ABSTRACT
Characterization of products from sawdust degradation using selected microbial culture isolated from it was carried out. A composite sawdust waste sample was collected from MCC timber market in Calabar. The sample was diluted using serial dilution method and inoculated on Nutrient agar and Sabauraud dextrose agar using pour plate technique and incubated at 370C and 270C respectively. Five bacteria species and 3 species of fungi were identified which included Bacillus sp, Serracia mercescens, Micrococcus sp, Pseudomonas aeruginosa, Streptococcus sp, Aspergillus niger, Rhizopus sp and Penicillumsp. The bacteria and fungi species were individually and in combination inoculated into sterile sawdust containing sterile water. These were kept at 270C for 21 days. Protein, glucose contents and chemical components in the degraded sawdust samples were determined using GC-MS chromatography. It was observed that Aspergillus niger degradation had protein content 7.87g/l, Serracia mercescens and Aspergillus niger 6.56g/l, and Serracia mercescens 5.75g/l. These organisms used lignocellulolytic enzymes and similar enzymes in the degradation process. In the case of glucose, the combined degradation produced 2.24g/l, Aspergillus niger 2.05g/l and Serracia mercescens 1.94g/l. It was also observed that sawdust inoculated with Serratia marcescens produced 14 compounds while the combined species of Aspergillus niger and Serratia marcescens produced only 12 compounds. The following compounds Methylene cyclo propane carboxylic acid, Glycine N-Cyclopropylcarbonyl-methyl ester, acetic acid, propanoic acid, benzaldehyde, thiophene, tetrahydro, furfural and propanoic acid 2,2-Dimethyl-2-ethylhexyl ester were detected from the combination of Serratia marcescens and Aspergillus niger though in different concentrations. The percent concentrations of the compounds from Serratia marcescens range from n-Hexadecanoic acid 3.59 to Methylene cyclo propane carboxylic acid with 11.56 while Serratia marcescens and Aspergillus niger range from Butanoic acid, 4-chloro 3.59 to Benzaldehyde with 11.56. Microbial degradation of sawdust produced economically important products and therefore can contribute to the economy of this country at the same time reduce pollution caused by it which is a novel.
Key words: Sawdust, biodegradation, bioactive, extracts, microbial culture, lignocellulosic.
Microbial biodegradation is the use of bioremediation and biotransformation methods to harness the naturally occurring ability of microbial xenobiotic metabolism to degrade, transform or accumulate environmental pollutants including hydrocarbons (example oil).
Biodegradation is the process by which organic substances are broken down into smaller compounds by living microbial organisms or their enzymes (Godliving and Yoshitoshi, 2002). These organic substances can either be degraded aerobically with oxygen, or anaerobically without oxygen. Interest in the microbial biodegradation of agricultural wastes has intensified in recent years (Koukkou and Anna-Irini, 2011).
Sawdust is a by-product of woodworking operations such as sawing, milling, planning, routing, drilling and sanding. These operations shatter lignified wood cells and break out whole cells and groups of cells. Shattering of wood cells creates dust known as sawdust. This dust causes a lot of health hazards (Baran and Teul, 2007; Zhang, 2004). It is composed of fine particles of wood made up of 3 major components, cellulose, hemicelluloses and lignin (Green, 2006; Alexander, 1997; Erikson et al., 1990). It is generally used for blotting material, hand cleaner, wood filler, compost, packing and for writing notes (Berendeohu, 2018). The general recalcitrance of cellulose, lignin and hemicelluloses and the importance of their biodegradation in the environment have received much attention for several years (Erikson et al., 1990). Raw sawdust takes an average of 180 days to decompose in the soil due to deficiency of nitrogen (Olayinka and Adebayo, 1989). Microbial degradation of sawdust has been very difficult due to the presence of lignin, a highly recalcitrant component that does not degrade easily (Erkson et al., 1990). Despite this, Lennox et al. (2010) were able to degrade sawdust at certain degrees using microorganisms. Shide et al., (2004) reported that wood sawdust was degraded by Lentinus squarrosolus (Mont) singer, a basidiomycete also known as a white rot fungi to form protein, glucose and ethanol. Cellulose fibrils have high tensile strength which is used in the textile industry, paper and miscellaneous materials like vulcanized fibre, plastic filters, filtering media and surgical cotton; other uses include adhesives, explosives, thickening agents, coated paper, cellophane, artificial leather, films and foils (Hitchner and Leatherwood, 1982). This is to say that numerous products can be gotten from biodegradation of sawdust. Fungi of the classes hyphomycetes, zygomycetes, pyrenomycetes, hymenomycetes and the actinomycetes and bacteria of the groups Cytophage, Erwinia, Pseudomonas, Sporolytophaga, Xanthamonas and Streptomonas species degrade hemicelluloses (Lennox et al., 2010; Wuyep et al., 2012).
