volume was applied, the ammonium nitrate solution did not reduce the pH to the required level. Therefore, neutralization with a mixture of 4% HNO₃ and 4% H₃PO₄ was performed. The results of the pH dynamics are presented in Table 1.

Table 1
Neutralization of BWW with 0.8% NH₄NO₃ and a mixture of 4% HNO₃ and 4% H₃PO₄
Content of NH₄NO₃, % vol.	рН	Content of HNO₃ and H₃PO₄, % vol.	рН
0.0	9.4	0.00	9.4
1.4	8.4	0.26	6.9
4.4	8.0	0.53	6.6
8.2	7.7	0.70	6.6
10.8	7.6	0.93	6.6
15.1	7.5	1.00	6.6
19.3	7.5	1.13	6.5
25.7	7.4

Two days after the start of the experiment, the pH was 6.1 for the experimental samples and 7.1 for the control samples. However, owing to the high buffer capacity of the substrates, the pH began to shift toward the alkaline region one week after neutralization. To increase the pH above 7.2, the substrates used in the laboratory experiments were additionally acidified with a mixture of 0.5% HNO₃ and 0.5% H₃PO₄ during watering. This low concentration was used to reduce the risk of adverse effects on microbial associations. Unfortunately, the pH of all the samples tended to increase. In the control samples, alkalinization of the medium occurred much faster reaching higher values. Watering with diluted acids was carried out twice a week. In all the experiments, the pH stabilized after two months (Fig. 1). Therefore, further acidification of the experimental samples was not carried out. During the third and fourth months of the experiment, the pH remained stable. In the room temperature experiments, the pH reached relatively high values during the first stage but stabilized at the level of the thermostated variants during the last month. This is most likely due to microorganisms being more active at elevated temperatures. Interestingly, the second wave of substrate alkalinization began in the fourth month of the experiment. Even in the experimental samples, the pH reached 8.1 (8.4 in the control) (Fig. 1). Moreover, the pH was higher in the thermostated samples than in the samples incubated at room temperature.

Notably, the samples cultivated at elevated temperatures presented abundant fungal growth and a pronounced fungal odor, as observed visually one week after the start of the experiment. Similar results for samples grown at room temperature were noted only after two months.

The toxicity class determined using Chlorella vulgaris (Table 2) increased in the thermostated samples in the middle of the experiment and decreased to the fifth class by the end of the third month. Moreover, the toxicity of the samples incubated at room temperature increased over time. These findings suggest that the observed effects are most likely attributable to delayed processing of compounds that are toxic to Chlorella vulgaris at room temperature. However, the toxicity level increased again by the fifth month of the experiment. This was attributed to an increase in substrate pH (according to preliminary experiments, alkalinization of the solution negatively affects the growth of Chlorella vulgaris).

Table 2
Toxicity class determined using *Сhlorella vulgaris*
Variant	Control		BWW		BWW/sawdust
Incubation time	R*	T**	R	T	R	T
2 months	5	4.5	5	4	5	4
3 months	4	4	4	5	5	5
4 months	4	4	5	5	5	4.5
5 months	4	3.5	4	3.5	4	4
* R – samples incubated at room temperature, ** T – samples incubated at 37°C in a thermostat

Loss of lignin and cellulose was observed even in the control samples (Table 3). Even simple pH optimization likely activates indigenous microflora capable of degrading wood polymers. The addition of microorganisms accelerated the degradation of lignin, especially cellulose. The introduction of sawdust to the mixture shifted the polymer degradation toward cellulose. In samples containing sawdust, the loss of cellulose is almost half that of the initial amount. It is logical that under thermostated conditions, the loss of cellulose is more pronounced. In contrast, the loss of lignin in the BWW/sawdust sample was rather small, especially when the sample was incubated at room temperature.

Table 3
Content of lignin and cellulose
Variant	Control		BWW		BWW/sawdust
Variant	R***	Т****	R	Т	R	Т
Lignin*	33.9	34.4	33.5	32.1	35.0	34.0
Cellulose**	21.3	20.1	19.2	16.8	21.8	18.4
* initial content of lignin in BWW – 37.0, in a mixture of BWW/sawdust – 35.6; initial content of cellulose in BWW – 28.0, in a mixture of BWW/sawdust – 32.8; * R – samples incubated at room temperature; **** T – samples incubated at 37°C in a thermostat

According to the calculations, the initial nitrogen and phosphorus contents were 0.13 and 0.18%, respectively (Table 4). By the end of the experiment, the amount of phosphorus had decreased by approximately half. This may indicate the transition of soluble phosphorus to its inactive insoluble form or its deposition in the biomass of microorganisms. The degree of nitrogen loss depended on the experimental conditions. For the variants incubated at room temperature, 70% of the losses occurred. In the thermostated samples, losses ranged from 24–32%. Interestingly, this parameter was independent of the presence of microorganisms. Consequently, nitrogen losses are either abiogenic or catalyzed by indigenous microflora.

