DESC:Field of Invention:
The current invention is in the field of bioremediation of water bodies. More particularly, the current invention relates to use of immobilised biofilms for bioremediation of static and moving water bodies like lakes, ponds, rivers, nallah (rivulet), sewage drain, effluent treatment plant, sewage treatment plant etc.

Background of Invention:
Bioremediation and phytoremediation are two techniques used to clean up contaminated soil and water. Bioremediation involves the use of microorganisms to degrade or transform pollutants, while phytoremediation uses plants following the translocation strategy to remove contaminants. Both the techniques have advantages as well as disadvantages and can be used in different scenario depending on the type and extent of contamination.

Bioremediation is a natural process that can completely destroy a wide variety of contaminants and is perceived as an acceptable waste treatment method. It can often be carried out on-site, reducing the need for transportation and potential threats to human health and the environment.

Microbes are used in the technique rather than any chemical degradants. Therefore, there are no waste products that could contaminate the environment. As a result, the technology can be regarded as a “green technology”.

Bioremediation as a developing technology has recently received attention as this method often uses a variety of living or dead microbial catalysts to remove contaminants from the air, water or soil.

The State of Israel filed Indian Patent Application ? IN201927053232 and IN201827047319, both of which discuss utilization of biofilms. These patent applications describes the use of biofilms to enhance or maintain a subject's health, including giving a therapeutic dose of a probiotic product made up of a combination of lactobacilli and bacilli to the subject.

Due to a greater degree of resistance to pollutants and environmental stress, the biofilms containing multiplicity of microorganisms are often found to be more advantageous for bioremediation than free-floating planktonic cells, particularly, due to their capability of metabolizing a variety of harmful chemicals through several catabolic and metabolic pathways.

Heavy metals, petroleum-based products, explosives, herbicides and insecticides are just a few of the toxic contaminants from the soil and water, which are detrimental to free microbial population that biofilms' microbial communities effectively resist.

Wastewater typically contains a significant amount of organic matter, including substances like food waste, human and animal waste, oils, fats, proteins and plant residues. These organic compounds can undergo biological degradation and serve as a source of energy for microorganisms in wastewater treatment processes. Additionally, the physical characteristics of wastewater include colour, odour, temperature, turbidity and total solid content. For instance, a typical sample of sewage has a water content of 99.9 percent and a solids content of 0.1 per cent.

Therefore, the current inventors propose a novel biofilms which can effectively remediate wastewater and replenishes water bodies.

Summary of Invention:
In view of above, in one aspect, this invention provides a novel immobilised biofilm which is effective in bioremediation of liquid waste from stagnant or flowing water.

The immobilised biofilm of the invention is immobilised by laying the biofilm over porous particles.

The immobilised biofilm is self-generating and sustainable. It continuously replenishes the microbial population in the environment and thus becomes a sustained release source for beneficial microbes for bioremediation of water bodies. The immobilised biofilm is effective in reducing COD and BOD of effluent water.

Detailed Description of Invention:
In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood by the person of ordinary skill in the art to which this invention pertains.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may not only mean “one”, but also encompasses the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

The current invention describes immobilised microbial biofilm which is supported on porous material. The described biofilm is effective in reducing BOD and COD of effluent water or wastewater. In accordance with one of the embodiment, the immobilised microbial biofilm is used for bioremediation of stagnant and/or flowing water bodies like lakes, ponds, rivers, nallah (rivulet), sewage drain, effluent treatment plant, sewage treatment plant etc.

In an embodiment of the invention, the biofilm of the invention consists of a mixed microbial population which consists of at least one primary film forming spore bearing bacteria and at least one secondary non-spore forming bacteria.

In preferred embodiment, the primary film forming bacteria is selected from at least one aerobic or facultative aerobic spore forming bacillus species having film forming capabilities. The preferred film-forming bacteria are bacteria of Bacillus genus.

In one of the embodiments, the primary biofilm forming bacteria are mixed with additional strains of bacteria having a film-forming capability that helps in formation of mixed biofilms.

