Biochars Originating from Different Biomass and Pyrolysis Process Reveal to Have Different Microbial Characterization: Implications for Practice

: Sustainable technologies are increasingly promoted in various production areas. Protection of natural resources, as well as rational waste management, may lead to better optimization of technologies. Biochar, a product of pyrolysis of organic residues has found wide applications in waste management, agriculture, energy and construction industry. In the present study biochar samples produced in Poland and in Brazil were analysed for microbial content using three substrates: Plate Count Agar, Malt Agar, and Potato Agar. Both qualitative and quantitative measurements were done. Microscopic analysis of the biochar structure was also performed. We found that microbial cultures in both biochars represented a wide range of biodiversity of microorganisms genera and species. We demonstrate that the biochar samples differ depending on the botanical origin as well as on the production technology. Structure of the tested samples also varied depending on the botanical origin. Sample 1 ‐ PL (pine) was characterised by a compact and regular structure, while sample 2 ‐ PL (oak) showed porous and irregular structure. Sample from Brazil (1 ‐ BR) showed a more delicate structure than Polish biochars. Obtained properties may suggest a range of implications for practice.


Introduction
Biochar, carbonous substrate originating from the process of pyrolysis [1] has been demonstrated to have various potential benefits such as increasing agricultural yields [2][3][4], soil and sediment remediation [5] or improving animals health in livestock farming [6]. There has been also a recent interest in using biochar in construction [7] with potential benefits for diminishing humidity inside buildings, thermal isolation and fungus development control [8][9][10][11][12]. Biochar or microporous carbon is also used for humidity control, a property that has been extensively researched by Nakano et al. (1996) and Abe et al. (1995). Humidity control by microporous carbon can reduce asthma and

Materials and Methods
The research material consisted of two types of biochar coming from Brazil and Poland: 1. biochar from Brazil obtained from Gliricidia plant (1-BR), 2. biochar from Poland obtained from Pine woodchips (1-PL), 3. biochar from Poland obtained from Oak woodchips (2-PL).
Biochar samples provided for the research were placed in sterile conditions. Similarly, throughout the study, extensive precautions were put in place to limit microbial contamination.
1. Brazilian biochar characterization The Brazilian biochar was produced in home-made drum stove from Gliricidia sepium at the temperature of approximately 350°C. Gliricidia is a commonly used plant in organic farming for nitrogen-fixing. It is also used as 'living fence' at agricultural farms and pasturelands. Despite its benefits, it grows fast and may provide excessive shadow, limiting plant growth. In certain circumstances, there is, therefore, a need for cutting off the upperground branches that do not have an alternative use. Moreover, at the site where the biochar was produced (Brazilian Agricultural Research Station, Embrapa Agrobiologia, Seropedica km 47), Gliricidia become an invasive species that disseminated rapidly entering native forest fragments. The source of biomass for biochar production in Brazil, therefore, did not compete with alternative biomass uses, as at times criticised in literature [28]. Branches of Gliricidia were dried for two weeks before pyrolization. Cut and dry biomass was put into the stove, fired up and closed with drum cup and isolated with a layer of sand. The pyrolization time was 24 hours. After that time biochar was cooling for another 24 h (with open stove) and sieved at 4 mm ( Figure 1).

Figure 1.
Steps of production: Rio de Janeiro state where biochar was produced, Gliricidia tree, dry Gliricidia before being cut at put into the stove, drum stove, biochar (before being sieved), table with biochar properties [4].

Polish biochar characterization
Biochar was produced according to Fluid SA company technology. The process consisted in thermal refining of plant biomass and other post-production biomass residues through their autothermal roasting at the temperature 260°C in reduction atmosphere and without the use of additional energy, catalysts, and chemical additives.
Carbonising products consisted mainly of biochar (from 65% to 70% of energy compared to the energy of the feed) and process gases (from 20% to 35% of energy compared to the energy of the feed). During the process, a significant increase of the carbon element (C) in relation to the biomass of the feed was recorded (1.5 to 2.0 times) as well as an increase of energy density, on average by 4.0 times, reduction of the amount of hydrogen (H), on average by 2.5 times, and reduction of the amount of oxygen (O2), on average by 3.0 times. Polish samples of biochar were produced from pine woodchips (1-PL) and oak woodchips (2-PL).
The research was carried out at the Innovation Research Center (CBNI) and Regional Center of Agriculture, Environmental and Innovative Technology (EAT) Pope John Paul II State School of Higher Education in Biała Podlaska, Poland.

Microbiological Analysis of Biochar Samples Consisted of Qualitative and Quantitative Determination of Bacteria and Fungi Species
For the research three standardized substrates were used in the assessment process: a) The total number of microorganisms cultivated on PCA substrate (PLATE COUNT LAB-AGAR™) in temperature of 30°C and time period 72 h. b) The total number of fungi colonies cultivated on PDA substrate (POTATO DEXTROSE LAB-AGAR™) in temperature of 30°C and time period 72 h, after that time the temperature was decreased to 21°C for the next 72 h. c) The total number of fungi colonies cultivated on MA substrate (MALT EXTRACT LAB-AGAR™) in a temperature of 24°C and time period 144 h.
The qualitative and quantitative assessment was done on the basis of microbial flora species composition using macroscopic and microscopic methods, as well as taxonomic keys and atlases. It was expressed in CFU/g units [30][31][32][33][34][35].

