Classifications

• • C—CHEMISTRY; METALLURGY
  • C05—FERTILISERS; MANUFACTURE THEREOF
  • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
  • C05F11/00—Other organic fertilisers
  • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/38—Pseudomonas
  • C12R2001/385—Pseudomonas aeruginosa
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/425—Serratia
  • C12R2001/43—Serratia marcescens

Definitions

• the present invention relates to biofertilizers and their optimum composition.
• Nutrients such as Nitrogen (N), Phosphorus (P) and Potassium (K) are essential and they should be adequately supplied for the growth of plants.
• P is essential for photosynthesis and, therefore, the assimilation of carbohydrates, such as sugars.
• phosphorus In its many compounds, phosphorus has roles in cell division, in stimulation of early root growth, in hastening plant maturity, in energy transformations within the cells and in fruiting and seed production.
• mineral fertilizers account for more than 90 percent of the fertilizers used (Sabri, M.A. (2009/ Evolution of fertilizer use by crops in Malaysia: Recent trends and prospects, Paper presented at IFA Crossroad Asia- Pacific in Kota Kinabalu, Sabah, Malaysia. Dec 2009).
• Biofertilizer is a low cost technology, eco-friendly and harmless to environment with the ability of supplementing nutrients Many definitions and interpretations of the term biofertilizer exist (Vessey, J.K. (2003). Plant growth promoting rhizobacteria as biofertilizers. Plant and Soil. 255: 57 1-586).
• biofertilizer is a product that consisting of selected and different types of living microorganism, which are known to improve plant growth through the supply of available plant nutrients through biological processes (Hegde, D.M., Dwiv, B.S. and Sudhakara, S.N. ( 1999). Biofertilizers for cereal production in India— a review. Indian Journal of Agricultural Science. 69: 73-83; Vessey, 2003). Development of biofertilizer requires carrier material to be mixed with microorganisms. Such carriers being used are peat (Albareda, M., Rodriguez-Navarro. D.N.. Camacho. M. and Temprano, F.J. (2008).
• biofertilizers containing an arbuscular mycorrhizal fungus ⁇ Glomus mosseae or Glomus intraradices) with or without N-fixer ⁇ Azotobacter chroococcum), P sokibilizer ⁇ Bacillus megaterium) and K solubilizer ⁇ Bacillus mucilaginous was experimentally proven to significantly increase the growth of Zea mays.
• the aim of the present invention is to select bacteria isolates that are capable of soliibilizing P in a liquid medium at the amount of at least 100 ⁇ g/mL, and to develop an optimum biofertilizer composition for these bacteria.
• the object of the present invention is a biofertilizer comprising at least one bacterial strain selected from the group comprising Pseudomonas aeruginosa strain AGKT 1 , Serratia marcescens strain AG T4 and Bacillus amyloliquefaciens.
• the bacteria are grown and used in a culture medium comprising peptone, KC1, MgS0 4 .7H 2 0, MnS0 4 .H 2 0, FeS0 4 .7H 2 0, NaCl and a sugar selected from glucose and fructose, and having a pH between 6 and 10.
• a particularly suitable culture medium for Pseuclomonas aeruginosa strain AG T 1 comprises glucose 0.5 g, peptone 0.325 % (w/v), KC1 0.02 g/L, MgS0 4 .7H 2 0 0.1 g, MnS0 4 .H 2 0 0.002, FeSO 4 .7H 2 0 0.002 g and NaCl 0.2 g in 1 L, pH 9.0. Inoculation of the bacterial inoculum is preferably at 2.750% (v/v).
• a particularly suitable culture medium for Serratia marcescens strain AGKT4 comprises fructose 60 g, peptone 6 g, KC1 0.02 g, MgS0 4 .7H 2 0 0.1 g ,MnS0 4 .H 2 0 0.002 g, FeS0 4 .7H 2 0 0.002 g and NaCl 0.2 g in 1 L, pH 7. Inoculation of the bacterial inoculum is preferably at 1 .875% (v/v).