The secondary film forming bacteria are aerobic or facultative anaerobic non-bacillus species comprising of Pseudomonas spp. and Lactobacillus spp. particularly,
Lactobacillus rhamnosus ATCC 53103, and
Pseudomonas fluorescens NCIM 5226.

In a preferred embodiment, the primary film forming bacteria are bacterium or a bacterial consortium consisting of Bacillus pumilus ATCC 14884, Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 and Bacillus megaterium ATCC 9885, Bacillus subtilis ATCC 6633.

In accordance with above embodiment, the biofilm containing primary film-forming bacteria and the secondary film forming bacteria is laid on surface of porous material. Said porous material is selected from polymeric granular gel, preferably Polyvinyl Alcohol-based beads, foam sponge clay aggregates, foam brick pieces, clay brick pieces, alginate beads, sago, seeds of sweet basil etc., collectively referred to as “micro bead(s)”. The microbial biofilm is immobilised over said micro beads, which are particulate, porous substrates

The said biofilm is immobilized over porous base material, such that the anaerobic microbial population is entrapped in the aerobic biofilm which is exposed to air.

Further, the immobilized biofilm of the invention are self-regenerative and, thus, when applied in water bodies, it becomes a sustained release source of beneficial microbes for bioremediation of water bodies. The micro beads release a microbial population of live viable microbial cell, in range of 101 to 109 cfu/gm of micro beads.

In accordance with this invention, the biofilm, while regeneration utilizes the organic matter present in the waste water thereby reducing the organic content of the water. As consequence of this utilization by microorganism in biofilm, there is a decrease on COD and BOD of the wastewater. In accordance with one of the embodiments, the biofilm of the invention reduced COD and BOD upto 99% by 24 -120hrs under aerobic, anaerobic and anoxic condition.

In accordance with one of the embodiments, the micro beads are prepared with different combinations of film forming bacteria (spore bearing as well as non-spore bearing) such as:
(a) Facultative anaerobic microbial population entrapped inside the core of substrate followed by aerobic one on the outer surface of the substrate gel;
(b) only aerobic microbial population entrapped in entire substrate, and
(c) only facultative anaerobic microbial population entrapped in entire substrate.

The micro beads can be used for liquid waste management, bioremediation of water bodies and as a deodourizer and /or a decolourizer, both under aerobic and facultative anaerobic conditions.

In an embodiment, the process for generation of immobilised biofilm of the invention comprises:
a) Preparing of microbial culture;
b) sequential immobilizing each culture of step (a) under aerobic, anaerobic and anoxic conditions;
c) decanting the culture media from the microbeads of step (b), and
d) storing the microbeads of step (c).

Example 01: Selection of strains for entrapment
Following bacterial strains were screened for following attributes:
Bacillus subtilis ATCC 6633 (BS 6633),
Bacillus subtilis 11774, (BS02)
Bacillus pumilis ATCC 14884, (PB02)
Bacillus megaterium ATCC 9885, (CN02)
Bacillus licheniformis ATCC 14580, (LB02)
Lactobacillus rhamnosus ATCC 53103, (RL03)
Pseudomonas fluorescens NCIM 5226 (PF)

The microbial strains selected for immobilization on beads in bioremediation applications were chosen based on their ability to produce biofilms, synthesize critical enzymes, and exhibit aerobic or facultative aerobic metabolism, ensuring consortium efficacy in degrading complex pollutants.
Biofilm-forming bacteria play a pivotal role in wastewater treatment by enabling efficient pollutant removal, system stability, and operational resilience.

Biofilm production by bacteria was determined by using Congo red agar.

Media used: Congo red agar
Test culture used: Proteus vulgaris ATCC 13315
Protocol: Cultures were streaked on nutrient media containing 0.8% congo red and 3% sucrose. Black color colonies indicated biofilm production while red colonies indicated negative for biofilm production. .