Microscopic examination of the structure.
Examination of the biochar sample structure was done using a research microscope Nikon Eclipse E-200 with fluorescence attachment and SCA image analysis.

Results
In the sample, 1-BR cultured on PCA substrate dominated strains of Bacillus sp. (the average number of colonies from UNC to 204 colonies for dilutions ranging from 1 to 10 -5 . The plates were overgrown on the entire surface with the colonies of Bacillus sp. On two plates confluent growth was observed, which made it impossible to perform a quantitative count (Table 1).  On malt substrate (Table 2) (3), and an overgrowth of Bacillus sp. on the entire surface plate was recorded. In the case of dilution of 10 -3 , on average 7.3 colonies of Aspergillus sp., Aspergillus versicolor, Aspergillus flavus, and Penicillium sp. were isolated, as well as Bacillus sp., in the case of which confluent growth was observed.
Aver (18) Afum (1) Psp (8) Ʃ (27) Aver (36) Asp. For the dilution of 10 -4 , in the first repetition, Bacillus sp. was isolated, characterised by a confluent growth, while in the second and third repetition Aspergillus versicolor species were recorded with the average number of colonies equal to 1.0. After averaging the readings the average number of fungi colonies ranged from UNC to 45. On POTATO substrate (Table 3) for the dilution of 1, no colonies of microorganisms were recorded.  (2) Afl (2) Asp. (2) Bsp (*) Acan (2) Anig (1) Afl (UNC) Ʃ(UNC) Aver (2) Acan (1) Afl (1) Ʃ (4) Aver (3) Bsp (*) Aver (6) Acan (1) Anig (1) Afum (1) Afl (1) Psp (1) Bsp (*) Ʃ(11) 3.7 In the case of dilutions from 10 -1 to 10 -5 , in all repetitions, strains of Bacillus sp. overgrown on the entire surface of the plate were observed, which made a quantitative count impossible to perform. Additionally, the following genera or species were isolated: Aspergillus sp., Aspergillus niger, Aspergillus flavus, Aspergillus versicolor, Aspergillus candidus, Aspergillus fumigatus, as well as Penicillium sp. (the average number of colonies ranging from 0.3 for the dilution of 10 -4 to 3.7 for the dilution of 10 -3 ). However, for the dilution of 10 -5 , no colonies of microorganisms were isolated. In the case of sample no. 1-PL cultured on PCA substrate ( Table 4) dominated strains of Bacillus sp. The average number of colonies ranged between 545.5 for the dilution of 10 -1 and for the dilution of 1 also uncountable growth of Micrococcus sp. For the dilution of 10 -5 , no colonies of microorganisms were isolated. On MALT substrate (Table 5), for the dilution of 1, strains of Aspergillus flavus were isolated, and the average number of colonies count was 1.7. Within the range of dilutions between 10 -1 and 10 -5 , no colonies of microorganisms were isolated. On POTATO substrate (Table 6) strains of Bacillus sp. overgrown on the entire plate were observed (average number of colonies from UNC for the dilution of 1 to 1 colony for the dilution of 10 -4 ). For the dilution of 10 -5 , no colonies of microorganisms were isolated. In the case of sample no. 2-PL on PCA substrate for the dilution of 1, only in the second of the three repetitions, 1 colony of Aspergillus flavus was isolated. On the plates, with the dilutions ranging from 10 -1 to 10 -5 , no colonies of microorganisms were isolated. On the MALT substrate for the dilution of 10 -1 1 colony of Penicillium sp. was isolated. Meanwhile, for the dilutions ranging from 10 -2 to 10 -5 no colonies of microorganisms were isolated. On POTATO substrate, for the dilution of 10 -1 , 1 colony Aspergillus versicolor was isolated, while for the dilution of 10 -2 1 colony of unidentified fungi was recorded. On the plate, with the dilution of 10 -3 , no microorganisms were recorded. In contrast, on the plate with the dilution of 10 -4 one yeast colony was isolated. On the plate, with the dilution of 10 -5 , no microorganisms were recorded. After the microbiological analysis of biochar samples, it was noted that sample 2-PL was the least microbiologically polluted. However, it should be stated that in Polish samples biologically different microorganisms were present. In the sample, 1-PL dominated strains of Bacillus sp. While in sample 2-PL dominated, though in small amount, strains of fungi species: Aspergillus flavus, Aspergillus versicolor, and Penicillium sp. In the case of biochar sample from Brazil, the same microorganisms were present as the ones isolated and identified in Polish samples.  The microscopic analysis, carried out using research microscope Nikon Eclipse E-200 with fluorescence attachment and SCA image analysis, shows the structure of biochar 1-BR ( Figure. 2) as irregular, but this structure is not as dense as in the case of biochar 2-PL (oak) (Figure. 4). The structure of biochar sample 1-BR (Brazil) is undulating and porous. However, this sample seems to have the most irregular structure when compared with samples 1-PL (pine) (Figure. 3) and 2-PL (oak).
Oak (sample 2-PL), being hardwood, in the microscopic images shows a hard and porous structure, while pine (sample 1-PL) has regular, slightly porous, and delicate structure.