• a particularly suitable culture medium for Bacillus amyloliquefaciens comprises fructose 60.0 g, peptone 6.0 g, MgSO 4 .7H 2 0 0.10 g, KC1 0.2 g, MnS0 4 .H 2 0 0.002 g, FeS0 4 .7H 2 0 0.002 g and NaCl 0.2 g in 1.0 L distilled water, pH 8.0. Inoculation of the bacterial inoculum is preferably at 2.750% (v/v).
• the biofertilizer further contains a carrier, which is selected from cocoa pod husk, cocoa bean shell and peat moss, more preferably the carrier is cocoa bean shell.
• a carrier which is selected from cocoa pod husk, cocoa bean shell and peat moss, more preferably the carrier is cocoa bean shell.
• the biofertilizer in both dry and liquid forms may be stored at temperatures of 10 °C to 30 °C. In these conditions, the biofertilizer compositions were capable of maintaining the number of cfu at the levels sufficient for the use in the application. The number of cfu were above 5 logio cfu/g for dry formulation and 5 logio /mL for liquid formulation after 6-month storage. Generally, the dry formulations displayed superioriority over the liquid formulations in terms of the survivability of bioinoculant.
• the dry formulations exhibit the desirable characteristic of pH above 5 and near to the neutral pH.
• the moisture content of above 37% in all the formulations at both temperatures were satisfactory.
• the liquid formulations were also satisfactory as the number of cfu were in the suitable range for a formulation.
• the biofertilizer can be administered in combination with an N P fertilizer.
• the biofertilizer may be used in the agriculture, in particular for crop and soil improvement, as biofungicide or plant growth regulator . Examples of carrying out the Invention
• Example 1 Isolation and characterization of bacteria
• Bacterial isolates were isolated from soil with dumped palm oil shell, rhizosphere soil of cocoa, soil under dumped cocoa pods, dumped cocoa pods, liquid sample from cocoa fermentation box, healthy cocoa pods, rhizosphere soils of grass, corn and groundnut. All sources of bacteria samples were taken from Tawau, Sabah. All samples of 10 g each was put in 90 mL of Nutrient Broth in a 200 mL conical flask shaken for 24 to 48 h. Solution of soil or sample of 1 mL was diluted in a serial dilution technique. Exactly, an aliquot of 0.1 mL was put on the nutrient agar (NA) for isolation of bacteria.
• NA nutrient agar
• the nutrient agar plates were incubated at 28 °C for one to six days. Isolated, predominant, morphological distinct colonies were selected, purified by repeated culturing and maintained on NA slants.
• the Bacillus amyloliquefaciens was isolated from rhizosphere soil of cocoa at 25 °C at the depth of sampling point 0-15 cm.
• the AGKT 1 bacterial strain was isolated from soil under dumped oil palm shell at 30 °C at the depth of sampling point 0-1 5 cm.
• the AGKT4 bacterial strain was isolated from rhizosphere soil of groundnut at 30 °C at the depth of sampling point 0- 15 cm.
• the bacterial inocula were prepared by inoculating a loopful of bacterial stock cultures from NA slants into 10 mL of sterile LB broth in a universal bottle. The bottles were incubated for 18 h at 28 °C in an oven. After 18 h, a loopful of inoculum from each bacterial inoculum was aseptically streaked on the plate assay. The plates were incubated at 28 °C in an incubator for one to four days. Observation on the growth of bacteria, clearing zone surrounding the colonies of bacteria as well as the change of bromophenol blue color was recorded as potential PSB.
• the NBRIY medium (Nautiyal, 1999) was used which comprised Ca 3 (PO 4 ) 2 5 g, glucose 10 g, MgSO 4 .7H 2 0 0.10 g, KC1 0.2 g, (NH 4 ) 2 SO 4 0.5 g, MnS0 4 .H 2 0 0.002 g, FeS0 .7H 2 0 0.002 g/ NaCl 0.2 g, 1.0 L distilled water, pH 7.0.
