Lipase-producing Microorganisms Research Articles

Biodegradation of Oil by a Newly Isolated Strain Acinetobacter junii WCO-9 and Its Comparative Pan-Genome Analysis.

Waste oil pollution and the treatment of oily waste present a challenge, and the exploitation of microbial resources is a safe and efficient method to resolve these problems. Lipase-producing microorganisms can directly degrade waste oil and promote the degradation of oily waste and, therefore, have very significant research and application value. The isolation of efficient oil-degrading strains is of great practical significance in research into microbial remediation in oil-contaminated environments and for the enrichment of the microbial lipase resource library. In this study, Acinetobacter junii WCO-9, an efficient oil-degrading bacterium, was isolated from an oil-contaminated soil using olive oil as the sole carbon source, and its enzyme activity of ρ-nitrophenyl decanoate (ρ-NPD) decomposition was 3000 U/L. The WCO-9 strain could degrade a variety of edible oils, and its degradation capability was significantly better than that of the control strain, A junii ATCC 17908. Comparative pan-genome and lipid degradation pathway analyses indicated that A. junii isolated from the same environment shared a similar set of core genes and that the species accumulated more specific genes that facilitated resistance to environmental stresses under different environmental conditions. WCO-9 has accumulated a complete set of oil metabolism genes under a long-term oil-contamination environment, and the compact arrangement of abundant lipase and lipase chaperones has further strengthened the ability of the strain to survive in such environments. This is the main reason why WCO-9 is able to degrade oil significantly more effectively than ATCC 17908. In addition, WCO-9 possesses a specific lipase that is not found in homologous strains. In summary, A. junii WCO-9, with a complete triglyceride degradation pathway and the specific lipase gene, has great potential in environmental remediation and lipase for industry.

Hydrolyzable microplastics in soil\u2014low biodegradation but formation of a specific microbial habitat?

Microplastics (MP, plastic particles between 0.1 and 5000 μm) contaminate agricultural soils through the application of organic fertilizers, sewage sludge, and plastic mulch. MP surfaces and the MP-soil interface provide specific habitats for soil microorganisms—the plastisphere. Microorganisms in the plastisphere may benefit from utilizing MP as a carbon (C) source. Hydrolyzable MP with ester bonds are susceptible to enzymatic depolymerization by hydrolysis. In a microcosm experiment, we investigated MP biodegradation of small and large (< 0.5 mm and 0.5–2 mm respectively), hydrolyzable (a poly(lactic acid)/poly(butylene co-adipate terephthalate) blend, PLA/PBAT) and non-hydrolyzable (low-density polyethylene, LDPE) polymers, and the effects of these MP on microorganisms in dry and wet MP-amended soil. MP affected neither abundance and composition of the main soil microbial groups (fungi, Gram-negative, and Gram-positive bacteria), specific activities of ß-glucosidase, ß-xylosidase, lipase, and phenoloxidase, nor respiration in MP-amended soil. Only large PLA/PBAT particles in dry soil were significantly mineralized (15.4% of initial PLA/PBAT-C after 230 days). PLA/PBAT mineralization coincided with enhanced lipase and ß-glucosidase activities on the surfaces of individual PLA/PBAT particles extracted from the soil after incubation (compared to LDPE and non-incubated PLA/PBAT particles). We detected cracks on the surfaces of PLA/PBAT particles using scanning electron microscopy, indicating initiation of MP biodegradation, presumably due to depolymerization by lipases. Results suggest that the PLA/PBAT plastisphere is a polymer-specific habitat for lipase-producing soil microorganisms. Our study demonstrates that analyzing biogeochemical interactions within polymer-specific plastispheres is essential to assess MP fate and their impacts on microbially driven soil processes.

