WO2005118488A1 - Identification of calcium carbonate dissolving bacteria and its possible industrial utilization - Google Patents
Identification of calcium carbonate dissolving bacteria and its possible industrial utilization Download PDFInfo
- Publication number
- WO2005118488A1 WO2005118488A1 PCT/TR2004/000028 TR2004000028W WO2005118488A1 WO 2005118488 A1 WO2005118488 A1 WO 2005118488A1 TR 2004000028 W TR2004000028 W TR 2004000028W WO 2005118488 A1 WO2005118488 A1 WO 2005118488A1
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- WIPO (PCT)
- Prior art keywords
- calcium carbonate
- dissolving
- caco
- identification
- agrobacterium
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/06—Contaminated groundwater or leachate
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Definitions
- the present invention relates to a calcium carbonate dissolving bacteria and a novel method for treatment of clogging in drip irrigation system, by utilization of this kind of bacteria.
- drip irrigation also called trickle irrigation or micro-irrigation
- trickle irrigation is a localized irrigation method that slowly and frequently provides water directly to the plant root zone (Evans, 2000). It is known as low cost water delivery system. Due to limited water resources and environmental consequences of common irrigation systems, drip irrigation system is getting more attraction and playing important role for agricultural production, particularly with high value cash crops such as greenhouse plants, ornamentals and fruit production. Therefore, use of drip irrigation system is rapidly increasing around the world.
- Microorganisms with CaCO 3 dissolving activity may be useful for development of an environmentally friendly method in controlling of chemical clogging of emitters in drip irrigation system.
- objects of the present invention are; 1 )to isolate and identify microorganisms with CaCO 3 dissolving activity, and 2) to develop a biological method for treatment of chemically clogged emitters in drip irrigation systems.
- a II p lates were i ncubated a t 30 °C f or 3 -5 days. After incubation period, sub-culturing on the same media used for isolation purified the grown bacterial and fungal colonies. In this study, a total of 257 bacterial strains and 133 fungi isolates were isolated. Bacterial strains were maintained for long- term storage in nutrient broth with 15% glycerol at -80 °C. Fungi isolates were kept in PDA slants at 4 °C in refrigerator.
- a modified agar is prepared for the determination of biologically dissolving calcium carbonate bacterial microorganisms.
- Yeast Dextrose Calcium Carbonate (YDC) agar medium which is generally using in the pigmentation of bacteria, is used in the isolation of b acteria, by increasing the quantity of calcium carbonate in it. All of the bacteria and fungi isolated in the present invention were separately plated on Yeast Dextrose Calcium Carbonate Agar (YDC) medium with a little modification (yeast extract, 12g; dextrose, 20g; calcium carbonate (light powder), 25g (can be changed between 25 - 30 gr); agar, 15g; distilled water, 1 L) and incubated at 30 °C for 7 days.
- the CaCO 3 dissolving microorganisms were screened and determined based on a visual observation of clearing zones around the colonies of the organisms grown on the modified YDC medium after incubation. Of all microorganisms screened, only one bacterial strain was discovered with CaCO 3 dissolving ability in our plate assay repeated 3 times. Preparation and analysis of FAMEs from whole cell fatty acids from bacterial strains was performed according to the method described by manufacturing manual (Sherlock Microbial Identification System Version 4.5, MIDI, inc., Newark, DE).
- Approximately 40 mg of living cells from the bacterial strain was harvested, and added to 1 ml 1.2M NaOH in 50% aqueous methanol with 5 glass beads (3mm dia) in a screw cap tube, then incubated at 100 °C for 30 min in a water bath. After the saponified samples were cooled at room temperature for 25 min, they were acidified and methylated by adding 2 ml 54% 6 N HCI in 46% aqueous methanol and incubated at 80 °C for 10 min in a water bath. After rapid cooling, methylated fatty acids were extracted with 1.25 ml 50% methyl-tert butyl ether (MTBE) in hexane.
- MTBE methyl-tert butyl ether
- FAMEs were separated by gas chromatography with a fused-silica capillary column (25m by 0.2mm) with cross-linked 5% phenylmethyl silicone.
- the operating parameters for the study were set and controlled automatically by computer program.
- the chromatograms with peak retention times and areas were produced on the recording integrator and were electronically transferred to the computer for analysis, storage and report generation. Peak naming and coloumn performance was achieved through the use of calibration standard mix containing nC9-nC20 saturated and 2&3 hydroxy fatty acids. Cellular fatty acids were identified on the basis of equivalent chain length data.
