GB2342924A - Microbial composition and its use in the neutralisation of alkaline waste waters - Google Patents
Microbial composition and its use in the neutralisation of alkaline waste waters Download PDFInfo
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- GB2342924A GB2342924A GB9823332A GB9823332A GB2342924A GB 2342924 A GB2342924 A GB 2342924A GB 9823332 A GB9823332 A GB 9823332A GB 9823332 A GB9823332 A GB 9823332A GB 2342924 A GB2342924 A GB 2342924A
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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
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- 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, e.g. protozoa; 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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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
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/26—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof
- C02F2103/28—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof from the paper or cellulose industry
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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
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/30—Nature of the water, waste water, sewage or sludge to be treated from the textile industry
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- 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
Abstract
The invention relates to a microbial composition for the neutralization of alkaline waste waters by biological means and a method of neutralization of alkaline waste waters using a synergistic mixture of the bacterial strains of <I>Bacillus alkalophilus and Bacillus sp</I>.
Description
MICROBIAL COMPOSITION AND ITS USE IN THE
NEUTRALISATION OF ALKALINE WASTE WATERS
Field of the Invention
The present invention relates to a microbial composition and a
process for the preparation of the said microbial composition useful for
the neutralization of alkaline waste-waters.
Description of the prior art
Rapid urbanization of developing countries as well as
industrialization has adversely affected the environment, particularly with
respect to water pollution. Water is polluted mainly by wastes generated
through industrial processes, chemical agents from fertilizers & pesticides,
silt from degraded. catchments and traditional organic wastes. Waste-water
generated from industries is a major cause of surface water pollution.
Some of the waste-waters from industrial processes pose a major
problem for treatment as they are alkaline in nature. industries producing
alkaline waste-waters include textile industries, paper and pulp industries
and many others. These alkaline waste-waters have a high pH ranging . from 8.0-11.0. Since the pH of alkaline waste-waters is very high (more
than 9), so it is not permissible to drain out these waste-waters as such.
Further, for the biological treatment of such waste-waters, prior
neutralization is essential as most of the microbial strains present in
activated sludge process work well near neutral pH. Therefore, alkaline waste-waters need to be first neutralized and then either sent to drainage
or to treatment processes for further treatment.
The studies reveal that the neutralization of alkaline waste-waters is
being done by purelychemical means where lots of acid is used which is often economically not feasible. Use of large quantities of acids is unsafe as it poses serious health hazards to the workers. There is no prior art proposa for the neutralization of alkaline waste-waters by biological means. Therefore, a microbial composition is formulated for the neutralization of alkaline waste-waters.
The alkelophilic bacteria used in the present invention are able to neutralize the alkaline environment and thus may find wide applications in industries emanating alkaline waste-waters.
The main advantages of the present invention are economy and safety.
From economic point of view, the use of prepared microbial composition has advantages, as tonnes of acid is being used everyday to neutralize the alkaline waste-waters. Such a process becomes expensive to the extent that the industries find it economically non-feasible. A better alternative to the existing problem is to neutralize such waste-waters by biological means using the formulated microbial composition.
The utitization of acid in large quantities for the neutralization of wastewaters is also not safe for the industry as the strong acid has dangerous effect on the health of workers as well as on the industrial processes. Besides this, use of a large quantity of acid also increases the volume of industrial waste-waters to be drained out in the main stream.
For solving the aforementioned problem, the inventors have realized that there exists a need to provide an economic and safe process to neutralize the alkaline waste-waters which are emanating. from different industries.
Alkalophilic bacteria used in present invention have been foQnd to be adapted to the alkaline environment up to a pH 11.0. During the exponential phase, the growth rate immediately altered to the phenotypic characteristic of bacteria at higher pH indicating rapid adaptation. Due to such adaptation, the occurrence of the different pH optima is unusual and may reflect the operation of different limiting physiological systems at these pH values. The response of exponentially growing cultures to alteration in pH values in the medium indicates that these optima were not due to the presence of
more than one genetically different variants. On the laboratory scale
experiments, the microbial composition selected are able to neutralize
alkaline medium as well as alkaline waste-water from textile industry. The
microbial composition chosen for the invention produces more acid in presence of carbohydrates than without carbohydrates. Higher acid
production in the presence of carbohydrates may be due to utilization of carbohydrates by bacteria as a source of carbon. Some industrial wastewaters may be rich in high carbohydrate content (e. g. starch) and may help in producing acid by using the available carbohydrates in the waste-waters. By using the alkalophilic microbial composition, the addition of sugar industry waste-waters (rich in carbohydrate content) in the alkaline waste-water (without carbohydrate content) may be advantageous for neutralization of these combined waste-waters.
Obiects of the invention
The main object of the present invention is to provide a microbial composition and a process for the preparation of microbial composition useful for the neutralization of alkaline waste-waters.
Another object of the present invention is to provide a safe as well as economical process for the neutralization of alkaline waste-water.
The formulated microbial composition of the present invention comprises of cultures of following bacteria: (a) Bacillus alkalophilus (b) Bacillus sp..