Table 4
Content of labile forms of biogenic elements in substrates
Sample	Content of biogenic elements, %
Sample	nitrogen	phosphorus
Calculated	0.130	0.180
Control R*	0.041	0.064
Control Т**	0.092	0.070
BWW R*	0.041	0.078
BWW Т**	0.096	0.070
BWW/sawdust R*	0.042	0.073
BWW/sawdust Т**	0.096	0.085
* R – samples incubated at room temperature, ** T – samples incubated at 37°C in a thermostat

Thus, the best results were observed for the BWW/sawdust variant. This is most likely due to the presence of sawdust in the mixture stimulating the growth and development of the mycelium of wood-destroying fungi in the initial stage. The accumulation of microbial biomass resulted in the acceleration of substrate processing.

Next, we carried out a semi-industrial experiment on BWW transformation. A 3:1 mixture of BWW and sawdust was used, which was neutralized with a mixture of nitric and phosphoric acids. The pH of the substrate was measured to be 7.2 immediately after acidification. The substrate was then left for one week under these conditions to allow for uniform neutralization. After two days, the pH decreased to 4.5 and remained at this level for 4 days (Fig. 2). Thereafter, the pH gradually shifted toward neutrality. As the pH of the substrate was less than 7 before the introduction of microorganisms, it was decided not to acidify it further but rather to control the pH weekly. The microorganisms were introduced on the 8th day after acidification of the substrate.

The pit began to warm up a day after the application of microorganisms and reached a maximum temperature of 56.1°C after 12 days (Fig. 3). A smooth decrease in temperature was observed from day 16 of the experiment onward. However, the decrease was not critical, with the temperature of the pit remaining at least 40°С.

The pH of the pit stabilized within the neutral range and never exceeded 6.9 (Fig. 4). This finding indicates the ability of the introduced microorganisms to stabilize the main monitored process parameters.

Samples were collected from different depths at 1, 2 and 3 months of fermentation to determine the degree of toxicity to Chlorella vulgaris (Table 5).

Table 5
Dynamics of changes in the BWW environmental hazard class in barns at different stages of pre-processing and fermentation
Type of sample	Hazard class
Combined sample of the initial substrate (BWW + sawdust) from the pit	3.8
Combined sample after processing with acids	4
Incremental samples after processing with acids and addition of microorganisms
Horizon 10 cm	4
Horizon 50 cm	3,5
Horizon 80 cm	3,5
Incremental samples after 1 month of fermentation
Horizon 10 cm	3,5
Horizon 50 cm	4,0
Horizon 80 cm	3,5
Incremental samples after 2 months of fermentation
Horizon 10 cm	4,0
Horizon 50 cm	4,5
Horizon 80 cm	4,5
Incremental samples after 3 months of fermentation
Horizon 10 cm	5
Horizon 50 cm	5
Horizon 80 cm	5

The table shows that the original hazard class of the substrate was 3.8. Acidification reduced it to the fourth class. However, the substrate becomes more toxic under the action of microorganisms. Only after three months of treatment was the substrate was completely non-toxic (fifth hazard class).

A slowdown in the germination of cress seeds was observed at all depths of the pit at all sampling dates by the third day of the experiment (Table 6). However, 100% of the seeds subsequently germinated. The average root length and total weight of the seedlings increased with increasing duration of BWW composting, indicating a decrease in substrate toxicity. Consequently, by the end of the processing period, a non-toxic substrate into which seeds could be sown directly was obtained.

Table 6
Dynamics of phytotoxicity in the pit at different stages of fermentation
Variant		Germinated seed, %	Root length, cm	Germ length, cm	Dry weight, g
Variant		1 month
depth	10	95	3,93	1,24	0,0325
	50	95	3,49	1,15	0,026
	80	80	3,90	1,23	0,0255
		2 months
depth	10	95	5,06	1,66	0,038
	50	90	4,91	1,22	0,031
	80	95	4,81	1,28	0,034
		3 months
depth	10	95	5,51	0,97	0,041
	50	90	6,53	1,05	0,039
	80	90	6,08	0,9	0,039

Table 7 shows the main agrochemical characteristics of the target product. For convenience, literature data for high-moor peat (the optimal soil substitute substrate) and gray forest soils (typical of Eastern Siberia) are provided.

