Plate Screening Method Research Articles

Antitumor activity of urease-free l-asparaginase with low glutaminase coactivity produced by marine-derived Aspergillus flavus

l-asparaginase, which has a high therapeutic effect in various cancer diseases, hydrolyzes l-asparagine to aspartate and ammonia and is mainly used in treating Acute Lymphoblastic Leukaemia. This study aims to investigate the l-asparaginase production potential of marine filamentous fungi and the partial purification and characterization of the l-asparaginase. The rapid plate screening method was used to measure the l-asparaginase activity of 94 marine fungi. l-asparaginase producers were then screened for glutaminase and urease activity. The selected extracellular l-asparaginase producer, Aspergillus flavus MF10, showed 2.82 U/mL l-asparaginase activity in small-scale fermentation. l-asparaginase was concentrated by ultrafiltration followed by acetone precipitation. The specific activity of the enzyme, purified 4,8 fold by partial purification, was determined as 259 U/mg. There were no urease and glutaminase activities in the fermentation broth, but low glutaminase activity (1.27 U/mL) was detected in the concentrated fermentation broth. The optimum pH and temperature of the l-asparaginase were 8.0 and 40 °C, respectively. l-asparaginase was stable at pH 7.0–8.0 and can maintain its activity at 70% at 40 °C after 1 h. While the enzyme decreased the viability of SH-SY5Y (neuroblastoma) to 43% and LNCap (prostate cancer) to 61%, it did not show cytotoxic effects in healthy cell line L929 (fibroblast). It was also determined that l-asparaginase showed low cytotoxicity in HaCaT (keratinocyte) cells. In conclusion, it was shown that urease-free l-asparaginase with low glutaminase coactivity, produced from marine-derived A. flavus MF10, has the potential for medical use.

A High-Throughput Method Based on Microculture Technology for Screening of High-Yield Strains of Tylosin-Producing Streptomyces fradiae.

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 μg/ml) and UN-C137 (6889.72 ± 70.25 μg/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 μg/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases.

β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers – they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Searching for new microbial enzymes for plastic polymer degradation: Polyurethanes and Lycra

New enzymes and biocatalysis could provide routes to help limit global plastic pollution, as suggested by the example of PETases for the PET degradation. Experiments were conducted to screen soil microorganisms for enzymes able to break down the plastic polymer Lycra, which contains polyurethane linkages and could be utilized as a carbon and nitrogen source. The methodology is based on a plate assay, which compared the growth of bacteria at 50°C and 65°C on minimal media and minimal media with a top layer, that contained DMF-dissolved Lycra. The medium provided the main nutrients required for bacterial growth, with Lycra being the only substantial carbon source. Promising candidates were identified as bacterial colonies with visibly enhanced growth on the Lycra-enriched minimal medium plate. The selected soil microorganisms were then tested with a liquid culture assay, where the strain was cultivated at 50°C or 65°C in liquid broth medium, that contained dissolved Lycra. The liquid culture samples were collected at 4 time points between 0-48h, and the aliquots tested on HPLC for detection of released Lycra monomers. Around 50 different bacterial strains from 6 various soil and compost sources were tested with the described plate screening method and 7 bacterial thermophilic species were detected, that showed visibly enhanced growth on the Lycra-enriched plates. These results could support the development of Lycra enzymatic degradation. New enzymes could be engineered, optimized and yield a sustainable solution for the textile industry, which would not require toxic chemicals or contribute to landfill.

Preliminary Investigation of Amylase Producing-Bacteria from Soil in Gombe Metropolis

Amylases are enzymes that are able to hydrolyse starch or glycogen molecules into polymers of glucose units. They have great potential applications in various industrial processes like in pharmaceutical, fermentation and food industries. Research on starch degrading enzymes has resulted into increased applications of amylases in different industrial processes. These enzymes occupy a greater space in the current biotechnological processes such as detergent, starch degradation, pharmaceutical, foodstuff, textile, and paper manufacturing. In fact, amylases constitute nearly 25% of the total sale of global enzymes. Amylases have been screened and identified from various sources, both eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria, respectively. To further isolate novel amylases with enhanced desirable properties for such diverse industrial application, more organisms need to be screened. In this study, a total of 27 bacterial isolates were isolated from soil samples in Gombe metropolis. The bacteria were screened for amylase production using plate screening method. Each isolate was streaked onto a 1% starch agar plate and incubated for 24h at 37 °C. The plates were covered with iodine solution and observed for positive amylase isolates based on the formation of clearing zones against the blue black background. The results confirmed eight (8) isolates of amylase-producing bacteria which include Bacillus subtilis, Escherichia coli, Streptococcus spp., Salmonella spp., Pseudomonas spp., Serratia spp., Proteus vulgaris, and Klebsiella spp. In conclusion, bacterial isolates capable of amylase production have been successfully screened and identified. This research may serve as a stepping stone to isolating functional amylase enzymes from these bacteria for promising industrial applications.