This research is therefore aimed at characterization and identification of products of biodegradation of lignocellulosic material, sawdust using microbial culture isolated from it.
Samples collection
Fresh and dry undecomposed sawdust composite samples were collected from MCC timber market, Calabar, Cross River State, Nigeria in sterile polythene bags and transported to the laboratory for analysis.
Isolation and identification of indigenous bacteria and fungi involved in sawdust degradation
Bacteria were isolated and identified by carrying out a ten-fold dilution of the wet decomposing sawdust as described by Lennox et al. (2010). Antifungal agent, nystatin was incorporated into nutrient agar. One milliliter of the diluted solution of the sawdust was plated and incubated at 37°C for 18 to 24 h. The discrete colonies were sub-cultured and stock cultures were prepared from the pure cultures and stored at 4°C until needed. The method of Anderson et al., (1973) was used in the isolation of fungi. One milliter from the dilution above was inoculated on potato dextrose agar and incubated at 27°C for 7 days. The discrete colonies were sub-cultured and the pure cultures were stocked and stored until needed. The isolated colonies were characterized and identified.
Purification and maintenance of isolates
Each discrete colony on a Petri dish was transferred using a sterile inoculating loop into plates containing freshly prepared nutrient agar and were incubated at 37°C for 24 to 48 h, respectively. After incubation, the cultural characteristics of the isolates were recorded and compared with descriptive features contained in Bergey's Manual for Determinative Bacteriology (Holt et al., 1994). The isolates were then preserved on nutrient agar slants stored in the refrigerator at 4°C for biochemical characterization and identification.
Biochemical characterization and identification of isolates
The method of Oranusi et al., (2004) as modified was employed for the identification of the bacteria isolates while the fungal isolates were identified based on the taxonomic schemes described by Fawole and Oso (1988). Biochemical and sugar fermentation tests were used for the identification of the bacteria isolates. The biochemical tests carried out were: Catalase, methyl-red, oxidase, citrate utilization, coagulase, indole, coagulase and oxidase tests. The identities of the bacteria were confirmed using the identification aid outlined in Bergey's Manual for Determinative Bacteriology (Holt et al., 1994) as well as that of known taxa as described by Cheesbrough (2003). The fungal isolates were characterized based on colonial morphological features and microscopic examination using the Lactophenol cotton blue stained slide cultures and the results compared with fungal atlas.
Preparation of bacterial and fungal inoculum
The identified bacteria and fungi strains isolated from sawdust sample were transferred from stock agar slant cultures to nutrient agar broths and incubated until a sizable inoculum size of 107 cfu/ml was obtained at 35°C.
Sawdust inoculation and degradation
The collected sawdust samples were first autoclaved to kill all indigenous bacteria and fungi present in it. Three sterile conical flasks were obtained and 25 g of sawdust was introduced into each and properly labeled; bacteria, fungi and combined (bacteria + fungi). 150 ml of distilled water was added into each of the conical flasks followed by the addition of the inoculums size of 10% v/v (17.5 ml) into each of the sawdust solutions. The conical flasks were then incubated at room temperature (26 to 28°C) for 21 days. At the end of the 21 days, the supernatants in the conical flasks were carefully filtered out and the residue sawdust was analysed.
Estimation of protein and glucose contents of fermented sawdust
Protein and glucose contents of the fermented sawdust samples were determined using ASTM standard method (1974) as reported by Bakulin et al., (2007).
Gas chromatography mass spectrometry (GC-MS) analysis
The analysis for chemical components present in sawdust degradation extracts was carried out using GC-MS analyzer (BRUKER SCION 436-GC SQ). Extracts were dissolved in methanol (high-performance liquid chromatography grade) and filtered through Whattman TM FILTER DEVICE (0.2 μm). Helium (99.99%) was used as carrier gas, at a flow rate of 1 ml/min in split mode. RESTEK Rtx®-5 (Crossbond® 5% diphenyl/95% dimethyl polysiloxane) with 30 m length, 0.25 μmdf, and 0.25 mm ID column was used for separation of chemical components. 2 μL of sample was injected into the column. The injector temperature was 320°C. The temperature of oven starts at 70°C and hold for 2 min, and then, it was raised at a rate of 7°C per minute up to 320°C, hold for 1 min. Temperature of ion sources was maintained at 250°C. The mass spectrum was obtained by electron ionization at 70eV and detector operates in scan mode 30 to 500 Da atomic units. Total running time was 38.71 min including 3 min solvent delay. The spectrum of the unknown component was compared with the spectrum of the known components using computer searches on a NARICT Ver.2.1 MS data library. The name, molecular weight, retention time and structure of the components of the test materials were ascertained and results were recorded.