The microbial composition is obtained by acclimatizing both the bacteria at higher pH, inoculating a suspension individually, incubating, mixing in equal proportions based on optical density and centrifuging. The resultant pellet is lyophilized to get a mixed powder of both the bacterial strains.
Summary of the invention
The present invention provides a process for the development of microbial composition of alkalophilic bacterial useful for the neutralization of alkaline waste-waters which is very economical and safe.
Detailed of the invention
The composition provided according to the present invention contained bacteria consisting of : 1. Bacillus alkalophilus CBTCC/Micro/8 (having characteristics similar to ATCC
No. 27647).
2. Bacillus sp. CBTCC/Micro/9 (having characteristics similar to ATCC
No. 27557).
The alkalophilic bacteria used for the above stated composition were isolated from sewage having slight alkaline pH (7.5-8.0) by following the standard method. A selective medium, Alkaline Bacillus broth medium, was used for the isolation of alkalophilic bacteria. Alkaline Bacillus broth medium contained 10.0 gm glucose, 5.0 gm peptone, 5.0 gm yeast extract, 10.0 gm K2HPO4 and 0. 2 gm MgSO4 7H2O p ! er litre. For solid medium, 20.0 gm agar was added. Aseptically added 100 ml of 10% sodium carbonate to the medium. The medium was sterilized at 120 C for 20 min. The pH of alkaline Bacillus broth medium was adjusted to 8.0 to 11.0 using Tris-HCI buffer and NaOH-Na2CO3 buffer or NaHCO3 ONa2C03 buffer under sterile conditions. Sewage sample was diluted serially from 10'1 to 10'1 . 0.1 ml of serially diluted sewage sample was streaked onto agar plates of the medium. Incubated the inoculated plates at 37 C. Several alkalophilic colonies were isolated and streaked on separate agar plates. The isolates were grown in broth medium of different pH values ranging between 8.0 to 11.0 for 24 hrs. at 37 C. The isolated pure cultures were used for further study.
The main characteristic features of Bacillus alkalophilus (CBTCC/Micro/8) used for the invention are: -Bacillus alkalophilus is endospore forming and Gram positive rod.
-Bacillus alkalophilus is aerobic in nature.
-Bacillus alkalophilus shows its optimum growth at 37 C.
-Bacillus alkalophilus grows at high pH range i. e. 8.0 to 11.0 but
not able to grow at neutral pH value. This shows its alkalophilic nature.
-Bacillus alkalophilus produces acid in the presence of carbohydrates
i. e. glucose and sucrose.
-Bacillus alkalophilus is catalase positive.
-Baallus alkalophilus hydrolysescasein, gelatin and starch but unable
to hydrolyse urea.
-Baa'llus alkalophilus is able to utilize citrate and propionate.
-Bacillus alkalophilus is able to reduce methylene blue.
All the above characteristics of Bacillus alkalophilus used for the
invention are similar to Baallusalkalophilus corresponding to ATCC
No. 27647.
The main characteristic features of Bacillus sp. (CBTCC/Micro/9) used for the invention are : -Baallus sp. is endospore forming and Gram positive rod.
-Baa'llus sp. is aerobic in nature.
Bacillus sp. shows its optimum growth at 37 C.
-Bacillus sp. grows at high pH range i. e. 8.0 to 11.0 but not able to
grow at neutral pH value. This shows its alkalophilic nature.
-Bacillus sp. Produces acid in the presence of carbohydrates i. e.
glucose and sucrose.
-Bacillus sp. is catalase positive.
-Bacillus sp. hydrolyses casein, gelatin and starch but unable to
hydrolyse urea.
-Bacillus sp. is unable to utilize citrate.
-Bacillus sp. is able to reduce nitrate.
All the above characteristics of Bacillus sp. used for the invention are similar to Bacillus general having characteristics similar to ATCC No. 27557.
The microbial composition of the present invention is useful for the neutralization of alkaline waste-waters.
The microbial composition is formulated by inoculating the individual strains of the above mentioned bacteria separately in Alkaline Bacillus broth medium containing 10.0 gm glucose, 5.0 gm peptone, 5.0 gm yeast extract, 10.0 gm K2HP04 & 0.2 gm MgS04 7H20 per litre. For solid media 20.0 gm agar was added. Aseptically added 100 ml of 10% sodium carbonate to the medium. The medium was sterilized at 120 C for 15 min. Morphological properties and taxonomic characteristics of bacteria were investigated according to the Bergey's
Manual of Determinative Bacteriology. These cultures were grown in two different media i. e. Nutrient Broth (NB) medium and Modified Tryptone Soya
Broth (TSB) medium for obtaining better growth. Nutrient broth medium contained 1.0 gm beef extract, 2.0 gm yeast extract, 5.0 gm peptone, 5.0 gm sodium chloride per litre at pH about 7.0. The cultures were grown by inoculating them in
the same medium but at different pH, ranging from 8.0 to 11.0. Tris-HCI buffer was use for adjusting the pH upto 9. 0. Buffer NaHC03-Na2C03 was used to adjust pH values from 9.0 to 11.0. Modified Tryptone Soya Broth medium comprised of 5.6 gm pancreatic digest of casein, 1.6 gm sodium chloride, 0.83 gm dibasic potassium phosphate, 0.83 gm dextrose per litre.
