Soil science
S. Arabteymori; A. Halajnia; A. Lakzian; F. Nikbin
Abstract
Introduction Surfactants as surface-active substances with combined hydrophobic and hydrophilic properties are widely used in various fields. In soil remediation processes these substances can be used to increase the availability of organic and inorganic contaminants to improve microbial decomposition ...
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Introduction Surfactants as surface-active substances with combined hydrophobic and hydrophilic properties are widely used in various fields. In soil remediation processes these substances can be used to increase the availability of organic and inorganic contaminants to improve microbial decomposition of organic pollutants or heavy metals adsorption. In recent years, researchers have been seeking to produce and use surfactants that are more environment friendly. In this regard, produced biosurfactants by microorganisms are of special importance due to their environmental benefits. Microorganisms produce a wide range of biosurfactants. Biosurfactants are extracellular compounds that can combine with metals such as zinc, copper, and cadmium and can increase the solubility of these metals and reduce their toxicity. Negatively charged anionic biosurfactants such as rhamnolipids and lipopeptides can increase heavy metals availability by combining to metals and changing the properties of soil solution. In this study, the effect of surfactant application from Pseudomonas putida and Bacillus subtilis and some chelators include sodium citrate, humic acid and Na2-EDTA on soluble cadmium in a contaminated calcareous soil was investigated.Materials and MethodsThis study was conducted as factorial in a completely randomized design in laboratory conditions at several steps separately. A calcareous soil sample was contaminated with 15 mg kg-1 cadmium from the source of Cd (NO3)2. Contaminated soil incubated for 4 weeks at field capacity. Acid deposition method was used for surfactant extraction from culture medium of Pseudomonas putida KT-2440 and Bacillus subtilis 1795. The structure of extracted biosurfactants was investigated by FTIR. Equilibrium time was obtained by determining the amount of soluble cadmium at times 6, 12, 24, 36, 72 hours by adding 1mM sodium citrate, humic acid and Na2-EDTA to the contaminated soil (ratio of 1 to 5 soil to solution).The concentrations of 0, 0.1, 0.25, 0.5, 1 and 2 mM of humic acid, sodium citrate and Na2-EDTA were used to determine the appropriate concentration of each chelator. To investigate the interaction of chelators and biosurfactants on soluble cadmium, an experimental was conducted as a completely randomized design with factorial arrangement design. Experimental treatments consisted of three types of chelating agents (sodium citrate, humic acid, Na2-EDTA and control), two types of surfactants from Pseudomonas putida and Bacillus subtilis, and five concentration levels of the biosurfactants (0, 25, 50, 100 mg L-1).Results and DiscussionThe highest amount of soluble cadmium (11.59 mg L-1) was observed in Na2-EDTA treatment at 72 hours, which was significant compared to the other treatments. The lowest amount of soluble cadmium was obtained through application of sodium citrate (0.205 mg L-1) at 36 hours. In all studied concentrations, Na2-EDTA had the greatest effect and sodium citrate had the least effect on soluble cadmium. While the use of Na2-EDTA at all concentrations caused a significant increase in soluble cadmium, sodium citrate had no significant effect on soluble cadmium at studied concentrations. Humic acid at concentrations higher than 0.5 mM significantly increased the soluble cadmium. Increasing the concentration of humic acid and citrate from 1 to 2 mM did not show any significant impact on soluble cadmium. At all levels of biosurfactant application, Na2-EDTA and humic acid caused a significant increase in soluble cadmium concentration. In control and sodium citrate treatments, application of biosurfactants did not cause significant difference in the concentration of soluble cadmium. The highest amount of soluble cadmium was obtained as a result of the application of Bacillus subtilis surfactant and Na2-EDTA. However, increasing the concentration of Bacillus subtilis surfactant from 25 to 100 mg L-1 had no significant effect on increasing the efficiency of Na2-EDTA. Pseudomonas putida surfactant had no significant effect on soluble cadmium in Na2-EDTA application. While in humic acid treatment, the application of the Pseudomonas putida surfactant at the highest concentration (100 mg L-1) increased the concentration of soluble cadmium. Using Bacillus subtilis surfactant did not have effect on soluble cadmium in application of humic acid.ConclusionAmong the studied chelators (sodium citrate, humic acid and Na2-EDTA), Na2-EDTA had the greatest effect on soluble cadmium. While sodium citrate had no significant effect on soluble cadmium. Surfactants from Pseudomonas putida and Bacillus subtilis had different effects on increasing the efficiency of studied chelators and soluble cadmium in the studied soil. In Na2-EDTA and humic acid application, surfactant from Bacillus subtilis at a concentration of 25 mg L-1 and surfactant produced by Pseudomonas putida at a concentration of 100 mg L-1 had a significant effect on soluble cadmium, respectively. It seems using biosurfactants and chelators on increasing soluble cadmium in soil can be useful for phytoremediation purposes to increase its uptake by plant. However, further research is needed.