Cultural and morphological characterization of fungal isolates
The cultural and morphological characteristics of the fungal isolates results are shown in Table 1. Based on the colony and microscopic morphology, three species of fungi were identified.
Morphological and biochemical characterization and identification of the bacteria isolates
Table 2 shows the results of morphological and biochemical characterization and identification of the isolates from the sawdust sample analyzed. Five bacteria species were identified.
Estimation of protein and glucose contents
The available protein and glucose contents in the degraded sawdust sample were estimated as presented in Table 3. From the result, it was observed that the fungus degradation had higher protein content available followed by the combined (bacteria and fungi) with the bacterium degradation having the least available protein. However, in the case of glucose, the combined degradation recorded the highest glucose content while the least was seen in the bacterium degradation.
Sawdust degradation (bacteria) components identified by GC-MS analysis
The results of GC-MS analysis of sawdust sample degraded by inoculated bacteria isolate identified fourteen bioactive chemical constituents present in the extracts. The gas chromatogram shows the relative concentrations of various compounds getting eluted as a function of retention time as presented in Table 4 and Figure 1 respectively. The heights of the peak indicate the relative concentrations of the compounds of the components present in the plant extracts. The mass spectrometer analyzed the compounds eluted different times to identify the natures and structure of the compounds. The compounds which were identified by GC-MS analysis were 12 in total (Table 5). Identification of the bioactive compounds was carried out by comparison of their mass spectra and retention time with those of reference standard and published data in NIST MS library (Figure 2).
A total of 8 microbial species were isolated from the sawdust; 5 bacteria and 3 fungi. Microbial degradability of sawdust through the isolation of autochthonous bacterial and fungal species utilizing sawdust as their carbon and energy sources and the observation of differences in sawdust before and after degradation have been demonstrated by Lennox et al. (2010). This corroborates the findings of Godliving and Yoshitoshi (2002) and Zhang et al., (2018) that bacteria and fungi degrade wood sawdust by producing enzymes such as carboxymethyl cellulose, lignin peroxidase and xylanase. Beguin and Aubert (1994) also reported the biodegradability of cellulose. This agrees with the finding of Dosoretz et al., (1990), when they reported the reduction in carbon content of sawdust when subjected to microbial degradation. Hitchner and Leatherwood (1982) reported the ability of cellulase enzyme in the degradation of cellulose. Microbial species have different capabilities of degradation of sawdust (Lennox et al., 2010). This indicates that only few of the isolates are capable of utilizing sawdust as its source of carbon and energy for growth. The microbial isolates, Bacillus sp, Streptococcus sp., P. aeruginosa, Micrococcus sp., S. marcescens, A. niger, Rhizopus sp. and Penicillium sp. in this study are similar to those isolated by Lennox et al. (2010) except Cellulomonas sp., Cytophaga sp., Flavobacterium sp., Xanthomonas sp., for bacteria, but only 2 fungi species, Rhizopus sp. and Penicillium sp. agree with the findings of Lennox et al. (2010). Two out of the eight microbial species isolated from the sawdust were used for this research. S. marcescens and A. niger used in the biodegradation showed their capability to utilize saw dust to produce important products. The efficacy of fungi in cellulose degradation has also been reported (Deeble and Lee, 1985; Kelsey and Shafizadeh, 1980). These reports have provided insight into the possibility of degradation of sawdust using indigenous microorganisms, thereby paving way for enhanced natural attenuation of sawdust polluted sites (Lennox et al., 2010). The saw dust inoculated with S. marcescens produced 14 compounds while the combined species of A. niger and S. marcescens produced only 12 compounds. The following compounds, Methylene cyclo propane carboxylic acid, Glycine N-Cyclopropylcarbonyl-methyl ester, acetic acid, propanoic acid, benzaldehyde, thiophene tetrahydro, furfural and propanoic acid 2,2-Dimethyl-2-ethylhexyl ester were detected in both the S. marcescens and A.niger/S. marcescens inoculated saw dust samples though in different concentrations. Methylene cyclo propane carboxylic acid with 11.56% was the highest produced by S. marcescens but only 9.38% from the combined. This could be as a result of some antagonistic activities of the organisms by producing several hydrolytic enzymes like chitinase, protease, lipase and cellulase (Purkayastha et al., 2018).