Effectiveness of Aeromonas hydrophila for the removal of oil and grease from cattle slaughterhouse effluent

Little is known about the use of microbial lipases for the treatment of beef industry effluent. It is essential to develop techniques for the sustainable management of this waste, given its high polluting potential, especially due to the high concentrations of oils and greases (O&G) present. Therefore, this study investigates the pretreatment of slaughterhouse effluent using lipase-producing microorganisms isolated from the waste itself. Firstly, a physicochemical characterization of the effluent was performed, followed by isolation and identification of a lipase-producing bacterium, which was used in a pretreatment step. A central composite rotational design (CCRD) was applied, considering the independent variables pH, temperature, and inoculum concentration, resulting in 17 assays, with lipolytic activity, O&G, and free fatty acids (measured during 72 h) as the response variables. The slaughterhouse effluent presented high polluting potential, with an O&G concentration of 2553.3 mg L−1 and COD of 8914.4 mg L−1. The isolated bacterium was identified as Aeromonas hydrophila. After 72 h of the experiment, lipolytic activity of 1.40 U mL−1, 59% O&G removal (pH: 7.0, temperature: 30.0 °C and inoculum concentration: 10%), and production of 36.0 μmol mL−1 of free fatty acids were obtained. It could be concluded that cattle slaughterhouse effluent is an excellent source of lipase-producing microorganisms and that the use of enzymes for the treatment of this waste is a promising technology.

Optimization of organic solvent-tolerant lipase production by Acinetobacter sp. UBT1 using deoiled castor seed cake.

Organic solvent-tolerant lipase-producing microorganisms were isolated from petrol spilled soil. From ten morphologically distinct lipase-producing bacterial isolates, highest amount of lipase-producing isolate UBT1 was identified as Acinetobacter sp. using 16S rRNA gene sequencing (NCBI Accession No: MH879815). An increase in lipase production from 42 U/mL to 243 U/mL was obtained when different deoiled seed cakes were supplemented instead of olive oil in the medium. Further optimization of media components by the statistical approach assisted in discerning the main influencing media components and their optimum concentrations. Nine components glucose, castor seedcake, potassium nitrate, gum arabic, calcium chloride, magnesium sulphate, potassium di-hydrogen phosphate, dipotassium hydrogen phosphate, and ferric chloride were selected for Plackett-Burman design. The optimum concentrations of three significant selected components for the lipase production were found to be 0.025 gm% glucose, 0.002 gm% calcium chloride, and 0.2 gm% potassium di-hydrogen phosphate as determined by Response Surface Methodology. Increase in lipase production with 292.29 U/mL was achieved in the media containing optimized components and 2 gm% deoiled castor seed cake. Purification studies with ammonium sulphate precipitation, dialysis, and gel permeation chromatography resulted in 77.54% recovery with 5.77-fold partially purified lipase. The residual activity of lipase in 50 and 75% concentration of n-hexane among other solvents after 24h was 105.05 and 90.42%, respectively, indicating its solvent tolerance. The present study reports the isolation of organic solvent-tolerant lipase-producing Acinetobacter sp. UBT1, optimization of the culture media for lipase production using the deoiled castor seed cake, and its partial purification.

STANDARDIZATION OF THE CULTIVATION OF THE ISOLATED FILAMENTOUS FUNGUS A4 FOR LIPASE PRODUCTION / PADRONIZAÇÃO DO CULTIVO DO FUNGO FILAMENTOSO ISOLADO A4 PARA A PRODUÇÃO DE LIPASES

Lipases are produced by microorganisms to form an important biotechnological group of enzymes with different properties and specificities. Among the lipase-producing microorganisms are the filamentous fungi, which are abundant and widespread in nature. Because of their catalytic versatility, lipases are highly commercialized, being applied mainly in the production of detergents, food, animal feed, paper and cellulose, pharmaceutical and biodiesel industries. This work sought to select a filamentous fungus that produces lipases and optimize the conditions for cultivation so as to increase lipolytic activity. Initially, 21 filamentous fungi were grown in a solid medium for lipases for four days in a bacteriological incubator at 30 oC. The fungus with the largest halo representing lipase activity was incubated in five different submerged media at 30 oC for eight days, and the lipase activity of crude extracellular enzyme extracts was evaluated every 24 hours. After defining the culture medium, its composition was varied, analyzing different sources of nitrogen, salt solutions, the influence of the initial pH of the medium and the carbon source. The isolated fungus, identified as A4, exhibited promising lipase synthesis in Khanna submerged culture medium containing urea, peptone and KNO 3 as the nitrogen source, Vogel salts solution, initial pH of 4.5 and cotton oil as a carbon source. This work demonstrated the importance of standardizing chemical conditions for the growth and production of lipases from filamentous fungi, thereby contributing to a greater understanding of the lipases produced by the selection of the fungus with a potential for enzyme production.