Different pH values of TSB medium were obtained by using Tris-HCI buffer and NaOH-NaHCO3 or NaHCO3-Na2CO3 under sterile conditions.
Compared the growth of bacterial strains in NB medium and modified
TSB medium. On the basis of better growth of bacterial strains, modified
TSB medium was selected for further study. The bacterial strains unable to grow at pH 11.0 were acclimatized at pH 11.0. Production of acid by acclimatized bacteria was observed in modified NB medium. Modified NB medium contained 1.5 gm peptone, 5.3 gm NaCI, 0.12 gm yeast extract, 0.016 gm beef extract, 0.3 gm AHPO4 per litre. The different pH value ranging from 9. 0 to 11.0 of modified NB medium was adjusted by using
NaOH-NaHCO3 buffer or NaHC03-Na2C03 under sterile conditions. The
Bacterial were grown in modified Nutrient Broth medium for 2 to 5 days.
The acid produced by the bacteria was observed by measuring the decrease in pH of the incubated mediahvaste-waters with the help of a pH electrode at different time intervals. A dye, phenol red indicator, was added to the media to observe the change in colour of the media due to. acid production. Change in colour of phenol red from red to orange and orange to yellow was also an indication of acid production by the bacteria.
Accordingly, the present invention provides a microbial composition comprising a synergistic mixture of at least following two bacterial strains present in about equal proportion for the neutralization of alkaline wastewaters: a. Bacillus alkalophilus b. Bacillus sp.
The invention further provides a process for the preparation of microbial composition useful for the neutralization of alkaline wastewaters which comprises:
The invention further provides a process for the preparation of microbial composition useful for the neutralization of alkaline waste-waters which : j' a) isolating the bacterial strains Bacillus alkalophilus and Bacillus sp.
from sewage by standard method; b) inoculating the said individual bacterial strains in an Alkaline
Bacillus broth medium containing MgS04 ; c) growing the said individual bacterial strains for 16-24 hrs; d) inoculating the said individual bacterial strains from step (c) in
a modified Tryptone Soya Broth medium, having original pH value
about 7.0, the said pH value of the medium being adjusted to
different pH values ranging between 8.0-11.0, using Tris-HCI buffer
and NaOH-Na2CO3 buffer or NaHCO3-Na2CO3 buffer under sterile
conditions; e) growing the said individual bacterial strains obtained from step (d) in
modified Tryptone Soya Broth medium for 16-24 hrs. at different pH
values ranging from 8.0-11.0 to select the bacterial strains growing
at pH 11. 0 and the remaining bacterial strains unable to grow at pH
11.0 being acclimatized at pH 11.0 ; f) inoculating the said selected acclimatized individual bacterial trains
from step (e) in a modified Nutrient Broth medium, having pH
values about pH 7.0, the said pH being adjusted to different pH
values ranging from 9.0-11.0 using NaOH-Na2CO3 buffer or NaHCO3-Na2CO3 buffer under sterile conditions; g) adding a dye, phenol red indicator and optionally 1 %
carbohydrate to the said inoculated medium of individual bacterial
strains obtained from step (f) to observe the change in colour
for acid production and to identify the acid producing strains at pH 9. 0-11. 0; h) growing the said inoculated bacterial strains obtained from step
(g) for a period of at least 2 days and thereafter observing acid
production by the change in colour of phenol red in the said
medium from red to orange, orange to yellow and by measuring
the decrease in pH of the said medium; i) selecting the acid producing bacterial strains at pH 11.0; j) mixing the said selected bacterial strains from step (i) to obtain mixed
bacterial suspension; k) centrifuging the mixed suspension of bacterial strains obtained from
step (j) at 8,000-12,000 rpm to obtain pellet ; I) washing the obtained pellet from step (k) by suspending the pellet in
triple distilled water and recentrifuging at 8,000-12,000 rpm; m) collecting the pellet from step (I) and lyophilizing the obtained
pellet to store at 1 to 4 C for longer use;
The invention further provides a method for the neutralization of alkaline
waste-waters using a microbial composition comprising a
synergistic mixture of at least following two bacterial strains present
in about equal proportion for the neutralization of alkaline waste
waters:
a. Bacillus alkalophilus
b. Bacillus sp.;
the said method comprising: a) reconstituting lyophilized bacterial powder by adding 50 ml distilled
water ; b) adding 10% reconstituted bacterial strains in alkaline medium and
alkaline waste-waters containing phenol red indicator and optionally 1% carbohydrate; c) observing acid production by the change in colour of phenol red in
modified Nutrient Broth medium and in the alkaline waste-waters
from red to orange, orange to yellow and by measuring the decrease
in pH of the said medium as well as of alkaline waste-waters
to neutral after a period of at least 2 days;
In an embodiment of the present invention, the formulated microbial composition is obtained by inoculating a suspension of the following bacterial strains: a) Bacillus alkalophilus & b) Bacillus sp., individually.