• the inoculum of each bacterial isolate was prepared by inoculating a loopful of bacterial stock culture into the LB broth and incubated at 28 °C in an incubator for 18 h.
• Isolate of AGKT1 could solubilize phosphate at the amount of 50.2 ng/mL, 121.2 Hg/mL, 128.1 ⁇ g/mL, 129.1 ⁇ g/mL, 145.8 ⁇ /mL, 138.7 ⁇ g/mL, 133.2 ⁇ g/mL and 103.2 ⁇ / mL at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively.
• Isolate of AGKT4 could also solubilize phosphate at the amount of 99.0 ⁇ g/ mL, 125.1 ⁇ g/mL, 94.7 ⁇ g/mL, 163.6 ⁇ g/mL, 122.4 ⁇ g/mL, 128.0 ⁇ g/mL, 144.9 ⁇ g/mL and 108.6 ⁇ g/ m at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively.
• Bacillus amyloliquefaciens could solubilize phosphate at the amount of 103.4 ⁇ g/ mL, 121.1 ⁇ g/mL, 142.8 ⁇ g/mL, 123.8 ⁇ g/mL, 143.9 ⁇ g/mL, 129.4 ⁇ g/mL, 123.7 ⁇ g/mL, and 127.1 ⁇ g/ m at at the first day, the second day, the third day, the fourth day, the fifth day, the sixth day, the seventh day and the eighth day, respectively.
• the control was used using NBRIY medium containing all the rock phosphate sources without bacterial inoculation.
• the inoculum of each bacterial isolate was prepared by inoculating a loopful of bacterial stock culture into the LB broth and inctibated at 28 °C in an incubator for 18 h.
• One millilitre of each bacterial inoculum which had an optical density at 600 nm of 0.8 ( « 10 12 cfu) was inoculated into a 50 mL medium in a 100 mL conical flask.
• the flasks were incubated for four days at 28 °C and agitated at 70 rpm on an orbital shaker (Lab-Line Orbit Shaker, Lab-Line Instrument, Inc.).
• the degree of ability of the bacterial isolates to solubilize insoluble phosphate on various poorly rock phosphates was diverse.
• CRP CRP
• sequence of the ability of bacterial isolates to solubilize phosphate was AG T4>BA>AGKT1
• CIRP, NCRP and FMP the sequences were BA>AGKT1>AGKT4, BA>AGKT4>AGKT1 and AGKT1>BA>AGKT4 / respectively.
• the ability of bacterial isolates to solubilize the rock phosphates was due to the reduction of pH in the medium.
• the sequences of pH reduction in the medium for CRP, CIRP, NCRP and FMP were AGKT4 ⁇ BA ⁇ AGKT1, AGKT4 ⁇ BA ⁇ AGKT1, AGKT4 ⁇ BA ⁇ AGKT1 and AGKT4 ⁇ BA ⁇ AGKT1, respectively.
• AGKT4 Rod Negative Absent 16s rDNA sequencing Further, the bacteria were identified using 16s rDNA sequencing. Two bacterial isolates from AGKTl and AGK.T4 were used for genomic DNA extraction to identify the isolates. The genomic extraction was done according to DNeasy Blood and Tissue Kit (QIAGEN) manual book. An approximately 1.5 kbp band was obtained.
• PCR Polymerase Chain Reaction
• the reaction mixture was then subjected to PGR reactions containing the above forward and reverse primers using a PCR machine.
• the reaction mixture was initially heated for 4 min at 94 °C, followed by 30 PCR cycles (94 °C 1 min, 58 °C 1 min and 72 °C 1 min); then, one cycle of 7 min at 72 °C and held at 4 °C.
• the primers of AGKT l and AGKT4 were amplified.