Isolation and Identification of Magnusiomyces capitatus as a Lipase-Producing Yeast from Olive Mill Wastewater

Olive mill wastewaters (OMW) are effluents originated from olive oil extraction. As an oil-rich residue, OMW is a potential source of lipase-producing microorganisms and a complex medium potentially suitable for lipase production. The aim of the present study was to isolate yeasts with the ability to produce extracellular lipases from OMW. Thirty-two yeast isolates were obtained and screening for esterase/lipase activity using rapid plate detection methods allowed the selection of five isolates. Subsequently, extracellular lipolytic activity was determined in shake-flasks, and the best activity was found in the isolate JT5 (0.85 U/mL). This isolate was identified as Magnusiomyces capitatus by DNA sequencing. Growth and lypolytic activities by M. capitatus JT5 were assessed in undiluted OMW, and optimization of lipase production was achieved by a positive interaction of two factors (oxygen availability and nitrogen concentration). The highest lipase activity (1.4 U/mL) was obtained at NH4Cl concentration of 2.8 g/L and kLa of 0.65 min−1. The growth of M. capitatus JT5 in a stirred tank bioreactor, using undiluted OMW, allowed the improvement of lipase production (up to 3.96 U/mL) by increasing olive oil concentration in the medium, under the selected conditions of nitrogen concentration and oxygen availability. This study highlighted the isolate M. capitatus JT5 as a lipase-producing microorganism that is able to grow in undiluted OMW under controlled conditions. Results obtained in shake-flasks have been reproduced satisfactorily in the stirred tank bioreactor.

Microbial Production of Ethanol From Sludge Derived From an Urban Wastewater Treatment Plant.

A collection of lipase-producing microorganisms was isolated from sludge derived from an urban wastewater treatment plant. The microorganisms with the highest levels of lipase activity were selected in order to use triglycerides present in the sludge effectively and were then transformed with pdc:adhB genes for the production of ethanol. The transgenic strains showed high growth rates in diluted sludge and produced lipase protein in order to utilize fat present in the sludge, which provides an abundant source of carbon. Using sludge derived from treated wastewater as nutrient source, ethanol was produced by certain transgenic species belonging to the genera Proteus. Different forms of sludge were tested for maximal ethanol production, with dehydrated sludge being found to produce the best performance.

Production of lipase extrated from aqueous waste: enzymatic activity kinetics

Lipases are an important group of enzymes with various applications in the food, chemical and pharmaceutical industry, besides having great interest for the treatment of effluents with high lipid content. The objective of this study was to isolate, characterize and select lipolytic bacteria that produce lipase from aqueous waste effluents and to study the enzymatic activity kinetics of the extract obtained via submerged fermentation. The results obtained are promising, being possible to isolate and characterize 23 lipase-producing microorganisms, mostly gram-positive bacteria, but after the fermentation step in a liquid medium, gram negative bacteria showed the highest enzymatic activity (56.72 U.L-1 for STP 2A` bacterium and 81.99 U.L-1 for R2B). In the enzymatic activity kinetic study with the selected bacterium (R2B), among the six variables (temperature, pH, minimal mineral medium, soybean oil, glucose and sodium nitrate), temperature was the one that most positively influenced the enzymatic activity, and the best results were obtained at 40°C. It was concluded that the enzyme extract obtained from environmental waste may be used to treat the effluent and contribute to reduce environmental impacts.

English

The aim of this study was to isolate and select lipase-producing microorganisms originated from different substrates, as well as to optimize the production of microbial lipase by submerged fermentation under different nutrient conditions. Of the 40 microorganisms isolated, 39 showed a halo around the colonies and 4 were selected (Burkholderia, Bacillus sp., Penicillium lanosum and Corynebac glutamyl), where strains showing lipolytic halo Radius (R) / colony radius (r) ratio greater than 2.0 were selected. The results of submerged fermentation expressed as enzymatic activity revealed that the genera of microorganisms significantly influenced the enzymatic reaction, and lipase obtained from Burkholderia cepacia was the most promising, with activity of 0.0058 U.mL-1. It was also observed in the optimization step of lipase production that the sodium nitrate content (NaNO3) had a positive effect on enzyme production, and its increase was indicative of higher enzymatic activity. The addition of sources of organic nitrogen (corn steep liquor, p = 0.2398), carbon (soybean oil, p = 0.3379), magnesium MgSO4.7H2O (p = 0.4189) and potassium KH2PO4 (p = 0.8562) had no significant effects on the lipase production and could result in decreased production of extracellular lipases. Key words: Burkholderia cepacia, hydrolytic enzymes, residue, submerged fermentation.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Screening, identifying and medium optimization of a lipase-producing filamentous fungus from soil for high chiral resolution of 1-phenylethanol