S. marcescens produced 10.6% of pentanoic acid 2-ethylhexyl ester which was absent in the combined. This also could be some antagonistic or use of the compound by A. niger. Benzaldehyde concentration was 11.56% from the combined but only 5.91% from S. marcescens. Thiocyanic acid, methylene ester 10.6% produced by the combination was not produced by S. marcescens. The results here show that selective utilization of these organisms in the degradation of saw dust can lead to the production of economically important products. These compounds produced by these microorganisms are the first of its kind in scientific world and therefore a major contribution to knowledge.
The protein and glucose analysis revealed an increase in protein content by 9.5, 49.9 and 25% using
Sarratia marcescens, Aspergillus niger and combination of
S. marcescens and
A. niger respectively. For the glucose, there were decreases of 48.6, 45.2 and 40% using
S. marcescens, A. niger and combination of
S. marcescens and
A. niger respectively. This is to say that these microorganisms were able to utilize saw dust to increase the protein content while at the same time utilized the glucose contained in the saw dust. This is contrary to the findings of Bohdan and Yaser (2011), Reddy and Yang (2009), Van Wyk (2001) and Ndukwe et al., (2012) who detected an increase in glucose levels, though the saw dust samples were pretreated. This is pretreatment of the lignocelluloses wastes to a greater extent must have removed lignin from the different saw dust materials. This research has shown that saw dust waste can be bio converted to useful products without pretreatment as has always been believed. Most of the products produced by the fermentation of the sawdust are very useful. Glycine, N-Cyclopropylcarbonyl-methyl ester produced by
S.marcescens is used as additive in pet food and
animal feed, in foods and pharmaceuticals as a sweetener/taste enhancer, or as a component of food supplements and protein drinks. It is also used in cosmetics. Benzaldehyde, 11.56% produced by
S. marcescens and
A. niger is also an important compound used chiefly in the organic synthesis of dyes, perfumes, and flavors, and as a solvent. Methylene cyclo propane carboxylic acid (11.56%) and Pentanoic acid 2-Ethylhexyl ester (10.6%) also produced from the degradation of sawdust by
S. marcescens have been found to be very useful as an intermediates in the production of pesticides, pharmaceuticals and other agrochemicals. Acetic acid (7.58%) produced by
S. marcescens and
A. niger has several health benefits which include reducing inflamation, lowering blood pressure, and keeping blood sugars spikes at bay. Acetic acid is also used as a condiment and in the pickling of raw vegetables and other foods
The direct fermentation of sawdust without pretreatment with selected indigenous microbial flora is a novel and the amounts of economically important products resulting from the fermentation are also novels. The use of sawdust for the production of these compounds will lead to reduction of pollution caused by sawdust.
The authors have not declared any conflict of interests.
REFERENCES
|
Alexander M (1997). Soft rot bacteria. Introduction to Soil Microbiology 6th Edition, John Wiley and Sons, New York pp. 148-162.
|
|
|
|
Anderson NA, Wang SS, Schwandt JW (1973). The pleurotus ostreatus sapidus species complex. Mycologia 6:28-35.
Crossref
|
|
|
|
|
ASTM (1974). Standard Test Methods for Moisture, Ash, and Organic Matter ofPeat and Other Organic Soils, ASTM International, West Conshohocken, PA; 2007.
|
|
|
|
|
Bakulin MK, Grudtsyna AS, Pletneva A (2007). Biological fixation of nitrogen and growth of bacteria of the genus Azotobacter in liquid media in the presence of Perfluoro carbons. Applied Biochemistry and Microbiology 4:399-402.
Crossref
|
|
|
|
|
Baran S, Teul I (2007). Wood dust: occupational hazard which increases risk of respiratory disease. Journal of Physiology and Pharmacology 58 Suppl 5(Pt 1):43-50.
|
|
|
|
|
Beguin P, Aubert P (1994). The biological degradation of cellulose. FEMS Microbiology Reviews 13(1):25-58.
Crossref
|
|
|
|
|
Berendeohu R (2018). Six things you can do with sawdust and wood shavings. Popular Mechanics 28(7):14-20.