In an another embodiment of the present invention, the individual strains of the above mentioned bacteria are inoculated separately in NB medium and modified Trytone Soya Broth medium having original pH about 7.0, adjusted to different pH values ranging from pH 8.0 to 11. 0.
In a still another embodiment of the present invention, pH of
Nutrient Broth medium is adjusted using Tris-HCI buffer and NaOH- Na2CO3 buffer or NaHCOs-Na2CO3 buffer.
In one of the embodiments of the present invention, pH of modified Tryptone Soya Broth rnedium is adjusted using Tris-HCI buffer and NaOH-Na2CO3 buffer or NaHCO3-Na2CO3 buffer.
In an embodiment of the present invention, the incubation of bacterial strains. is carried out by gentle agitation at approximately 75-100 rpm
In one of the embodiments of the present invention, the growth of incubated bacterial strains is carried out at a temperature ranging between 35 C40 C for a period of 16-24 hrs.
In an another embodiment of the, present invention, the growth of the bacterial strains is compared in Nutrient Broth medium and modified
Tryptone Soya Broth medium by observing optical density at 650 nm.
In a further embodiment of the present invention, the modified
Tryptone Soya Broth medium supporting the growth of the individual bacterial strains is selected for further study.
In an another embodiment of the present invention, the individual bacterial strains are inoculated in modified Tryptone Soya Broth medium having original pH value 7.0 which is adjusted to different pH values ranging from 8.0 to 11.0 to select the bacterial strains growing at higher pH values. The bacterial strains, unable to grow at pH 11.0 were acclimatized upto pH 11.0.
In a still another embodiment of the present invention, the selected acclimatized bacterial strains are inoculated in modified Nutrient Broth medium having original pH value 7.0 which is adjusted to different pH values ranging from 9.0 to 11.0 using NaOH-Na2CO3 buffer or NaHC03- Na2CO3 under sterile conditions to observe acid production.
In one of the embodiments of the present invention, a dye, phenol red indicator and 1 % glucose and 1 % sucrose are added into modified
Nutrient Broth medium to measure the change in colour and decrease in pH of the medium.
In an another embodiment of the present invention, acid production is observed by bacterial strains by change in colour from red to orange, orange to yellow and measuring the decrease in pH of modified
Nutrient Broth medium.
In a still another embodiment of the present invention, the suspension of the individual selected bacterial strains are mixed thoroughly in equal proportions based on optical density.
In one of the embodiments of the present invention, the mixed suspension of the bacterial strains is centrifuged at appropriate rpm preferably at 8,000-12, 000 rpm for a period of approximately 20-30 minutes.
In an another embodiment of the present invention, the resultant pellets of mixed bacterial strains are washed by dissolving in an appropriate quantity of distilled water and recentrifuging at an appropriate rpm in the range of 8,000-12,000 rpm for a period of approximately 20-30 minutes at a temperature less than or equal to 4 C.
In a still another embodiment of the present invention, the mixed microbial composition is suspended into modified Nutrient Broth medium and alkaline waste-waters of textile industry separately having different pH values ranging from 9.0 to 11.0. In an another experimental setup, 1% glucose as well as 1% sucrose are also added separately into the medium as well as in the alkaline waste-waters having different pH values ranging from 9.0 to 11.0 to provide the addition carbon source to both the bacteria used in the study which lead to more acid production by the bacteria.
In a yet another embodiment of the present invention, phenol red indicator, a dye used as a indicator of acid production, is added to modified Nutrient Broth medium as well as alkaline waste-waters to observe the change in colour due to acid production.
In a further embodiment of the present invention, all the suspensions of bacterial strains growing at different pH values containing different carbohydrates as well as phenol red indicator are incubated for 2-5 days.
In one of the embodiments of the present invention, acid production is observed by the change in colour of phenol red from red to orange, orange to yellow and by measuring the decrease in pH of the modified Nutrient Broth medium as well as of alkaline wastewaters after 2-7 days of incubation time.
In a another embodiment of the present invention, the prepared microbial composition is useful for the neutralization of alkaline wastewaters with the optional addition of 1% cabohydrates.
The invention is illustrated with the help of the following examples and should not be construed to limiting the scope of the invention.
Example 1
Two loops from agar plates of Bacillus alkalophilus and Bacillus sp. were inoculated separately in plates of NB medium and modified TSB medium containing 2% agar having original pH about 7.0 which is adjusted to different pH values ranging from 8.0-11.0. For adjusting the different pH values of NB medium, different buffers were used. Tris-HCI buffer was used for adjusting pH values upto 9.0, whereas, Na2CO3 NaHCO3 buffer was used to adjust the pH values ranging from 9.0-11.0.