The kinetic resolution of racemates by biocatalysis is one of the major routes to manufacture optically pure compounds. Lipase-producing microorganisms were screened from soil for chiral resolution of 1-phenylethanol in the study. One microorganism with high chiral resolution ability was isolated and identified as Rhizopusstolonifer by the sequence similarity analysis and construction of phylogenetic tree. The media for the microorganism were optimized using Plackeet-Burman, Steepest Ascent and Box-Benhnken experiments. After optimization, the composition of the optimized medium used for lipase production was as follows: soluble starch (7.60 g/L), yeast extract (22.59 g/L), K2HPO4 (5.55 g/)L, NH4Cl (1.80 g/L), MgSO4·7H2O (2.40 g/L), NaCl (1.00 g/L), olive oil (25.00 mL/L), Tween-80 8.00 (mL/L), pH 4.59, and the lipase activity increased nearly by 2.04-fold, from 0.46 to 1.40 U/mL. The optimization was significant for the isolation and purification of the lipase and for the research of enzymatic characteristics. Key words: Screening, lipase, identification, Rhizopus stolonifer; optimization, 1-phenylethanol.

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca2+, Zn2+, Mg2+) - with the exception of Co2+— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.

Screening of thermophilic neutral lipase-producing Pseudomonas sp. ZBC1 and improving its enzymatic activity

From oil-contaminated soil, three lipase-producing microorganisms were selected as good lipase producers using rhodamine B-olive oil plate agar and they were identified as from Pseudomonas, Burkholderia and Klebsiella genera by morphology, biochemical characterization and 16S rRNA gene sequencing. Among the three strains, Pseudomonas sp. ZBC1 was found to produce thermophilic neutral lipase which exhibited maximum activity at 80oC and pH 7.0. Plackett-Burman design (PB design) and response surface methodology (RSM) analysis showed that the optimum values of the most significant factors are MgSO4 (0.85 g/L), inoculum size (65.9 ml/L) and volume of medium (51.57 ml). Under the optimal conditions, lipase production by Pseudomonas sp. ZBC1 was enhanced by approximately 2.30 fold (from 8.50 to 19.50 U/ml). Keywords : Pseudomonas , lipase, thermophilic, response surface methodology African Journal of Biotechnology Vol. 12(9), pp. 949-957

QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms

The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R2adj) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R2cv) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

Bacterial diversity of lipase-producing strains in different soils in southwest of China and characteristics of lipase

Due to the rapid development observed in industries, environmental pollution became more and more critical. Lipase-producing strains played a key role in the enzymological remediation of polluted soils. Diversity of culturable lipase-producing bacteria in the southwest of China was studied with 198 samples collected from different points, along the transect from Tibetan Plateau-Ya An–Sichuan Basin. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) technology was used to amplify the isolated lipolytic producer’s DNA samples and the amplified results were analyzed by NTSYS-pc2.1 to identify the similarity of isolates. A total of 54 bacterial strains were isolated from samples: 21 strains from Sichuan Basin (38.9%), 15 strains from Ya An (27.8%) and 18 strains from Tibetan Plateau (33.3%). The result of enzyme activity analysis showed that intracellular enzyme activity was nearly three folds than extracellular enzyme activity, and the intracellular enzyme activity was 6.79, 6.23 and 5.59 U/ml from Sichuan Basin, Ya An and Tibetan Plateau, respectively. Based on 16S rRNA gene sequence analysis, 12 strains were identified and belonged to Bacillus, Klebsiella, Pseudomonas and Enterobacter. The results showed that the Bacillus strains were the preponderant strains in the Tibetan Plateau and the Klebsiella strains were predominant isolates in Ya An. It suggested that the strains of Klebsiella were the transitional lipase-producing strains from Tibetan Plateau to Sichuan Basin. It could be concluded that the diversity of lipase-producing strains showed a transitional tendency: diversity of lipase-producing strains in Sichuan Basin were significantly higher than that of other two places and the diversity decreased gradually from plain region to Plateau region. Key words: Enterobacterial repetitive intergenic consensus-polymerase chain reaction(ERIC-PCR), lipase-producing microorganisms, diversity, Tibetan Plateau

Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production

The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.