- a FAME profile of bacterial strain tested was identified by comparing the commercial databases with the MIS software package. Identity of the bacterial strain was relieved by computer comparison of FAME profile of the unknown test strain with those in library.
- the bacterial strain isolated and identified as CaCO 3 dissolving activity was grown on nutrient agar. A single colony was transferred to a 500-mL flasks containing nutrient broth (NB), and grown aerobically in flasks, on a rotating shaker (150 rpm) for overnight at 30 °C. Bacteria-grown nutrient broth was then diluted in sterile distilled water containing 0.025% Tween 20 to a final concentration of 10 CFU/mL, and used for biological treatment of chemically clogged emitters in drip irrigation system.
- NB nutrient broth
- Tween 20 0.025%
- An experimental model of drip irrigation system was designed on a workbench at the Laboratory.
- a hydraulic structure was formed with two 12-m-long drip laterals, one of which was treatment and the other was control lateral.
- Each lateral with emitter spacing, operation pressure and flow rate was 0.33 m, 0.5 atm, and 23L/h per emitter, respectively.
- the irrigation system was operated 8h daily for 20 days.
- the water with pH value of 7.86 based on the LSI calculation was used, and all emitters were partially or totally clogged due to CaCO 3 precipitation during the time of the experiment.
- 500mL of bacterial suspension and sterile distilled water was applied to the treatment and control lateral, respectively. This application was repeated two times with 24 h interval.
- the irrigation system was operated and flow rate of each emitter was measured daily.
- All of the microbial organisms including 257 bacterial strains and 133 fungi isolates recovered from the microflora of 30 lateral samples with clogged emitters, were screened for CaCO 3 dissolving activity on the modified YDC plate assay. Only one bacterial strain was selected with CaCO 3 dissolving ability in-vitro (plate) assay. The bacterial strain was identified as Agrobacterium FS-16 based on FAME analysis by
- the production of clearing zones around the colonies of bacteria grown on the modified YDC medium is an indication of the presence of the CaCO 3 dissolving activity determined based on a visual observation. It is an easy qualitative assay to screen CaCO 3 dissolving microorganisms.
- the mechanism of bacterial strain for the CaCO 3 dissolving activity has not been studied yet, but it is possible to explain by the production of organic acids resulting in the lowering of pH in the surrounding medium. In most of the commercial system, clogging of emitters with CaCO 3 precipitates is treated with acids such as sulfuric, hydrochloric, phosphoric and organic acids in order to lower water pH and prevent chemical precipitation.
- modified YDC medium may be used as a rapid and reliable plat assay for detection and identification of microflora with CaCO 3 dissolving activity from various environmental samples in the future studies.
- the bacterial strain, Agrobacterium FS-16 discovered in the present study could be utilized for industrial applications, like treatment of drip irrigation system where CaCO 3 precipitation is problem.
Abstract
A calcium carbonate dissolving bacteria and a process for treatment of clogging as a result of calcium carbonate precipitates in drip irrigation systems, floor heating systems, washing apparatus and medical treatment, by utilization of this kind of bacteria.
Description
IDENTIFICATION OF CALCIUM CARBONATE DISSOLVING BACTERIA AND ITS POSSIBLE INDUSTRIAL UTILIZATION
DESCRIPTION
FIELD OF THE INVENTION
The present invention relates to a calcium carbonate dissolving bacteria and a novel method for treatment of clogging in drip irrigation system, by utilization of this kind of bacteria.
BACKGROUND OF THE INVENTION
In prior art it is known that, drip irrigation, also called trickle irrigation or micro-irrigation, is a localized irrigation method that slowly and frequently provides water directly to the plant root zone (Evans, 2000). It is known as low cost water delivery system. Due to limited water resources and environmental consequences of common irrigation systems, drip irrigation system is getting more attraction and playing important role for agricultural production, particularly with high value cash crops such as greenhouse plants, ornamentals and fruit production. Therefore, use of drip irrigation system is rapidly increasing around the world.
Emitter clogging has often been recognized as inconvenient and one of the most important concern for drip irrigation systems, resulting in lowered system performance and water stress to the non-irrigated plants (Povoa and Hills, 1994; Capra and
Scicolone, 1998). Partial and total plugging of emitters is closely related to the quality of the irrigation water, occurs as a result of multiple factors, including physical, biological and chemical agent (Coelho and Resende, 2001 ; Gilbert et al., 1981 ; Pitts et al., 1990). Chemical precipitates have been acknowledged to be one of the most difficult clogging factors to control (Hills et al., 1989; Nakayama and Bucks, 1991 ). Iron, calcium, magnesium and manganese dissolved in irrigation water with higher pH (greater than 7) induce chemical precipitates to clog emitters (Hills et al., 2000; Gilbert and Ford, 1986). Particularly an increase in either pH or temperature reduces the solubility of calcium in water and result in calcium carbonate precipitation (Pitts et al., 1990). Potential risk for chemical clogging of emitters in drip irrigation system is
generally calculated based on the tendency for CaCO3 formation using Langelier Saturation Index (LSI) (APHA, 1995; Yuan et al., 1998).