Sr no Culture Biofilm production
1 LB02 +++
2 PB02 ++
3 BS02 +++
4 BS6633 +++
5 CN02 +++
6 PF +++
7 RL03 +++
+++ Strong biofilm, ++ moderate, + low, - negative

Enzymes serve as bio catalysts that drive the breakdown of complex organic pollutants. Strains were systematically screened for their ability to synthesize specific hydrolytic and oxidative enzymes (e.g., proteases, lipases, cellulases, amylases, ligninases, oxygenases, peroxidases and polyphenol oxidases) that target pollutants like proteins, fats, cellulose, starch, lignocellulose and organic pollutants.

Screening was done on agar plate method with respective substrates for each enzyme assay.
Sr no Amylase Cellulase Lipase Protease Ligninase Oxygenase Peroxidase polyphenol oxidases

LB02 + + + + + + + +
PB02 + - + + - + + +
BS02 + + + + + + + +
BS6633 + + + + + + + +
CN02 + - + + - + + +
PF + - + + + + + +
RL03 + + + + + + + +
+ positive , - negative

Cultures were screened for their capability to grown under anaerobic condition by streaking on Thioglycollate agar and were incubated in anaerobic jar for 3 days.
Sr no Culture Anaerobic growth
1 LB02 +
2 PB02 +
3 BS02 +
4 BS6633 +
5 CN02 +
6 PF ++
7 RL03 ++
++ luxurious growth, + moderate growth

Example 02: Compatibility of cultures
Compatibility testing between bacterial strains is critical when assembling microbial consortia because it directly influences the effectiveness, stability, and functional synergy of the consortium.

The selected cultures were evaluated for self- and cross-compatibility to facilitate their subsequent co-culturing in consortia.

All cultures were found to be compatible with each other.

Example 03: Entrapment process
3a. Aerobic
Process for individual entrapment: Aerobic bacterial strains (LB02, PB02, BS02, BS6633, CN02, and PF) each at 2% concentration were separately inoculated into basal broth medium along with microbeads. Microbeads were subsequently employed to entrap the cells under aerobic conditions, and bacterial proliferation within the beads was monitored at 24-hour intervals using the spread plate method.

The initial bacterial load was approximately10*5 CFU/gram of beads. A linear increase in bacterial count (log scale) was observed from 48 to 144 hours. By 144 hours, strains LB02, BS6633, CN02, and Pseudo reached 10*9 CFU/gram of beads, while PB02 and BS02 attained 10*8 CFU/gram of beads. A gradual and consistent increase in viable cell numbers for all strains during the incubation period was observed.
Total viable count (cfu/gm beads)
Strains 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs
LB02 1 x 10*5 9 x 10*7 1 x 10*8 6.2 x 10*8 8 x 10*8 1 x 10*9
PB02 1.4 x 10*5 2.7 x 10*7 1.3 x 10*8 2.1 x 10*8 3 x 10*8 1 x 10*8
BS02 2.7 x 10*5 4.5 x 10*7 6.3 x 10*8 5 x 10*9 6 x 10*8 5 x 10*8
BS6633 1.8 x 10*5 5.4 x 10*7 2 x 10*8 1 x 10*8 2 x 10*8 3 x 10*9
CNO2 4.5 x 10*5 6.3 x 10*7 4.5 x 10*8 1.1 x 10*9 5.1 x 10*9 1 x 10*9
PF 1.8 x 10*5 9 x 10*7 1 x 10*8 1 x 10*8 6 x 10*9 7 x 10*9

3b. Anaerobic
For anaerobic entrapment, actively growing RL03 and PF bacterial strains were each inoculated into basal media containing microbeads and were allowed to immobilize under strict anaerobic conditions, using an anaerobic chamber to ensure the absence of oxygen. The microbeads immobilized the bacteria, providing a supportive matrix for their growth and viability. At 24-hour intervals, samples were taken to monitor bacterial proliferation by dispersing the beads and plating serial dilutions onto anaerobic agar. Over the incubation period, both RL03 and PF showed a steady increase in viable cell counts, typically reaching up to 10? CFU per gram of beads, demonstrating the effectiveness of this method for maintaining anaerobic cultures.
Total viable count (cfu/gm beads)
Strains 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs
RL03 1 x 10*5 9 x 10*7 1 x 10*8 6.2 x 10*8 8 x 10*8 1 x 10*9
PF 1.4 x 10*5 2.7 x 10*7 1.3 x 10*8 2.1 x 10*8 5 x 10*8 2 x 10*9

Example 04: Co-culturing concentration optimization of each microbe
To optimize the concentration and ratio of each culture for effective co-cultivation, a consortium study was conducted employing a sequential elimination strategy based on relative growth rates. All seven bacterial strains were initially inoculated together in a nutrient medium and incubated under standard conditions for growth.