Different pH values of modified TSB medium were adjusted using Tris HCI and NaOH-NaHCO3 buffers under sterile conditions. Both the bacterial cultures inoculated at different pH values were incubated at 37 C for 24 hrs in an incubator shaker at 75 rpm. Growth of individual bacterial cultures in both the media was observed at different pH values.
Table 1 shows the growth of both the bacteria at different pH values ranging from 8.0-11.0.
TABLE 1 Comparison of growth of alkalophilic bacterial cultures on agar plates in NB medium and modified TSB medium
pH values NB medium TSB medium Modified Bacillus Bacillus sp. Bacillus Bacillus sp. alkalophilus alkalophilus 8.0 + + ++ ++ 8.5 + + +++ +++ 9.0 ++ ++ ++++ ++++ 9.5 - - +++ +++ 10.0--++ ++ 10.5--++ ++ 11.0--++ ++ + very poor growth
++ poor growth +++ good growth ++++ very good growth
It was observed that both the bacteria showed better growth in the plates of modified TSB medium containing agar than NB medium containing agar (Table 1). This may be due to better buffering capacity and optimum ingredients present in modified TSB containing agar for the growth of both the bacteria. So, modified TSB is selected for further experiment.
Example 2
Two loops from agar plates of Bacillus alkalophilus and Bacillus sp. were inoculated separately in 500 ml of modified TSB medium having original pH about 7.0 which is adjusted to different pH values ranging from 8.0-11.0. Different pH values of modified TSB medium were adjusted using Tris-HCI and NaOH-NaHCO3 buffers under sterile conditions. Both the bacterial cultures inoculated at different pH values were incubated at 37 C for 24 hrs. in an incubator shaker at 100 rpm.
Growth of individual bacterial cultures in terms of optical density in modified TSB medium was observed at different pH values.
Table 2 represents the growth of both the bacteria at different pH values ranging from 8.0-11.0.
TABLE 2
Growth of the alkalophilic bacteria in terms of optical density (OD)
at 650 nm at different pH values in modified TSB medium
pH values Optical Density of bacteria at 650 nm Bacillus alkalophilus Bacillus sp.
8. 0 0. 372 0. 421 8. 5 0. 380 0.
9. 0 0. 512 0. 586 9. 5 0. 384 0. 489 10. 0 0. 299 0. 385 10. 5 0. 294 0. 343 11. 0 0. 281 0. 293 It was observed that both alkalophilic cultures showed optimum growth at pH 9.0. This represents the facultative nature of micro-organisms used for the study, which states that microbes show optimal growth at pH 9.0 or above but which can grow well in the near neutral pH range.
Acclimatization of both the bacterial strains is required at higher pH values.
Example 3
Two loops from agar plates of Bacillus alkalophilus and Bacillus sp. were inoculated separately in 500 mi of modified TSB medium of about pH 7.0. The inoculated bacterial cultures were incubated at 37 C for 24 hrs. in an incubator shaker at 75 rpm Optical density of both the bacterial cultures was observed.
Acclimatization of both the bacterial strains to different pH values in the modified TSB medium was accomplished by subculturing the bacterial cultures a number of times at pH values in increasing order. Once the optimum growth was obtained at pH 11.0,500 mi of growth suspension was centrifuged at 8, 000 rpm and the pellet after washing was suspended in carbonate buffer of pH 11.0. The suspended pellet was freeze dried to obtain bacterial powder of both bacterial strains, individually. The lyophilized bacterial powder was stored at about 1 C
Example 4
Once Bacillus alkalophilus and Bacillus sp. were acclimatized at pH 11 both the bacterial cultures were inoculated in modified NB medium of different pH values ranging from pH 8.0-11.0. To observe acid production by both alkalophilic bacterial strains, phenol red indicator was added to the medium. Both the bacterial strains were incubated at 37 C for 2-5 days in an incubator shaker at 75 rpm Change in the colour of phenol red indicator from red to orange and orange ; to yellow was observed which is an indicator of acid production by bacteria. The acid production by bacterial strains was also observed by measuring the pH. of the incubated modified NB medium at different time intervals.
Table 3 indicates the acid production by both the bacterial strains at different pH values. The acid production was observed by measuring the decrease in pH of the medium.
TABLE 3
Change of pH in modified NB medium by alkalophilic bacteria aftertwo and five days incubation
Alkalophilic Original pH of pH of the medium pH of medium bacteria the medium after 2 days after 5 days incubation incubation 9. 07 7. 91 7. 10 Bacillus 9. 53 8. 57 7. 85 alkalophilus 9.96 9.07 8. 64 10.48 9. 75 9. 30 9. 07 8. 09 7. 54 Bacillus 9.53 8.69 8.08 sp. 9. 96 9. 17 9 28 10.48 9.94 9.95 The overall change in pH was observed in the range of 0.5 to 2.0 pH units. This change in pH of the medium may possibly be attributed to the production of an enzyme amino acid deaminase which tends to decrease the alkalinity of the media.