A novel design to screen chlorogenic acid-producing microbial strains from the environment

The present study aimed to develop a plate-screening method, based on the specific color development of complexes formed between chlorogenic acid, a valuable plant-derived compound, and aluminum (III), to detect chlorogenic acid-producing microbial strains. Modified media with 0.75 mM aluminum chloride were developed to identify CGA-producing bacteria (based on beef extract agar medium) or fungi (based on the potato dextrose agar medium). Compared with conventional screening, the modified media let to 3.3 times more CGA producers from plants, at 90.9% selective accuracy. Novel chlorogenic acid-biosynthesizing strains included Brevibacillus borstelensis B14, Bacillus amyloliquefaciens B17, Bacillus badius B19, Sphingomonas yabuuchiae N21, Enterobacter tabaci N22, and Lodderomyces elongisporus S216 and P212. Strain S216 produced the highest chlorogenic acid yield (23.39 mg L−1). This study provides a highly efficient and low-cost tool for quick detection and subsequent identification of several newly isolated strains with chlorogenic acid-producing potential.

Screening and Identification of Trichoderma Strains Isolated from Natural Habitats with Potential to Cellulose and Xylan Degrading Enzymes Production.

A total of 123 Trichoderma strains were isolated from different habitats and tested for their ability to degrade cellulose and xylan by simple plate screening method. Among strains, more than 34 and 45% respectively, exhibited higher cellulolytic and xylanolytic activity, compared to the reference strain T. reesei QM 9414. For strains efficiently degrading cellulose, a highest enzyme activity was confirmed using filter paper test, and it resulted in a range from 1.01 to 7.15 FPU/ml. Based on morphological and molecular analysis, the isolates were identified as Trichoderma. The most frequently identified strains belonged to Trichoderma harzianum species. Among all strains, the most effective in degradation of cellulose and xylose was T. harzianum and T. virens, especially those isolated from forest wood, forest soil or garden and mushroom compost. The results of this work confirmed that numerous strains from the Trichoderma species have high cellulose and xylan degradation potential and could be useful for lignocellulose biomass conversion e.g. for biofuel production.

Enhanced microalgae growth through stimulated secretion of indole acetic acid by symbiotic bacteria

In many microalgal cultivation systems, microalgae co-exist with bacteria, while little is known about the characteristics of their symbiotic relationships. In this study, twenty-six microalgae growth-promoting bacteria were isolated from a culture system of Scenedesmus sp. LX1 cultivated in the secondary effluent from domestic wastewater by using the high-throughput multiple well plate screening method. Ten strains were found to produce and secrete indole acetic acid (IAA), promoting the growth of microalgae. Meanwhile, the microalgae might have secreted signal substances to induce IAA production in bacteria, which was amplified in the tryptophan abundant environment. This indicates that bacteria may mainly promote the growth of the co-existing microalgae through secreting IAA, and microalgae would selectively enhance IAA secretion in turn. Microalgae cultured with microalgal growth-promoting bacteria would be a new potential strategy for improving large-scale microalgal cultivation in an economic and environmentally-friendly way.

Enhanced production of l-lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high-linear energy transfer heavy ion mutagenesis.

The aim of this study was to improve l-lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy-ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l-lactic acid producers, a scale-down from shake flask to microtiter plate was developed. The results showed that 24-well U-bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale-down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l-lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed-batch fermentation, the A69 mutant can accumulate 114.2g/L l-lactic acid at 96h. Hence, the proposed traditional microbial breeding method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain lactic acid-overproducing strain.

Using Cellulolytic Nitrogen Fixing Bacterium, Azomonas agilis for Effective Degradation of Agricultural Residues.