Discussion
The microbiological analysis of the three biochar samples demonstrated that samples 1-PL and 2-PL were less microbiologically contaminated than sample 1-BR. In the case of sample 1-BR, similar microorganisms were isolated as from samples 1-PL and 2-PL. In sample 1-BR, among others, the following were present: Bacillus sp. Aspergillus sp. Aspergillus niger, Aspergillus flavus, Aspergillus versicolor, Aspergillus candidus, Aspergillus fumigatus, as well as Penicillium sp. and Rhizopus sp. Such a broad range of microorganisms was isolated neither from sample 1-PL nor 2-PL. At the same time, there is a certain similarity between the biochar from Poland and Brazil, namely the strains isolated from Brazilian biochar were present in two types of biochar from Poland, originating from two botanically different sources. When analysing the two samples from Poland opposite observations should be made, namely that biologically different microorganisms were present in these two samples. In sample 1-PL (pine) strains Bacillus sp. dominated. In sample 2-PL (oak) dominated, though in small amounts, strains of fungi: Aspergillus flavus, Aspergillus versicolor, as well as Penicillium sp. In the sample from Brazil strains Bacillus sp., Aspergillus sp., and Rhizopus sp. were the most numerous ones. After the microbiological and microscopic analysis, it can be concluded that the two samples have very interesting characteristics that can be used depending on the purpose. It was observed that sample 2-PL was less microbiologically contaminated. However, it should be stated that in the tested samples biologically different microorganisms were identified. In the sample 1-PL strains Bacillus sp. dominated. In the sample 2-PL dominated, thought in small amount, strains of fungi: Aspergillus flavus, Aspergillus versicolor, as well as Penicillium sp. Additionally, differences were observed in the microscopic images, where sample 1-PL (pine) was characterised by a compact and regular structure, while sample 2-PL (oak) had irregular and porous structure. At the same time, the significantly differing structure was observed in the case of biochar 1-BR (Brazil). It was more delicate than the other test samples. The porous structure and specific surface of biochar are its most important physical properties and they are responsible for the course of various processes in the soil. Biochar has high internal porosity, which affects the water absorption, sorption capacity, and retention of nutrients in the soil. Therefore, it improves physicochemical properties of the soil, facilitates the use of nutrients by plants, and prevents nutrients leaching. Biochar may improve soil structure (water-air properties) [36,37]. Presence of certain genera and species in the test samples may be associated with the generic traits of the raw materials from which they were derived. In addition, the manner, in which test samples were produced, might also be important. The biochar from Poland came from controlled, patented, and repeatable production. On the other hand, the biochar from Brazil was produced in a simple way reflecting the possibility to be repeated by the farmers, wherein not every production parameter can be controlled. This type of dependency is directly proportional to the microbiology of test samples. Summing up, all the samples have very interesting properties that may be used depending on the intended purpose. Implications for practice are now widely used in many research centres around the world. Herrman et al. (2019) concluded that the addition of biochar from rubber tree caused a raise of the soil pH value as well as its nutrient content. In the research cited higher sensitivity to the activity of applied biochar was recorded in the case of fungi occurring in the soil as compared to bacteria present there [38]. Similar studies were performed in Bulgaria [39] using biochar in the cultivation of wheat and maize in crop rotation. The stimulating effect of biochar on soil microflora was observed, in particular on the number of bacteria. The research was considered to be a promising method for preserving soil fertility. Azis et al. (2019) concluded that in the case of soil on which wheat is cultivated addition of biochar, in experimentally determined amount, improves the physicochemical properties of the soil and soil microflora, which in turn has a positive effect on the productivity of the soil and increased yields [40]. In other studies, Hardy et al. investigated the long-term application of biochar in order to improve the quality of soil microorganisms. For this purpose, specialized models imitating soil conditions were designed. Biochar was shown to modify the quantity and quality of soil microorganisms, however, in order to properly design a successful modification of the soil using biochar its properties should be considered in conjunction with the soil conditions [41]. Unfortunately, in the available literature of the subject, there is no similar research with which one could compare the results obtained in this work. Based on the definition of sustainable development, strategies of environmental protection should consist of continuous, integrated and anticipatory actions undertaken in production processes. Such actions lead to an increase in production efficiency and reduce risks to humans and the natural environment. Sustainable production aims at the manufacture of goods which uses processes limiting environmental pollution. In this context, biochar production based on waste-free use of organic biomass has the traits of sustainable development.

Conclusions
6. The results obtained can be used as relevant utilitarian data for implications for practice.
Funding: This research received no external funding.