E. Ghadiri; N.S. Naghavi; K. Ghaedi
Abstract
Introduction: Cellulase enzymes are the second largest group of the enzymes with many industrial applications such as in textile industries, production of detergents, animal and human food processing, paper industries and biofuel production. Many microorganisms are capable for production cellulases, ...
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Introduction: Cellulase enzymes are the second largest group of the enzymes with many industrial applications such as in textile industries, production of detergents, animal and human food processing, paper industries and biofuel production. Many microorganisms are capable for production cellulases, but only a small number of them produce significant amounts of this enzyme. The main sources of cellulases production are microorganisms including fungi and bacteria. Among the cellulose degrading aerobic and anaerobic bacteria, most of the studies have been done on Cellulomonas spp. and Clostridium spp., respectively. Also Bacillus spp. has been used for production of cellulase in a homologous manner. Expression of cellulases in some bacterial genera such as Bacillus, Pseudomonas, Ralstonia and Zymomonas, as well as some yeast species such as Saccharomyces cerevisiae and some fungal genera such as Aspergillus and Trichoderma has been reported too. Low levels of cellulase production has always been a major concern that leads to researches for finding of highly active microorganisms strains and employing biological technologies for identification of their enzyme coding genes suitable for probable transformation other organisms. The purpose of this study was the screening of the cellulose degrading bacteria in Mazandaran forest soils and detection of the enzyme coding gene in the isolate with the highest cellulase activity.
Materials and Methods: In order to isolate cellulase producing bacteria, soil samples were obtained from different regions of Mazandaran province forests including Nanoacla (A), Siaocla (B), Someesara (C), Namakabroud (D), Noor (E), Izadshahr (F), Sisanghan (G) and Sehezar (H) forests. Cellulase producing isolates were selected on carboxymethyl cellulose agar using congo red dye and the amount of their endoglucanase activities was measured by assessment of released glucose using dinitrosalicylic acid reagent. Each micromole of released glucose in 1 ml of enzyme solution per minute was considered as an enzyme activity unit (U/min.ml). Identification of bacterial species was performed by amplification and sequencing of a 1500 bp length fragment in 16S rDNA by using 1492R and 27F universal primers. Enzyme production by the selected isolates was also detected in different growth conditions. In order to investigate the effect of carbon source concentration, the amounts of 2-10 g/L of carboxymethyl cellulose were added to bacterial growth culture media. The effect of growth pH values in the range of 4 to 10 and sodium chloride at concentrations of 0 to 10 g/L were studied on endoglucanase production by the selected isolates in carboxymethyl cellulose media. Then the endoglucanase coding sequences in different strains of the bacterial sp. with the highest endoglucanase activity were investigated in Gen Bank and the primers were designed based on the obtained data for the gene amplification.
Results and Discussion: The results of heterotrophic bacteria counting showed the highest number at station B (Siakla forest). Subsequently, station A (Nanocla forest) and station G (Sisangan forest) had the highest number of heterotrophic bacteria, respectively. From the eight selected forest stations, eight top cellulase producing isolates were selected in carboxy methyl cellulose broth medium. The highest endoglucanase activities were belonged to the isolates A2 (1.92 U/min.ml), B2 (1.65 U/min.ml), and H3 (1.51 U/min.ml), respectively. The amplification of the 16SrRNA gene resulted in the formation of a 1500 bp band after electrophoresis in agarose gel electrophoresis. Sequencing results of the purified PCR products showed that B2 and A2 isolates belonged to Bacillus subtilis with 99% similarity. H3 isolate also belonged to Bacillus cereus with 99% similarity. In other studies in the forest soils of Iran for isolation of cellulase producing bacteria, Bacillus had been one of the most active cellulase enzyme producers. The present study showed that Noor forests are among the best places to isolate bacterial cellulase-producing strains. PCR amplification protocol was designed and the total sequence of endoglucanase with 1072 bp length was amplified. Molecular evaluation of endoglucanase gene in Bacillus subtilis (B2) showed 77% similarity to the endoglucanase gene (elgS) in Bacillus subtilis subsp. subtilis. Since the strain B2 had the highest production of endoglucanase among the isolated bacteria, it was attempted to enhance the production of the enzyme using this strain by changing the growth conditions. The isolate B2 was able to grow at different concentrations of carboxymethyl cellulose as a carbon source, but the highest endoglucanase activity was observed at the concentration of 8 g/L with a significant difference compared to other concentrations. The pH equal to 7 and the absence of sodium chloride salt was also led to significant highest endoglucanase production by this isolate.
Conclusion: The endoglucanase gene obtained in this study was reported for the first time with a new sequence. The enzyme showed more sustainable activity than other aerobic bacterial endoglucanases which had previously been studied. This sequence can be introduced into high expressional bacterial strains and used to produce high amounts of endoglucanase for bio-energy industries applications.