Following incubation, serial dilutions of the mixed culture were prepared and spread-plated onto agar to assess the relative abundance of each strain. The strain that produced the most colonies at the highest dilution-which indicated it was the fastest grower-was left out of the next round of consortium formation. This process was repeated to gradually remove the dominant, fast-growing strains, making it easier for the slower-growing strains to be included and establish themselves in the consortium.

The rationale for this approach was to prevent fast-growing strains from out competing slower-growing, yet potentially functionally significant, members during the initial establishment phase. Based on the outcomes of this study, the final inoculation ratio for the consortium was set as RL03 : BS6633 : CN02 : LB02 : PF : BS02 : PB02 = 1 : 1 : 1 : 1 : 0.5 : 0.5 : 0.5.

To promote balanced growth and functional synergy within the co-culture system, the initial concentrations of the faster-growing strains (LB02, CN02, Pseudo, BS6633) were inoculated at half the concentration used for the slower-growing strains (BS02, PB02, RL03).

This method allows each strain in the consortium to establish itself and contribute to the consortium’s overall effectiveness, especially during later entrapment and application stages.

Example 05: Entrapment process - CONSORTIA
6a. AEROBIC : Aerobic bacterial strains (LB02, PB02, BS02, BS6633, CN02, and PF) were sequentially inoculated into basal broth medium along with microbeads in ratio of 1:0.5:0.5:1:1:0.5 respectively. Microbeads were subsequently employed to entrap the cells under aerobic conditions, and bacterial proliferation within the beads was monitored at 24-hour intervals using the spread plate method.

The initial bacterial load was approximately10*5 CFU/gram of beads. A linear increase in bacterial count (log scale) was observed from 48 to 120 hours. By 120 hours, count of 10*9 cfu/g of beads was achieved and count exhibited presence of all inoculated strains in consortia. A gradual and consistent increase in viable cell numbers for all strains during the incubation period was observed.
Total count (cfu/gm beads)
24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs
Total aerobic viable count 1 x 10*5 9 x 10*7 1 x 10*8 6.2 x 10*8 1 x 10*9 5 x 10*9

Advantages of entrapment of bacterial consortia over individual strains:

*Higher Cell Loading Capacity: Achievement of high cell density is a key advantage of entrapping bacteria as a consortium, as it enables more rapid colonization and activity within the beads compared to individual strain entrapment.

A cell density of 10? cfu/g of beads was attained within 120 hours using consortia entrapment, whereas individual strain entrapment required 144 hours to reach the same cell concentration.

*Faster Entrapment and Enhanced Survival: Entrapment in consortium allows immobilization of all strains in a single step. This not only streamlines the process but also ensures that all strains are protected and stabilized together, accelerating the overall entrapment procedure compared to repeating the process for each strain individually

*Synergistic metabolic interactions: Co-entrapment of multiple bacterial strains within microbeads facilitates synergistic metabolic interactions among the consortium members.

Each strain contributes distinct enzymes, enabling the consortium to break down complex substrates more efficiently than individual strains alone.

* Functional redundancy: Consortia mitigate activity loss if one strain under performs, critical for wastewater treatment systems exposed to toxin fluctuations.
5b. ANAEROBIC
RL03 and PF were entrapped under anaerobic conditions due to their heightened susceptibility to viability loss and cellular damage. RL03 is a non-spore-forming bacterium, lacking the robust protective structures that endospores provide, while PF, as a Gram-negative strain, possesses a relatively fragile cell envelope that is more prone to environmental stresses.