Immobilized lipase from potential lipolytic microbes for catalyzing biodiesel production using palm oil as feedstock

Biodiesel has been regarded as a biodegradable and non-polluting fuel. Enzymatic transesterification reaction for manufacturing biodiesel from vegetable oils with alcohol is an attractive approach. However, the cost of enzyme remains a barrier for its industrial implementation. The aim of this research was the screening of lipase-producing microorganisms and the studies of potential lipase-mediated biodiesel production using palm oil as substrate. A total of 360 strains of bacteria, yeasts and fungi were isolated and screened from the samples of oil-contaminated soil and waste water. Among all the screened microbes, the potential lipolytic bacterium, Staphylococcus warneri , unicellular yeast , Candida rugosa and filamentous fungus , Fusarium solan i were selected because of their high specific activities. The lipase-producing conditions were subsequently optimized by using palm oil as an inducer and lipase activities were compared for both hydrolytic and synthetic catalysis. C . rugosa lipase, which exhibited the highest potential for catalyzing the biodiesel production, was further purified and immobilized on various hydrophobic supports. The catalysis of transesterification between methanol and palm oil by the C. rugosa immobilized lipases revealed that immobilized lipase from C. rugosa on Sepabeads EC-OD was the most promising for further development as a biocatalyst for the application of enzyme-catalyzed biodiesel synthesis. Key words : Screening, lipases, immobilization, biodiesel.

Selection of Lipase-Producing Microorganisms through Submerged Fermentation

Lipases are enzymes used in various industrial sectors such as food, pharmaceutical and chemical synthesis industries. The selection of microorganisms isolated from soil or wastewater is an alternative to the discovery of new species with high enzymes productivity and with different catalytic activities. In this study, the selection of lipolytic fungi was carried out by submerged fermentation. A total of 27 fungi were used, of which 20 were isolated from dairy effluent and 7 from soil contaminated with diesel oil. The largest producers were the fungi Penicillium E-3 with maximum lipolytic activity of 2.81 U, Trichoderma E-19 and Aspergillus O-8 with maximum activities of 2.34 and 2.03 U where U is the amount of enzyme that releases 1 micromol of fatty acid per min per mL of enzyme extract. The fungi had maximum lipolytic activities on the 4th day of fermentation.

Improved method for direct screening of true lipase-producing microorganisms with particular emphasis on alkaline conditions

A rapid and effective method for direct detection, selection and testing of microorganisms able to produce both cell-bound and extracellular true lipases is described. The method is based on formation of clearance zones on turbid solid media with emulsified olive oil around or under the colonies, cell fractions or culture supernatant of lipase-producing organisms. The method was successfully applied for the screening and isolation of microorganisms producing alkaline lipases.

A simple method for detection of lipolytic microorganisms in soils

Media selective for the isolation of bacteria, actinomycetes and fungi were amended with 0.1% sunflower oil emulsified with 0.01% Tween 80. Lipase-producing microorganisms produced clear zones on these media. When lipase-producing bacteria were cultured on a polycarbonate membrane laid on the selective medium for bacteria, clear zones were produced on the medium when the membrane along with bacteria was removed. The agar disc cut from the clear zone also produced a clear zone when placed on the fresh medium, indicating that clear zone formation is the result of the activity of extracellular lipases. The largest population of lipase-producing microorganisms in an agricultural soil was actinomycetes followed by bacteria and fungi. Ranging from 12 to 75% of bacteria, actinomycetes and fungi isolates from soils collected from three different locations were capable of producing lipases. In general, relatively small percentages of soil bacteria were lipase producers, and lipase producers were more common among soil actinomycetes and fungi. These three groups of microorganisms appear to be all important in decomposition of oils in organic matters in soils.