The determination of agrochemical characteristics enables the properties of soil (soil substitute substrate or fertilizer) to be assessed, revealing how they affect the growth and development of plants. It also reveals the nature and peculiarities of the soil interaction with applied fertilizers and substances originating from the atmosphere. Furthermore, it allows the amount of fertilizers and ameliorants to be calculated for application to the soil.

Table 7
Changes in the agrochemical characteristics of BWW during processing
Characteristics	Initial substrate	Final product	High-moor peat*	Gray forest soil*
Cation exchange capacity, mEq/100 g	5.16	8.46	10–12	12–30
Hydrolytic acidity, mEq/100 g	4.82	2.31	5–10	4.6–5.2
Sum of adsorbed bases, mEq/100 g	24.65	40.24	60–90	17–24
Mass content of humic acids in term of dry substance, %	21.3	42.33	9–14	1.9–3.8
Ass content	31.8	31.5	2–12	2.5–15
Mass content of total nitrogen (N) in term of dry substance, %	0.11	0.38	0.7–1.35	0.16–0.27
Content of ammonium nitrogen, mg/100 g	570	740	200	90
Mass content of phosphorus (Р₂О₅) in term of dry substance, %	0.31	0.57	0.1–0.3	0.08–0.2
Mass content of Р₂О₅, mg/100 g	134	141	0–15	86–319
рН	5.7	6.9	2–3	4.6–5.2
* literature data (Large information archive 2025)

One of the main agrochemical characteristics is the reaction of the soil medium: actual acidity, potential acidity and hydrolytic acidity. During BWW processing, the pH was shifted toward the neutral region. This allows the target product to reclaim acidic soils typical of Siberia without the need for additional neutralizing agents. A twofold decrease in hydrolytic acidity was also observed, which indicates the stabilization of the substrate's buffer properties.

Another important characteristic is the absorption properties of the soil. These include the cation exchange capacity and the sum of absorbed bases. These parameters reflect the ability of soil or substrate to absorb and provide plants with biogenic elements such as potassium, calcium and magnesium. These parameters increased throughout the experiment. This indicates an improvement in the quality of BWW as a soil. In the target product, these characteristics were only slightly lower than those of peat.

The third important parameter is the presence of humic substances, mineral nitrogen and phosphorus, as well as their mobile forms, in the soil or substrate. We have demonstrated that the content of humic acids increases sharply during the composting process. This is most likely due to the active transformation of long-term bark residues by the basidale fungi included in the inoculum. The low content of total nitrogen and phosphorus is because mineral acid solutions are used instead of mineral fertilizers, as is standard practice (Belovezhets 2019). However, a steady increase in all these parameters was observed by the end of the experiment. While this amount of nitrogen and phosphorus (in both their total and mobile forms) is insufficient for complete fertilizer, it is quite acceptable for the soil.

The high ash content of the product is due to the peculiarities of BWW itself and the use of mineral acids in the acidification process. This indicator does not affect the product properties and enables it to be used as a soil.

In conclusion, laboratory experiments have demonstrated that long-term stored BWW can undergo microbial degradation. The best results were obtained with the BWW/sawdust mixture. The substrate neutralization conditions were selected to simultaneously lower the pH and introduce the necessary mineral additives for microbial function. A semi-industrial experiment was conducted on a 20 m³ BWW/sawdust mixture (3:1 ratio). pH stabilization occurred more efficiently than it did in the laboratory experiments. The temperature increase characteristic of the aerobic processing of lignocellulosic waste was also observed. The target product was non-toxic to both plants and Chlorella vulgaris (hazard class 5). Processing improved the agrochemical parameters of the substrate, such as the hydrolytic acidity, cation exchange capacity and sum of absorbed bases. The increase in humic acid content indicates deep microbial degradation of the lignin component of BWW. Consequently, the microbial transformation of BWW was completed, resulting in non-toxic soil. This technology can be scaled up to process large volumes of BWW at storage sites.

Author contributions LA Belovezhets, ideas; formulation of overarching research goals and aims, preparation, creation and presentation of the published work, specifically data visualization, DS Belousov, сonducting a research and investigation process, performing the experiments, data collection, preparation, creation and presentation of the published work, specifically data presentation, NV Filinova, conducting a research and investigation process, specifically performing the experiments, data collection, preparation, creation and presentation of the published work by those from the original research group, specifically critical review, commentary or revision – including pre-or postpublication stages, EO Pristavka, conducting a research and investigation process, specifically performing the experiments, or data/evidence collection, preparation, creation and presentation of the published work, AA Pristavka, conducting a research and investigation process, specifically performing the experiments and data collection

Funding The authors declares that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability Data will be made available on request.

Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Application of wood sawdust microbial transformation for the utilization of long-term stored bark-wood waste

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