In prior art, clogging of emitters with chemical precipitates can often be reduced by acid treatment. Sulphuric, hydrochloric, phosphoric and organic acids are commonly used to lower water pH and prevent chemical precipitation (de Kreij et al., 2003; Yuan et al., 1998). Adding of metaphosphohc acid or a soluble metaphosphate, such as ammonium, sodium or potassium metaphosphate, to natural water containing a calcium compound in order to prevent or delay the precipitation of calcium carbonate, is disclosed in Patent No. GB479530. However, this type of acid treatment may cause harmful effect on agricultural crop production if soil pH is acidic (ASAE Standards, 2001 ). On the other hand, chemical treatments are banned for crop production in the area where organic farming is practiced (Lotter, 2003).
In prior art, there has been no study on the development and application of biological methods for treatment of emitter clogging. Microorganisms with CaCO3 dissolving activity may be useful for development of an environmentally friendly method in controlling of chemical clogging of emitters in drip irrigation system.
It is therefore objects of the present invention are; 1 )to isolate and identify microorganisms with CaCO3 dissolving activity, and 2) to develop a biological method for treatment of chemically clogged emitters in drip irrigation systems.
SUMMARY OF THE INVENTION
This s tudy was conducted to i nvestigate microbial organism, which can be used for preventing clogging in drip irrigation systems caused by calcium carbonate precipitation. A total of 1 33 fungi a nd 257 bacterial strains, isolated from p reviously plugged e mitters c ollected from tomato greenhouses were qualitatively screened for calcium carbonate dissolving ability on the modified Yeast Dextrose Calcium
Carbonate (YDC) agar medium, based on visiual observation. A bacterial strain (FS- 16) was demonstrated to have calcium carbonate dissolving ability under in-vitro conditions. Application of 500 ml bacterial suspension (109 cfu/ml) into one lateral of a pilot drip irrigation system designed in a laboratory showed that all emitters clogged due to calcium carbonate precipitation were cleaned and flow rate reached to
maximum within 48 hr after treatment. The bacterial strain was identified as Agrobacterium on the basis of conventional methods and fatty acid methyl (FAMEs) analysis using Microbial Identification System (MIS; MIDI Inc., Newark, Del.). This is the first study of discovering a calcium carbonate dissolving bacteria and its utilization for treatment of clogging in drip irrigation system.
DETAILED DESCRIPTION OF THE INVENTION
30 lateral samples with clogged emitters were collected from tomato greenhouses. Each sample was cut into the small pieces (15-20) and washed in 100 ml sterile peptone water by shaking in a Stomacher for 5 min. Then decimal dilutions of the resulting suspension in 9% (w/v) NaCI solution were prepared and plated on Potato Dextrose (PDA) agar, and Sabouraund Dextrose (SD) agar for fungal isolation and Man Rogosa Sharpe (MRS) agar, Violet Red Bile (VRB) agar, Baird-Paker agar and Nutrient (NA) agar for bacterial i solation. A II p lates were i ncubated a t 30 °C f or 3 -5 days. After incubation period, sub-culturing on the same media used for isolation purified the grown bacterial and fungal colonies. In this study, a total of 257 bacterial strains and 133 fungi isolates were isolated. Bacterial strains were maintained for long- term storage in nutrient broth with 15% glycerol at -80 °C. Fungi isolates were kept in PDA slants at 4 °C in refrigerator.
A modified agar is prepared for the determination of biologically dissolving calcium carbonate bacterial microorganisms. Yeast Dextrose Calcium Carbonate (YDC) agar medium, which is generally using in the pigmentation of bacteria, is used in the isolation of b acteria, by increasing the quantity of calcium carbonate in it. All of the bacteria and fungi isolated in the present invention were separately plated on Yeast Dextrose Calcium Carbonate Agar (YDC) medium with a little modification (yeast extract, 12g; dextrose, 20g; calcium carbonate (light powder), 25g (can be changed between 25 - 30 gr); agar, 15g; distilled water, 1 L) and incubated at 30 °C for 7 days. The CaCO3 dissolving microorganisms were screened and determined based on a visual observation of clearing zones around the colonies of the organisms grown on the modified YDC medium after incubation. Of all microorganisms screened, only one bacterial strain was discovered with CaCO3 dissolving ability in our plate assay repeated 3 times.