Actively growing culture, at concentration of 2% of each, was separately inoculated into basal broth medium along with microbeads. Microbeads were subsequently employed to entrap the cells under anaerobic conditions, and bacterial proliferation within the beads was monitored at 24-hour intervals using the spread plate method.

The initial bacterial load was approximately10*5 CFU/gram of beads. A linear increase in bacterial count (log scale) was observed from 48 to 144 hours. By 144 hours, strains reached 10*9 CFU/gram of beads. A gradual and consistent increase in viable cell numbers for all strains during the incubation period was observed.

Anaerobic entrapment offered several key advantages for these sensitive strains. By eliminating direct exposure to oxygen and fluctuating pollutant concentrations at the bead surface and periphery, the immobilization process created a stable micro-environment within the PVA matrix. This protected the cells from oxidative stress, desiccation, and abrupt changes in environmental conditions that could otherwise compromise their viability.

The maintenance of both viability and functionality is critical for downstream applications, such as bio remediation or waste-water treatment, where consistent and reliable microbial performance is essential. Thus, anaerobic entrapment proved to be an effective strategy for safeguarding and sustaining the operational potential of these vulnerable bacterial cultures.

Total count (cfu/gm beads)
24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs
Total anaerobic count 1 x 10*5 1 x 10*7 2 x 10*8 1 x 10*9 3 x 10*9 7 x 10*9

5c. Anaerobic followed by aerobic:
Aerobic microorganisms use oxygen to rapidly degrade many common organic pollutants, enabling faster bioremediation of easily biodegradable compounds. However, they are often unable to break down more complex or recalcitrant pollutants. Anaerobic microbes, which function without oxygen, specialize in degrading these tougher compounds through alternative metabolic pathways, although their activity is generally slower. Entrapping both aerobic and anaerobic strains together combines their complementary strengths. This co-immobilization allows for faster initial degradation by aerobes, followed by the breakdown of resistant pollutants by anaerobes. Additionally, close proximity within the beads facilitates metabolic cooperation, improving overall efficiency. Therefore, integrating both microbial types provides a versatile and robust approach to treating a wide range of environmental contaminants.

Process: The microbeads were employed to entrap the RL03 and PF culture under anaerobic conditions, resulting in a count of 10? CFU per gram of beads by 5th day of entrapment. Subsequently, the beads were surface-washed, and introduced into a consortium of aerobic strains (LB02, PB02, BS6633, CN02, and BS02) in 1: 0.5: 1 : 1:0.5 ratio respectively. The flasks were incubated under shaking conditions and bacterial entrapment was monitored at 24 hours interval by spread plate technique.

Total count (cfu/gm beads)
24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs
Total viable count (aerobic) 1 x 10*5 9 x 10*7 3 x 10*8 6.2 x 10*8 2 x 10*9 3 x 10*9
Total viable count (anaerobic) 3 x 10*5 5 x 10*7 1 x 10*8 3 x 10*8 1 x 10*9 2 x 10*9

Example 06: Application
6a. COD/BOD reduction by individual entrapped bacterial strain and bacterial consortium.
For industrial effluent:
Individual bacterial strains were entrapped within microbeads and evaluated for their ability to reduce COD independently. Simulated effluent was prepared, sterilized, and treated with the entrapped beads. COD levels were measured every 24 hours to assess the performance of each strain.

48 hrs 72 hrs 96 hrs 120 hr
COD (ppm) COD (%) reduction COD (ppm) COD (%) reduction COD (ppm) COD (%) reduction COD (ppm) COD (%) reduction
Control 2337 2314 2250 2145
BS02 Beads 1456 33% 590 75% 521 77% 489 77%
LB02 Beads 2013 14% 1884 19% 1764 22% 513 76%
PB02 Beads 1756 24% 528 75% 427 80% 385 83%
BS6633 Beads 1568 33% 1229 47% 1219 46% 638 70%
CN02 Beads 1934 17% 1532 33% 1219 46% 577 73%
RL03 Beads 1903 19% 1504 35% 518 77% 463 78%
PF Beads 1187 49% 526 77% 532 76% 577 73%