• the PCR products of 1442 kbp and 1445 kbp were obtained from AGKT l and AGKT4, respectively.
• the amplified products were examined by electrophoresis and extracted by using gel extraction kit (QIAquick) according to the manufacturer's instructions. The DNA purification was done according to QIAquick Gel Extraction Kit manual book.
• Sequencing and Blasting The extracted products were sent to First BASE Laboratories Sdn Bhd, Malaysia for sequencing. A homology search was conducted using a Basic Local Alignment Search Tool (BLAST) from the website address of http://www.ncbi.nlm.nih.gov/BLAST/.
• BLAST Basic Local Alignment Search Tool
• the optimized medium for Pseudomonas aeruginosa strain AGKT I comprised glucose 0.5 g, peptone 0.325 % (w/v), KCl 0.02 g/L, MgS0 4 .7H 2 0 0. 1 g, MnS0 4 .H 2 0 0.002, FeSO 4 .7H 2 0 0.002 g and NaCl 0.2 g in 1 L distilled water, pH 9.0. Inoculation of the bacterial inoculum was at 2.750% (v/v).
• the optimized medium for Serratia marcescens strain AGKT4 comprised fructose 60 g, peptone 6 g, KCl 0.02 g, MgS0 4 .7H 2 0 0.1 g ,MnS0 4 .H 2 0 0.002 g, FeS0 4 .7H 2 0 0.002 g and NaCl 0.2 g in 1 L distilled water, pH 7. Inoculation of the bacterial inoculum was at 1.875% (v/v).
• the optimized medium for Bacillus amyloliquefaciens comprised fructose 60.0 g, peptone 6.0 g, MgSO 4 .7H 2 0 0.10 g, KC1 0.2 g, MnS0 4 .H 2 0 0.002 g, FeS0 4 .7H 2 0 0.002 g and NaCI 0.2 g in 1.0 L distilled water, pH 8.0. Inoculation of the bacterial inoculum was at 2.750% (v/v).
• cocoa pods husks were dried in an oven at 70 °C for 7 to 14 days or sun dried for a few weeks. All dried cocoa pod husks, cocoa bean shell and peat moss were ground using a China Made Disk Mill Model FFC-45A manufactured by Qingdao (Dahua Double Circle Machinery, Co. Ltd., China) to pass through a 1 mm sieve (Testing Sieve, SANPO, Tokyo, Japan). Two hundred and ten grams of each carrier materials powder was packed in low density polyethylene plastic bags of 15 cm x 23 cm (0.05 mm gauge), sealed using a hot plastic sealer ( Hind, Malaysia) and sterilized at 121 °C and 15 psi for 3 h (1 h intervals).
• the carrier materials were then mixed with the liquid compositions.
• Example 4 Effect of the biofertilizer on cocoa seedlings Preparation of Soil for Potting and Seedlings: Non-sterile of low nutrient soil from the top of 15 cm of a soil from the Table Series was collected from QL Farms Sdn Bhd, Quoin Hill,
• N was made using Urea, P using Ca 3 (P0 4 ) 2 and K using C1 at the rate of 1.38 g N/plant, 0.56 g P/plant and 0.45 g K/plant, respectively. Fertilizers of N and K were applied at fortnightly intervals starting from 30 DAS up to the fifth month, while P was applied after 30 DAS as the only one-application.
• Treatment Tl was served as the control without any application of fertilizer.
• Treatment T2 was the standard nursery fertilizer application using NPK blue 12: 12: 12+ TE which was applied at the rate of 10 g/plant split to fortnightly intervals as recommended by Teoh ( 1980). All inorganic fertilizers were applied to the seedlings near the root zone of cocoa seedlings.
• NKP [Urea, KC1 and Ca 3 (P0 4 ) 2 ]
• N as Urea at 1.38 g N/ plant
• K as KC1 at 0.45 g K/plant
• P as Ca 3 (P0 4 ) 2 at 0.56 g P/plant
• Treatments T4-T12 applied at 37.5 g/ plants/ once at the adjacent of root zone in a circle furrow of 2 cm in depth and 5 cm apart from the basal stem.