Example 5
Both acclimatized bacterial cultures were used for acid production in the presence of different sources of carbohydrates. For this, 1% sucrose and 1% glucose were added in modified NB medium separately containing phenol red indicator. The change in colour of medium as well as decrease in pH was observed for both bacterial cultures at different pH values ranging pH 9.0-11.0.
Table 4 and 5 represent change in pH of medium by alkalophilic bacterial strains in the presence of 1% sucrose and 1% glucose respectively, after 2 and 5 days incubation.
TABLE 4
Change of pH in modified NB medium containing sucrose by
alkalophilic bacteria after two and five days incubation
Alkalophilic Original pH of pH of the medium pH of medium bacteria the medium after 2 days after 5 days incubation incubation 9. 02. 5.61 6. 00 Bacillus 9. 58 7. 81 6. 75 alkalophilus 9. 90 8. 22 7. 21 10. 42 8. 92 7. 80 9. 02 6. 86 5. 12 Bacillus 9. 58 7. 74 6. 61 sp. 9. 90 8. 22 7. 05 10.42 8.89 7.89 Presence of sucrose supported the growth of both the bacterial strains and enhanced the acid production by both the bacteria. After 2-5 days of incubatiori production by bacteria was indicated by the shift in pH values in the range of 1.5-4.0 pH units by both the cultures.
TABLE 5
Change of pH in modified NB medium containing glucose by
alkalophilic bacteria after two and five days incubation
Alkalophilic Original pH of pH of the medium pH of medium bacteria the medium after 2 days after 5 days incubation incubation 9. 25 5. 65 5. 39 Bacillus 9. 63 7. 48 6. 20 alkalophilus 9.98 8. 40 6.92 10.64 9. 09 7. 72 9. 25 7. 34 5. 67 Bacillus 9. 63 8. 02 6. 68 sp. 9. 98 8. 47 8. 02 10.64 9. 14 8. 80 There was a significant change in the original pH of the medium in
the range of 1.5- 4.0 pH units by both the bacteria. The decrease in pH of the medium was also indicated bycoiour change of phenol red from red to
orange to orangish yellow to yellow. The results of change in pH of medium in the presence of glucose were comparable with the results of change in original pH in the presence of sucrose.
From Table 4 and 5, it was concluded that presence of carbohydrates in the medium enhanced the acid production.
Example 6
Alkaline waste-waters (pH upto 12.0) from textile industry was selected for neutralization studies by using both the alkalophilic bacterial strains. The original pH of the waste-waters was measured.
For the neutralization of alkaline waste-waters by biological means, the acclimatized and lyophilized culture pf Bacillus sp. was reconstituted in 50 mi sterile triple distilled water, overnight. A 10% concentration of reconstituted culture was added in alkaline waste-waters for neutralization.
The alkaline waste-waters containing bacterial culture was incubated at 37 C fora periodof7 days in an incubator shaker at 100rpm.
Table 6 represents the neutralization of alkaline waste-waters from textile industry using Bacillus sp. The change in original pH of wastewaters was observed after 3 and 7 days incubation time.
TABLE 6
Neutralization of alkaline waste-waters from textile
industry by Bacillus sp.
Original pH of waste- After 3 days incubation @ After 7 days incubation water pH change in pH pH change in pH 10.67 10. 40 0. 27 9.75 0. 92 11.57 11. 21 0. 36 10. 72 0. 85 9. 88 9. 64 0. 24 8. 91 0. 97 12.01 11. 50 0. 51 10. 95 1. 06 9. 65 9. 20 0. 45 8. 75 0. 90 The overall change in original pH of the waste-waters was observed in the range of 0.24-1.06 units.
Example 7
The neutralization of alkaline waste-waters of textile industry was carried out using acclimatized and lyophilized culture of Bacillus alkalophilus. The lyophilized powder of Bacillus alkalophilus was reconstituted in 50 ml sterile triple distilled water, overnight. A 10% concentration of reconstituted culture was added in alkaline waste-waters for neutralization. The alkaline waste-waters containing bacterial culture was incubated at 37 C for a period of. 7 days in an incubator shaker at 100 rpm Table 7 represents the neutralization of alkaline waste-waters from textile industry using Bacillus alkalophilus. The change in original pH of waste-waterswas observed after 3 and 7 days incubation time.
TABLE 7
Neutralization of alkaline waste-waters from textile
industry by Bacillus alkalophilus
Original pH of waste- After 3 days incubation @ After 7 days incubation water pH change in pH pH change in pH 10.67 10. 36 0. 31 9. 79 0. 88 11.57 11.32 0.25 10.83 0.74 9. 88 9. 59 0. 29 8. 96 0. 92 12.01 11. 58 0. 43 10. 98 1. 03 9. 65 9. 27 0. 38 8. 69 0. 96 The overall change in original pH of the waste-waters using Bacillus alkalophilus was observed in the range of 0.25-1.03.