View
|
|
|
|
|
Bohdan V, Yaser D (2011). Assessment of pretreatments and enzymatic hydrolysis of wheat straw as a sugar source for bioprocess industry. International Journal of Energy and Environment 2(3):427-448.
|
|
|
|
|
Cheesbrough M (2003). Microbiological tests. Medical laboratory manual for tropical countries. Cambridge University Press, New Delhi, India pp. 22-36
|
|
|
|
|
Deeble MF, Lee JM (1985). Enzymatic hydrolysis of cellulosic substances in an attrition bioreactor. Biotechnology Bioengineer Symposium 15(2):277-293.
|
|
|
|
|
Dosoretz GG, Chenn HC, Greth HE (1990). Effect of Environmental conditions of extracellular protease activity in lignolytic cultures of Phanerochaete chrusosporium. Applied Environmental Microbiology 56(24):395-400.
|
|
|
|
|
Erikson KEL, Blanchette RA, Ander P (1990). Microbial and Enzymatic Degradation of Wood and Wood Components. Wood decomposition. Springerverlag, New York pp. 41-62.
Crossref
|
|
|
|
|
Fawole MO, Oso BA (1988). Laboratory manual of Microbiology. Spectrum Books Ltd, Nigeria pp. 26-31.
|
|
|
|
|
Godliving M, Yoshitoshi P (2002). Continuous production of lignin degrading enzymes by Bjer Kandora adusta immobilized on polyurethane foam. Biotechnology 24:173-1747.
|
|
|
|
|
Green H (2006). Wood, Craft, Culture, History. Penguin Books, New York. P 403.
|
|
|
|
|
Hitchner E, Leatherwood J (1982). Use of cellulose depressed mutant of cellulomonas in the production of single-cell protein from cellulose. Applied Environmental Microbiology 41:465-583.
|
|
|
|
|
Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST (1994). Bergeys manual of determinative bacteriology. Lippincott Wilkins. A Wolters Kluwer Company. Philadelphia.
|
|
|
|
|
Kelsey R, Shafizadeh F (1980). Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling. Biotechnology and Bioengineering 22(5):1025-1036.
Crossref
|
|
|
|
|
Koukkou I, Anna-Irini E (2011). Microbial Bioremediation of Non-metals: Current Research. Caister Academic Press pp. 312-320.
|
|
|
|
|
Lennox JA, Abriba C, Alabi BN, Akubuenyi FC (2010). Comparative degradation of sawdust by microorganisms isolated from it. African Journal of Microbiology Research 4(13):1352-1355.
|
|
|
|
|
Ndukwe NA, Okiel WO, Alo BI, van Wyke JPH, Mamabolo TM, Igwe CC (2012). Saccharification of delignified saw dust from 20 different trees in the Lagos area of Nigeria. African Journal of Biotechnology 11(100):16625-16629.
|
|
|
|
|
Oranusi SU, Oguoma OI, Agusi E (2004). Microbiological quality assessment of foods sold in student's cafeterias. Global Research Journal of Microbiology 3(1):1-7.
|
|
|
|
|
Olayinka A, Adebayo A (1989). Effect of pre-incubated sawdust based cow dung on the growth and nutrient uptake of Zea mys L. and on soil chemical properties. Biology and Fertility of Soils 7(2):176-179.
Crossref
|
|
|
|
|
Purkayastha GD, Mangar P, Saha D (2018). Evaluation of the biocontrol efficacy of a Serratia marcescens strains indigenous to tea rhizosphere for the management of root rot disease in tea. PLOS One 13(2):1-15.
Crossref
|
|
|
|
|
Reddy N, Yang Y (2009). Natural cellulose fibers from soybean straw. Bioresearch and Technology 100(14):3593-3598.
Crossref
|
|
|
|
|
Shide EG, Wuyep PA, Nok AJ (2004). Studies on degradation of wood sawdust by Lentinus squarrosulu (Mont.) singer. African Journal of Biotechnology 3(8):395-398.
Crossref
|
|
|
|
|
Van Wyk JPH (2001). A Review: Biotechnology and the utilization of organic waste as a resource for bioproduct development. Trend Biotechnology 19(5):172-177.
Crossref
|
|
|
|
|
Wuyep PA, Odunola MB, Balogun NA (2012). Purification and partial characterization of laccase from Lachnocladium sp. International Journal of Biological Chemistry 6(2):612-620.
Crossref
|
|
|
|
|
Zhang H, Krafft T, Gao D, Zheng G, Cai L (2018). Lignocellulose biodegradation in the biodrying process of sewage sludge and sawdust. Drying Technology, an International Journal 36(3):316-324.
Crossref
|
|
|
|
|
Zhang Y (2004). Indoor air quality. Engineering. Boca Raton, CRC Press, Florida P 18.
Crossref
|
|