Preparation and analysis of FAMEs from whole cell fatty acids from bacterial strains was performed according to the method described by manufacturing manual (Sherlock Microbial Identification System Version 4.5, MIDI, inc., Newark, DE). Approximately 40 mg of living cells from the bacterial strain was harvested, and added to 1 ml 1.2M NaOH in 50% aqueous methanol with 5 glass beads (3mm dia) in a screw cap tube, then incubated at 100 °C for 30 min in a water bath. After the saponified samples were cooled at room temperature for 25 min, they were acidified and methylated by adding 2 ml 54% 6 N HCI in 46% aqueous methanol and incubated at 80 °C for 10 min in a water bath. After rapid cooling, methylated fatty acids were extracted with 1.25 ml 50% methyl-tert butyl ether (MTBE) in hexane. Each sample was mixed for 10 min and the bottom phase was removed with a Pasteur pipette. The top phase was washed with 3 ml 0.3 M NaOH. After mixing for 5 min then the top phase was removed for analysis. Following the base wash step, the extract (FAMEs) is cleaned in anhydrous sodium sulfate and then transferred into a GC sample vial for analysis.
FAMEs were separated by gas chromatography with a fused-silica capillary column (25m by 0.2mm) with cross-linked 5% phenylmethyl silicone. The operating parameters for the study were set and controlled automatically by computer program. The chromatograms with peak retention times and areas were produced on the recording integrator and were electronically transferred to the computer for analysis, storage and report generation. Peak naming and coloumn performance was achieved through the use of calibration standard mix containing nC9-nC20 saturated and 2&3 hydroxy fatty acids. Cellular fatty acids were identified on the basis of equivalent chain length data. A FAME profile of bacterial strain tested was identified by comparing the commercial databases with the MIS software package. Identity of the bacterial strain was relieved by computer comparison of FAME profile of the unknown test strain with those in library.
Conventional methods have been also used for identification the bacterial strain as described previously (Forbes et al., 1998).
The bacterial strain isolated and identified as CaCO3 dissolving activity was grown on nutrient agar. A single colony was transferred to a 500-mL flasks containing nutrient broth (NB), and grown aerobically in flasks, on a rotating shaker (150 rpm) for overnight at 30 °C. Bacteria-grown nutrient broth was then diluted in sterile distilled water
containing 0.025% Tween 20 to a final concentration of 10 CFU/mL, and used for biological treatment of chemically clogged emitters in drip irrigation system.
An experimental model of drip irrigation system was designed on a workbench at the Laboratory. A hydraulic structure was formed with two 12-m-long drip laterals, one of which was treatment and the other was control lateral. Each lateral with emitter spacing, operation pressure and flow rate was 0.33 m, 0.5 atm, and 23L/h per emitter, respectively. The irrigation system was operated 8h daily for 20 days. The water with pH value of 7.86 based on the LSI calculation was used, and all emitters were partially or totally clogged due to CaCO3 precipitation during the time of the experiment. Then, 500mL of bacterial suspension and sterile distilled water was applied to the treatment and control lateral, respectively. This application was repeated two times with 24 h interval. The irrigation system was operated and flow rate of each emitter was measured daily.
All of the microbial organisms, including 257 bacterial strains and 133 fungi isolates recovered from the microflora of 30 lateral samples with clogged emitters, were screened for CaCO3 dissolving activity on the modified YDC plate assay. Only one bacterial strain was selected with CaCO3 dissolving ability in-vitro (plate) assay. The bacterial strain was identified as Agrobacterium FS-16 based on FAME analysis by
MIS and conventional tests. The production of clearing zones around the colonies of bacteria grown on the modified YDC medium is an indication of the presence of the CaCO3 dissolving activity determined based on a visual observation. It is an easy qualitative assay to screen CaCO3 dissolving microorganisms. The mechanism of bacterial strain for the CaCO3 dissolving activity has not been studied yet, but it is possible to explain by the production of organic acids resulting in the lowering of pH in the surrounding medium. In most of the commercial system, clogging of emitters with CaCO3 precipitates is treated with acids such as sulfuric, hydrochloric, phosphoric and organic acids in order to lower water pH and prevent chemical precipitation. Since it is thought that CaCO3 dissolving bacteria is working in similar way of chemical treatment, the CaCO3 dissolving bacteria was tested on a model drip irrigation system set up on a workbench of irrigation laboratory for additional data. Two application of 500mL of bacterial suspension at the concentration of 109 CFU/mL into one lateral of a pilot drip irrigation system showed that all emitters clogged due to calcium carbonate precipitation were cleaned and flow rate reached to maximum within 48 hr after
treatment. However, there was no reduction in the CaCO3 precipitants in the emitters of control lateral treated with sterile water. This finding was confirmed the in-vitro test result suggesting that Agrobacterium FS-16 is able to dissolve CaCO3 precipitants.