Example 8
Bacterial cultures of Bacillus alkalophilus and Bacillus sp., acclimatized at pH 11.0 were obtained individually as described in example 3. The acclimatized culture suspensions of both the individual bacterial strains of different pH were mixed inequal proportions based on optical density. The resultant growth suspension was centrifuged at 10,000 rpm for 30 minutes. The pellet was collecte and washed with triple distilled water. Recentrifuged the mixed suspension at 12,000 rpm for 20 minutes. The pellet was collecte and lyophilized. The lyophilized bacterial powder was stored at4 C.
Example 9
The acclimatized and lyophilized mixed bacterial powder of Bacillus alkalophilus and Bacillus sp. were reconstituted in 50 ml sterile triple distilled water overnight. The mixed reconstituted bacterial culture was used for the neutralization of alkaline waste-waters of textile industry.
Three different sets of alkaline waste-waters were prepared to observe the acid production. In the first set, . 10% of the mixed reconstituted bacterial suspension was added in the waste-waters. In the subsequent two sets, 10% of the mixed reconstituted bacterial suspension was added in waste-waters containing 1% glucose and 1% sucrose, respectively. All the three sets were incubated at 37 C for a period of 7 days in an incubator shaker at 75 rpm Acid production by microbial composition was observed in all the three sets after 2,4 and 7 days of incubation period.
Table 8 represents neutralization of alkaline waste-waters from textile industry by a microbial composition of alkalophilic bacteria in the presence as well as absence of carbohydrates.
TABLE 8
Neutralization of alkaline waste-waters from textile
industry by alkalophilic microbial composition
-------------------------------------------------------------------
Alkaline Original After 2 days After 4 days After 7 days
waste-pH of incubation incubation incubation
water waste
water
pH change pH change pH change
in pH in pH in pH
units units units
--------------------------------
With-10.67 10.52 0.15 10.14 0.53 9.65 1.02
out 11.57 11.35 0.22 11.12 0.45 10.62 0.95
carbo-9.88 9.76 0.12 9.40 0.48 8.90 0.98
hydrate 12.01 11.83 0.18 11.39 0.62 10.88 1.13
9.65 9.42 0.23 9.13 0.52 8.79 0.86
----
With 10.67 8.60 2.07 8.14 2.53 7.94 2.73 1 % 11.57 9.32 2.25 8.95 2.62 8.69 2.88
sucrose 9.88 7.75 2.13 7.50 2.38 7.36 2.52
12.01 9.82 2.19 9.43 2.54 8.96 3.05
9.65 7.63 2.02 7.26 2.39 7.02 2.63
With 10.67 8.30 2.37 7.61 3.06 7.24 3.43 1% 11. 57 8.96 2.61 8.24 3.33 7.83 3.74
glucose 9.88 7.72 2.16 7. 12 2.76 6.81 3.07
12.01 9.57 2.44 8.86 3.15 8.35 3.66
9.65 7.59 2.06 7.01 2.64 6.65 3.00
A significant change was observed in the original pH of the wastewaters of textile industry after regular time intervals. Decrease in original pH of waste-waters was less in the absence of carbohydrates as compared to that with carbohydrates. Among carbohydrates, original pH decreased markedly in the presence of 1 % glucose in comparison to 1 % sucrose.
From the examples explained above it is clear that neutralization of alkaline medium as well as alkaline waste-waters emanating from textile industry using microbial composition is a safe as well as economical biological process.
Avantages 1. The neutralization of alkaline waste-waters using microbial composition is a very economic biological process because tonnes of acid is being used everyday to neutralize the alkaline waste-waters. Such a process becomes expensive to the extent that the industries find it economically non-feasible. A better altemative to the existing problem is to neutralize such waste-waters by biological means using prepared microbial composition which will decrease the cost for the neutralization process drastically.
2. The neutralization of alkaline waste-waters by biological means is quite safe process as the utilization of acid in large. quantities for the neutralization of waste-waters is not safe for the industry as the strong acid
has dangerous effect on the health of workers as well as on the industrial processes. Besides this, use of a large quantity of acid also increases the volume of industrial waste-waters to be drained out in the main stream. The use of large quantities of acid can also be a reason for explosion hazard.
Claims (12)
- CLAIMS 1. A microbial composition comprising mixture of bacterial strains Bacillus alkalophilus and Bacillus sp.
- 2. A microbial composition comprising a first bacterial strain having characteristics similar to Bacillus alkalophilus deposited in culture collection ATCC as No.27647, or a mutant or variant thereof, and a second bacterial strain having characteristics similar to Bacillus sp., deposited in culture collection ATCC as No. 27557, or a mutant or variant thereof.
- 3. A microbial composition as claimed in claim 1 or claim 2, which comprises a synergistic mixture of the two bacterial strains, present in about equal proportions.