The study demonstrates that individual bacterial strains entrapped within micro-beads exhibit variable efficiencies in reducing chemical oxygen demand (COD) from simulated effluent over 96 and 120 hours. Among the individual strains, PB02 achieved the highest COD reduction at 96 hours (80%), followed closely by BS02 (77%), RL03 (77%), and Pseudomonas (76%). By 120 hours, all strains showed marginal improvements, with PB02 maintaining the highest reduction (83%) and other effective strains (BS02, RL03, Pseudomonas) reaching 77–78%. In contrast, LB02 consistently showed the lowest performance (22% at 96 hours, 70% at 120 hours).

When a bacterial consortium was used, COD reduction was significantly enhanced, exhibiting 64%, 67% after 48 and 72 hours reaching 92% at 96 hours and a maximum of 93% at 120 hours. This performance surpassed that of any individual strain, indicating a synergistic effect among the bacterial members in the consortium. The rapid and higher COD removal observed with the consortium demonstrates the advantage of microbial diversity and potential metabolic cooperation in complex substrate degradation, consistent with findings from other studies where mixed cultures or consortia outperformed single strains in bio-remediation applications

48 hrs 72 hrs 96 hrs 120 hr
COD (ppm) COD reduction(%) COD (ppm) COD reduction(%) COD (ppm) COD reduction(%) COD (ppm) COD reduction(%))
Control (N) 4023 3992 3890 3812
Test (entrapped beads) 1472 64% 1305 67% 311 92% 278 93%

For lake water
Simulated lake water was prepared, sterilized, and treated with the entrapped beads. COD levels were assessed for reduction.

24 hrs
COD (ppm) COD reduction(%)
Control (N) 300
Test (entrapped beads) 3.0 99%

COD was reduced by 99% in 24 hours in simulated lake water. This result highlights the potential of immobilized microbead technology for rapid and efficient remediation of polluted water bodies, offering a promising approach for improving lake water quality in a short time frame.

Example 6b: Application - Reduction of odour through bacterial consortia immobilized in microbeads.
Odour reduction is a crucial aspect of bioremediation, especially in wastewater treatment, as it not only improves community comfort but also signals efficient breakdown of organic pollutants. Persistent odours often indicate incomplete treatment or the presence of harmful gases like hydrogen sulfide and ammonia, which can pose health and environmental risks. Effective odour control reflects optimal microbial activity and is essential for regulatory compliance and public acceptance. Additionally, targeting odour-causing compounds through specific microbial strategies can enhance overall treatment efficiency, making odour reduction both a key indicator and a benefit of successful bioremediation processes.

The odour reduction capability of immobilized microbial beads was evaluated using a hedonic scale, which measures the subjective perception of odour intensity and pleasantness. To prepare the test sample, putrescible fruit waste was collected and submerged in water, then incubated under shaking conditions overnight to promote the release and accumulation of malodourous compounds. After incubation, the odour intensity of the resulting mixture was assessed as a baseline. The mixture was then filtered to remove solid residues, yielding a clarified, odour-rich solution. This filtrate was subsequently used as the test substrate in deodourisation experiments, where the effectiveness of the immobilized beads in reducing unpleasant odours was systematically studied and quantified using the hedonic scale.

Hedonic scale and score
Hedonic scale Index
Like extremely 7
Like very much 6
Like moderately 5
Dislike slightly 4
Dislike moderately 3
Dislike very much 2
Dislike extremely 1