Introduction: Azomonas agilis, a nitrogen-fixing bacterium, was isolated from rhizospheric soil in central Myanmar.Methods & Materials: The nitrogen-fixing activity of this bacterium was detected by plate screening method using glucose nitrogen free mineral medium and ammonium test-kit Cellulolytic activity was screened by plat assay and detected by Dinitrosalicyclic acid method (DNS).Results & Discussion: The isolated A. agilis grew in media containing 3-12% of NaCl, although the growth became poor when NaCl concentrations increased. Among various carbon sources, sucrose was the best source for ammonium accumulation of this bacterium, whereas arabinose was not the suitable carbon source. Although the nitrogen-fixing activity of A. agilis was highest after one week incubation, cellulase enzyme production was highest after 2-3 days of incubation. It was observed that cellulase enzyme activity of A. agilis for cellulose and sodium carboxymethyl cellulose (CMC) was almost the same. Three agricultural wastes were used to detect the cellulase enzyme activity of A. agilis, cellulase activity was better on filter paper as a substrate when compared to rice-straw and sawdust.Conclusion:So, the isolated A. agilis has high potential as an effective bacterial strain to use in sustainable agriculture and degradation of some agricultural residues.

Selective screening: isolation of fungal strains from contaminated soils in Austria

Summary Microorganisms are potent contributors to maintaining a safe environment as they are able to degrade organic toxicants. For environmental applications, mostly bacteria are used while fungal strains have received less attention. However, they are able to degrade highly persistent organic contaminants and survive extreme conditions, and may thus be promising organisms. To find new fungal candidates for these applications, twelve soil samples from polycyclic aromatic hydrocarbon (PAH) contaminated sites in Austria were used to isolate fungal strains. A microplate screening method using PAH contaminated soil as inoculant was set up to isolate fungal strains being able to live in presence of toluene, hexadecane, or polychlorinated biphenyl 126. Not many microbial strains are known that degrade these three contaminants, while the PAH contamination acted as selective pressure for the soil microbiota. After obtaining pure cultures, the fungal strains were further screened for their ability to live in the presence of one of the three contaminant substrates. The potential for technical application of the 11 best performing strains, identified using ITS and 18S rDNA, is discussed. The presented microtiter plate screening method is a cost efficient and quick approach to identify fungal strains for pollutant degradation and results in candidates with a high relevance for bioremediation techniques.

Isolation and Screening of Cellulolytic Filamentous Fungi.

Filamentous fungi are among the microorganisms that most efficiently are able to degrade plant biomass by secreting cell wall-degrading enzymes and they are therefore used extensively in the industry as workhorses for the production of enzymes, including cellulases for the use in second-generation biorefinery concepts. Fungi are therefore of interest both as resources for the search of novel cellulolytic enzymes and for production of enzymes and enzyme cocktails, which also can be carried out on-site using cheap lignocellulosic substrates for growth and enzyme production. Fungi can be isolated from different environmental niches, such as soil, compost, decaying wood, decaying plant material, building materials, and different foodstuffs. Selective isolation can be carried out using simple cellulosic or complex plant material in the media. In this chapter, methods used for the isolation and screening of cellulolytic fungi isolated from different ecological niches are presented. The screening assay presented in the chapter is an easy semiquantitative high-throughput agar plate screening method using azurine-cross-linked (AZCL) cellulose substrates.

ANALYSIS OF GUT FLORA FROM DAMP WOOD TERMITES (TRINERVITERMES SPP.) AND EXTRACTION, CHARACTERIZATION OF CELLULASE FROM THE ISOLATE

Objective: The objective of the study was to isolate, characterize, identify, and compare the potentials of cellulolytic strains isolated from the gut of damp wood termites (Trinervitermes species) collected from Chennai, Tamil Nadu, India.Methods: Termites were collected and used as hoard of cellulase producers and screened for the cellulase producers using carboxymethyl cellulose as the sole source of carbon and were authenticated using Congo red plate screening method. The isolates showing a significant zone of clearance were further confirmed by biochemically characterized.Results: Nine effective isolates were characterized and three strained were used for analysis. The organisms were subjected with substrate, temperature (25°C, 37°C, and 45°C), and pH to optimize the cultural condition. The enzyme activity was estimated using endoglucanase, FPase after incubating at appropriate conditions. Five isolates showing a significant zone of clearance were selected, out of which three belonged to Bacillus and one each to Staphylococcus. Optimization of media and genetic modification of the strains can further convalesce their competence. All the isolates have ensuring application in view of use in future.Conclusion: An effective strain of bacteria was isolated from the gut of termites can be used as a potential candidate for the production of cellulase in industries.