- 4. A process for the preparation of microbial composition useful for the neutralization of alkaline waste-waters, which comprises: (a) isolating the bacterial strain Bacillus alkalophilus and Bacillus sp. from sewage; (b) inoculating the said individual bacterial strain in an alkaline Bacillus broth medium containing MgSO4 ; (c) growing the said individual bacterial strains for 1624 hrs.; (d) inoculating the said individual bacterial strains from step (c) in a modified Tryptone Soya Broth medium, having original pH value about 7.0, the said pH value of the medium being adjusted to different pH values ranging between 8. 0-11.0, using Tris-HC1 buffer and NaOH-Na2CO3 buffer or NaHCO3-NazCO3 buffer under sterile conditions; (e) growing the said individual bacterial strains obtained from step (d) in modified Tryptone Soya Broth medium for 16-24 hrs. at different pH values ranging from 8.0-11.0 to select the bacterial strains growing at pH 11.0 and the remaining bacterial strains unable to grow at pH 11.0 being acclimatized at pH 11.0; (f) inoculating the said selected acclimatized individual bacterial strains from step (e) in a modified Nutrient Broth medium, having a pH value of about pH 7.0, the said pH being adjusted to different pH values ranging from 9.011.0 using NaOH-Na2CO3 buffer or NaHC03-Na2CO3 buffer under sterile conditions; (g) adding a dye, phenol red indicator and optionally 1% carbohydrate to the said inoculated medium of individual bacterial strains obtained from step (f) to observe the change in colour caused by acid production and to identify the acid producing strains at pH 9.0-11.0; (h) growing the said inoculated bacterial strains obtained from step (g) for a period of at least 2 days and thereafter observing acid production by the change in colour of phenol red in the said medium from red to orange, and from orange to yellow, and by measuring the decrease in pH of the said medium; (i) selecting the acid producing bacterial strains at pH 11. 0 ; (j) mixing the said selected bacterial strains from step (i) to obtain a mixed bacterial suspension; (k) centrifuging the mixed suspension of bacterial strains obtained from step (j) at 8,000-12,000 rpm to obtain a pellet; (1) washing the pellet obtained from step (k) by suspending the pellet in triple distilled water and recentrifuging at 8,000-12,000 rpm; and (m) collecting the pellet from step (1) and lyophilizing the obtained pellet.
- 5. A process as claimed in claim 4, which further comprises storing the pellet obtained in step (m) at 1 to 4 C.
- 6. A process as claimed in claim 4 or claim 5, wherein in step (i), the acid producing bacterial strains identified are Bacillus alkalophilus and Bacillus sp.
- 7. A process as claimed in any one of claims 4 to 6, wherein in step (d), the medium used is Tryptone Soya Broth modified by removal of Papaic digest of soyabean meal and changing the amount of ingredients.
- 8. A process as claimed in any of claims 4 to 6, wherein in step (d), the medium used is Nutrient Broth modified by addition of K2HPO4 and changing the amount of ingredients.
- 9. A method for the neutralization of alkaline wastewaters comprising adding to said waste-waters a microbial composition as defined in any of claims 1 to 3.
- 10. A method as claimed in claim 10, wherein the microbial composition is obtainable by a process as defined in any of claims 4 to 8.
- 11. A method as claimed in claim 10, comprising: (i) reconstituting lyophilized bacterial powder obtained in step (m) by adding 50 ml distilled water; (ii) adding 10% reconstituted bacterial strains to alkaline waste-waters containing phenol red indicator and, optionally, 1% carbohydrate; and (iii) observing acid production by the change in colour of phenol red in the alkaline waste-waters from red to orange, and from orange to yellow, and by measuring the decrease in pH of the said medium and the alkaline waste-waters to neutral after a period of at least 2 days.
- 12. A microbial composition substantially as described in any one of the Examples.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9823332A GB2342924B (en) | 1998-10-23 | 1998-10-23 | Microbial composition and its use in the neutralisation of alkaline waste waters |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9823332A GB2342924B (en) | 1998-10-23 | 1998-10-23 | Microbial composition and its use in the neutralisation of alkaline waste waters |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB9823332D0 GB9823332D0 (en) | 1998-12-23 |
| GB2342924A true GB2342924A (en) | 2000-04-26 |
| GB2342924B GB2342924B (en) | 2004-02-18 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9823332A Expired - Lifetime GB2342924B (en) | 1998-10-23 | 1998-10-23 | Microbial composition and its use in the neutralisation of alkaline waste waters |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305785C (en) * | 2003-01-22 | 2007-03-21 | 财团法人生物技术开发中心 | Zero-discharge of water glass effluents by alkaline biotreatment techniques |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0339092A (en) * | 1989-07-06 | 1991-02-20 | Kao Corp | Production of substance prepared by bacterium |
-
1998
- 1998-10-23 GB GB9823332A patent/GB2342924B/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0339092A (en) * | 1989-07-06 | 1991-02-20 | Kao Corp | Production of substance prepared by bacterium |
Non-Patent Citations (1)
| Title |
|---|
| WPI Abstract Acc. No. 91-096791/199114 & JP 03 039 092 A * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305785C (en) * | 2003-01-22 | 2007-03-21 | 财团法人生物技术开发中心 | Zero-discharge of water glass effluents by alkaline biotreatment techniques |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9823332D0 (en) | 1998-12-23 |
| GB2342924B (en) | 2004-02-18 |
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