This is the first invention of a bacterial strain with CaCO3 dissolving activity. Previously studies have reported the presence of microorganisms in nature causing CaCO3 precipitation, but not dissolving activity.
Our data suggested that the modified YDC medium may be used as a rapid and reliable plat assay for detection and identification of microflora with CaCO3 dissolving activity from various environmental samples in the future studies. Furthermore, the bacterial strain, Agrobacterium FS-16 discovered in the present study could be utilized for industrial applications, like treatment of drip irrigation system where CaCO3 precipitation is problem.
By above disclosed experiment, it is proved that utilization of Agrobacterium (FS-16) or its metabolites to be isolated from these bacteria, clogging in drip irrigation systems related with calcium carbonate precipitants can be treated biologically. In a similar way, other technological problems caused by calcium carbonate precipitation can be solved by utilization of Agrobacterium.
Nearby the cause of clogging of calcium carbonate precipitants in drip irrigation systems in agricultural irrigation, they cause important problems in many other fields like floor heating systems, washing apparatus needed detergent and fields requested medical treatment. This invention provides dissolving of calcium carbonate precipitates not only in agricultural irrigation, also in the fields mentioned in this paragraph.
Claims
What is claimed is; 1. A calcium carbonate dissolving bacteria strain.
2. A bacteria strain according to claim 1 is, Agrobacterium (FS-16).
3. A biological treatment in drain irrigation systems which are clogged by calcium carbonate precipitants.
4. The usage of calcium carbonate dissolving bacteria strain or its metabolites in biological treatment according to claim 3.
5. The calcium carbonate dissolving bacteria strain according to claim 4, is Agrobacterium (FS-16).
6. The utilization of Agrobacterium (FS-16) according to claim 5, is in the fields agricultural irrigation, floor heating systems, washing apparatus needed detergent and medical treatment.
7. An agar medium for detection and identification of microflora with CaCO3 dissolving activity.
8. An agar medium according to claim 7, is a modified Yeast Dextrose Calcium Carbonate Agar (YDC) medium, characterized by its increased quantity of calcium carbonate.
9. Calcium carbonate (light powder) content according to claim 9 is, 25 - 30g.
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/TR2004/000028 WO2005118488A1 (en) | 2004-05-31 | 2004-05-31 | Identification of calcium carbonate dissolving bacteria and its possible industrial utilization |
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| PCT/TR2004/000028 WO2005118488A1 (en) | 2004-05-31 | 2004-05-31 | Identification of calcium carbonate dissolving bacteria and its possible industrial utilization |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8143204B2 (en) | 2007-05-04 | 2012-03-27 | Ecolab Usa Inc. | Mg++ chemistry and method for fouling inhibition in heat processing of liquid foods and industrial processes |
| US8247363B2 (en) | 2007-05-04 | 2012-08-21 | Ecolab Usa Inc. | MG++ chemistry and method for fouling inhibition in heat processing of liquid foods and industrial processes |
| CN109997655A (en) * | 2019-03-29 | 2019-07-12 | 中国农业大学 | A method of inhibiting drip emitter microorganism clogging |
| CN110143661A (en) * | 2019-05-23 | 2019-08-20 | 华中科技大学 | A method of arsenic in rich calcium high-arsenic underground water is removed with charcoal |
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| US2897148A (en) * | 1954-03-03 | 1959-07-28 | Applic Biochimiques Soc Et | Process of purifying and utilizing residual waters resulting from the manufacture of paper pulp prepared by the soda process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8143204B2 (en) | 2007-05-04 | 2012-03-27 | Ecolab Usa Inc. | Mg++ chemistry and method for fouling inhibition in heat processing of liquid foods and industrial processes |
| US8247363B2 (en) | 2007-05-04 | 2012-08-21 | Ecolab Usa Inc. | MG++ chemistry and method for fouling inhibition in heat processing of liquid foods and industrial processes |
| CN109997655A (en) * | 2019-03-29 | 2019-07-12 | 中国农业大学 | A method of inhibiting drip emitter microorganism clogging |
| CN110143661A (en) * | 2019-05-23 | 2019-08-20 | 华中科技大学 | A method of arsenic in rich calcium high-arsenic underground water is removed with charcoal |
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