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into l-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

Development of cellulolytic nitrogen-fixing activity of Azotobacter spp. by transposon mutagenesis

The chemical mutagenized microorganisms used in this study were nitrogen-fixing bacteria; Azotobacter chrococcum and Azotobacter benjerinkii which were collected from the Microbiology Laboratory, Department of Biotechnology, Kyaukse. Transposon mutagenesis was studied on these strains to get dual activities such as nitrogen-fixing and cellulolytic activities by using recombinant Escherichia coli S17 carrying cellulase gene from Rhizobium leguminosarum. The targeted strains that carry the transposon were selected on the Glucose Nitrogen Free Mineral Medium which contains chloramphenicol (0.6 mg/20ml) and kanamycin (0.4mg/20ml). Nitrogen-fixing activity of Azotobacter spp. was detected by the colour changes in Glucose Nitrogen Free Mineral Medium (G-NFM) containing trace amount of Bromothymol Blue (BTB) as indicator and ammonium test kit. Cellulolytic activity of Azotobacter spp. before and after transposon mutagenesis was detected by both plate screening method and by Dinitrosalicylic colorimetric method. The nitrogen-fixing activities of the strains were almost the same before and after transposon mutagenesis. Cellulolytic activity measured in Glucose Nitrogen Free Mineral Medium was the highest in 5 days incubation.

Isolation of Phosphate Solubilizing Bacteria from White Lupin (Lupinus albus L.) Rhizosphere Soils Collected from Gojam, Ethiopia

Phosphorus is an essential macronutrient. A great portion of phosphorus from chemical fertilizers becomes insoluble and unavailable to plants because of its conversion into salts. Phosphate solubilizing microorganisms as a phosphorus biofertilizer improve soil fertility by solubilizing insoluble phosphate salts and increase crop production. This research aimed in isolation and characterization of phosphate solubilizing bacteria. Sixtythree soil samples collected from seven different parts of white lupin growing areas of Gojam, Ethiopia. Halo zone formation and plate screening method used for isolation. Based on halo zone formation; 152 phosphate solubilizing bacterial isolates obtained. Based on their solubilization index four isolates selected for subsequent experiments and characterization. The four selected isolates tested for their solubilization efficiency on solid media. Isolates HUPSB-35 and HUPSB-45 appeared maximum solubilization index (4.5 mm). Isolate HUPSB-57 was unique with its colony formation; salt tolerance (able to grow up to 10%) and utilization of wide range of carbon sources (utilize all the tested sugars). Isolate HUPSB-27 showed a wide range of pH and temperature preference (100% growth at 10, 15, 20, 25 and 30°C in all tested pH).

Co-production of Pectinase and Biosurfactant by the Newly Isolated Strain Bacillus subtilis BKDS1

Aim: To isolate pectinase producing bacterial strains from various samples and to investigate the co-production of pectinase and biosurfactant by this isolated strain. Study Design: Isolation, screening, selection and identification of pectinolytic bacteria. Production of pectinase and analysis of exo-pectinase types. Partial purification by ammonium sulphate precipitation and dialysis. The study also analyses the capability of the selected strain for biosurfactant production. Place and Duration of Study: Department of Life Sciences, University of Calicut, Kerala 673635, India, between July 2013 and November 2014. Methodology: The pectinolytic bacteria was isolated and screened by iodine plate assay method. The enzyme production was confirmed by 3,5-dinitrosalicylic acid (DNS) method. The potent enzyme producing strain was biochemically characterised and identified by 16S rRNA gene sequence analysis. Thiobarbituric acid (TBA) and DNS assay were performed to test the types of exo-pectinase produced. Partial purification of the enzyme was done by ammonium sulphate precipitation. Biosurfactant production by the strain was tested by methods like foam formation, Original Research Article Kavuthodi et al.; BMRJ, 10(2): 1-12, 2015; Article no.BMRJ.19627 2 drop collapse test, oil displacement test, microplate assay, hemolytic assay, penetration assay, emulsification activity and bacterial adhesion to hydrocarbon (BATH) assay. Results: Thirty six pectinolytic bacterial strains were screened from the collected samples. Four isolates were selected on the basis of zone size (20 mm to 26 mm) in well plate screening method. In which, the isolate showed maximum enzyme production in DNS assay (0.707 U/mL) is the same which exhibited larger pectin depolymerization area (26 mm) in well diffusion assay. This strain was identified as Bacillus subtilis BKDS1 using 16S rDNA based molecular technique. The optimum incubation time was found to be 72 h for the maximum enzyme production (1.288 U/mL). Assay of exo-pectinase revealed that, the organism was able to produce polygalacturonase (PG), pectin lyase (PNL) and pectate lyase (PEL). In ammonium sulphate precipitation, the enzyme activity was found in 40 -100% salt saturation fractions. On further analysis, it was observed that the organism also produced biosurfactant along with pectinase in the same culture medium. Conclusion: The present study indicates the possibility of an integrated process for obtaining pectinase enzymes and biosurfactants in the same culture media. So this could greatly increase the economic viability of the isolated strain.