• Treatments T13-T15 applied at 25 mL/ plant /month at the adjacent of the root zone 5 cm apart from the basal stem.
• Treatment T3 consisted of fertilizer from N and K source at the same rate as in treatments, T4 to T15.
• Treatments of T4 to T12, dry bioinoculant formulations were applied at 37.5 g/ plant once ( «10 14 cfu/ g), near the root zones of cocoa seedlings in a circular furrow of 2 cm in depth and 5 cm apart from the basal stem of cocoa seedling. The furrow was then covered with the soil.
• Treatments of T13, T14 and T15 which were liquid bioinoculant formulations, applied at 25 mL/ plant ( ⁇ IO 14 cfu/mL) by dispensing into the pots using a pipetor at 30-day interval.
• the liquid formulation was applied near the root zones of cocoa seedlings, 5 cm apart from the basal stem. All treatments were commenced after one month-old of cocoa seedlings. All pots were also amended with lime using CaCO 3 at the rate of 6 g/ pot as top dressing to raise the pH of soil. Filtered deionized water was used as watering the seedlings in the pots at the field capacity (22%) using ELGA deionized cartridge connected to the water supply. Regular maintenance of pest and disease was carried out during the course of the experiment.
• the growth parameters were measured.
• the plant height was measured from the cotyledon scar to the young shoot of cocoa seedlings using a measuring tape.
• the diameter of plant was measured using a digital clipper below the cotyledon scar of cocoa seedlings at the end of experiment.
• the number of leaves of cocoa seedlings was counted at the end of experiment. Only the recently matured leaves and matured leaves were counted. The results showed that the treatments had significant effect on the growth parameters of cocoa seedlings.
• Most of the treatments using phosphate-solubilizing bacteria in the formulation either in dry formulations or liquid formulation increased significantly the stem diameter, plant height and the number of leaves as compared to Tl (control).
• T7 (NKP + Pseudomonas aeruginosa strain AGKT1 + cocoa bean shell) and T8 (NKP + Serratia marcescens strain AGKT4 + cocoa bean shell) had the greatest effect on all growth parameters followed by T4 (NKP + Pseudomonas aeruginosa + CPH), T5 (NKP + Serratia marcescens + CPH),T9 (NKP + Bacillus amyloliqiiefaciens + peat moss) and T6 (NKP + Bacillus amyloliqiiefaciens + CPH).
• Table 7 The effects of treatments on stem diameter, height and number of leaves for 5-month old seedlings.
• treatments T7 and T8 showed the greatest effect on the number of leaves as compared to the other treatments, including control (Tl), standard fertilizer application (T2) and straight fertilizer NPK (T3).
• control Tl
• standard fertilizer application T2
• straight fertilizer NPK T3
• Table 8 shows that the treatments had a significant effect on the fresh weight of plant components.
• Treatments of T4, T5, T6, T7 and T8 resulted of significant increase of fresh weight of leaf, stem, root, total fresh of plant as well as shoot as compared to the other treatments.
• Most of these treatments had low ratio of root: shoot indicating that the effect of treatments was greater on the growth of the upper part of plant than the root growth. This case might explain that most nutrient uptake was on the upper part of vegetative growth, stem and leaves.
• Table 8 The effects of treatments on the fresh weight of leaf, stem, root, total weight, shoot and the ratio of roofcshoot of 5-month old seedlings.
• Dry weight of plant components was determined: The plants were harvested at twenty weeks of planting. The root, stem and leaves were separated and put into a handmade brown translucent paper (brown onionskin) envelop and oven dried separately at 70 ° C for about 5 days for a constant weight. Then the root, stem and leaves were weighed. The weight of dry root was divided by the weight of dry shoot (the total weight of dry leaves and stem) to get dry root:shoot ratio.

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