Decolorization and Biodegradation of Direct Blue 2B by Mix Consortia of Bacillus

Mix consortia of Bacillus sp was used to decolorize Direct Blue dye 2B. The bacterial cultures exhibited 60 % decolorization ability within 48 h at temperature 40 o C and pH 7. Maximum rate of decolorization was observed (60%) when starch was supplemented in the medium. Decolorization of Direct Blue dye 2B was monitored by TLC, which indicated that dye decolorization was due to its degradation into unidentified intermediates. Maximum, dye-decolorizing efficiency was observed at 200 mg/l concentration of Direct dye 2B. A plate assay was performed for the detection of decolorizing ability of bacteria. Clearing zone (decolorization) was formed surrounding the bacterial culture. Decolorization was confirmed by UV-VIS spectrophotometer. The initial dye solution showed high peak at the wavelength of 540nm. Plate screening method has shown dark brown zone observed around colonies producing ligninolytic enzyme responsible for dye degradation. The pot culture experiment revealed that the Direct Blue 2B dye show phytotoxicity against Allium sativum, but, after degradation of dye, the degraded products obtained show less toxicity.

English

The objective of this present study was to isolate, identify and screen for potential fungal isolates from local degrading wood chips with respect to glucanase and xylanase production. The fungal strains were isolated from degrading wood chips over a period of six months. Five hundred and twenty six isolates were obtained from the different degrading wood chips with different frequencies of occurrence. Using cultural and morphological observations, the isolates were identified and grouped into thirteen fungal species. All the fungal strains were screened for their ability to produce both glucanase and xylanase by plate screening method using Congo red as indicator. Twenty isolates were further selected among them and screened for the production of the enzymes in liquid medium. Among the twenty isolates, Trichoderma reesei TRAR had the highest glucanase activity of 10.18 U/ml while Fusarium compactum FCGA had the highest xylanase activity of 9.33 U/ml. Key words: Xylanase, glucanase, microfungi, degrading wood, screening.

Isolation of Plant Hormone (Indole-3-Acetic Acid - IAA) Producing Rhizobacteria and Study on Their Effects on Maize Seedling

18 rhizobacteria were isolated from various rhizospheric soils in Mandalay region, Myanmar and some of their biochemical characteristics were studied. Among them, 4 isolates belonged to Bacillus spp. and another 5 strains were recognized as Serratia spp. All 18 isolates were screened for indole-3-acetic acid (IAA) production and quantitative determination of IAA was done for all strains by UV-Vis spectrophotometer with 2 days interval during 10 days incubation. All isolates had different optimum IAA production periods and strain R1 was the best IAA producer strain with 121.1 ppm. It was observed that Bacillus spp. produced IAA ranging from 53.1 ppm to 71.1 ppm optimally and Serratia spp. can be regarded as poor IAA producer strains. Among these 18 strains, four of the best IAA producers (R1, R3, R5, R8) were selected for further study on maize plant and their activities of nitrogen fixation were also detected by plate screening method. Seed germination rate was studied with eight types of treatment and the treatment systems with isolates cannot show distinct positive effect on seed germination rate. At 20th day after sowing, dry root weight and fresh root weight, root length, number of adventitious roots, dry shoot weight and fresh shoot weight and shoot height were measured. In pot trial experiment, treatment R1 significantly increased the fresh shoot weight, shoot height and dry shoot weight whereas treatment R3 significantly increased fresh root weight, root length, dry root weight and number of adventitious roots and all treatments with bacterial isolates showed plant growth promotion than the control systems.