No. of volunteers Rating based on hedonic index Hedonic scale
Aerobic F. Anaerobic Aerobic + F. Anaerobic
1 3 3 3 Dislike moderately
2 3 3 3 Dislike moderately
3 3 3 3 Dislike moderately
4 3 3 3 Dislike moderately
5 2 2 2 Dislike very much
6 3 3 3 Dislike moderately
7 3 3 3 Dislike moderately
8 2 2 2 Dislike very much
Mean 2.75 2.75 2.75 The odour index lies between dislike moderately and dislike extremely
% odour reduction 29.16% 29.16% 29.16%
The results demonstrated a clear reduction in odour intensity across all treatment combinations, as assessed by the hedonic scale. Samples treated with aerobic culture-entrapped beads exhibited hedonic scale ratings between 4 and 6, corresponding to an odour reduction of 68.8%. Beads containing facultative anaerobic cultures achieved a 66% reduction in odour, while the combination of facultative anaerobes and aerobes resulted in the highest odour reduction at 75% within 96 hours. These findings indicate that the combined use of both aerobic and facultative anaerobic cultures is most effective for odour mitigation. Detailed hedonic scale scores and odour reduction percentages for all samples, after treatment are provided below.

No. of volunteers Rating based on hedonic index Hedonic scale
Aerobic F. Anaerobic Aerobic + F. Anaerobic
1 5 5 5 Like moderately
2 5 5 6 Like very much
3 5 6 6 Like very much
4 5 5 6 Like moderately
5 6 5 5 Like moderately
6 5 4 6 Like moderately
7 6 5 5 Like moderately
8 4 5 5 Like moderately
Mean 5.125 5 5.5 The odour index lies between like moderately and like very much
% odour reduction 68.8% 66% 75%

Example 6c: Application - Reduction of color through bacterial consortia immobilized in microbeads.
Various combinations of entrapped microbeads were introduced into artificial effluent supplemented with reactive red dye to evaluate their decolorization efficiency. The reduction in color intensity was systematically assessed at 24-hour intervals using spectrophotometric analysis. Over the course of the experiment, a progressive decrease in color intensity was observed in all treatments, indicating the active role of the immobilized microbial strains in dye degradation and breakdown of associated components.

By the 144th hour, microbeads entrapped with aerobic bacterial strains achieved a significant colour reduction rate of 70%, demonstrating their strong capacity for dye decolourization under oxygen-rich conditions. In comparison, microbeads containing facultative anaerobic strains exhibited a color reduction of 50%, suggesting moderate effectiveness in the absence or limited presence of oxygen. Notably, the combination treatment where both aerobic and facultative anaerobic strains were co-entrapped resulted in the highest decolourization efficiency, with an 76% reduction in color intensity observed within the same period.

These results highlight the enhanced performance achieved through the synergistic action of both aerobic and facultative anaerobic microbes, which likely facilitated a broader range of dye breakdown pathways.

Colour reduction
24 hours 48 hours 72 hours 96 hours 120 hours 144 hours
ppm %reduction ppm %reduction ppm %reduction ppm %reduction ppm %reduction ppm %reduction

Control 730 710 690 656 621 614
Entrapped beads (aerobic) 679 6.9% 624 12.7% 542 21.4% 321 51% 276 55% 187 70%

Entrapped beads(anaerobic) 694 4.9% 674 5.3% 604 12.4% 546 17% 387 38.1% 308 50%

Entrapped beads(combination of both) 692 5.2% 546 32% 423 40% 280 58% 265 59.5% 156 76%

Example 07: Sustained release and stability of beads
7a. Sustained release
After the entrapment process, microbial beads were decanted, gently dabbed and stored at ambient temperature for several weeks to months to simulate real-world shelf life and storage conditions. This approach ensured that the beads’ performance could be evaluated after extended storage, reflecting practical scenarios in bioremediation or wastewater treatment applications.

To assess the sustained release and viability of the immobilized bacteria, a batch of these stored beads was introduced into artificial effluent. At regular intervals specifically, every 24 hours samples of the medium were collected to monitor the release of viable bacteria from the beads. The spread plate method was used for quantification: serial dilutions of each sample were plated onto nutrient agar, incubated, and the resulting colony-forming units (CFU) were counted to determine the number of live bacteria released.
This release experiment was conducted continuously over a one-month period, with sampling at each interval providing a detailed profile of bacterial release dynamics.

The data demonstrated trends such as including an initial surge in bacterial release, followed by phases of consistent release, and eventually a gradual decrease in viability over time.

Total viable count (cfu/gm beads)
24th hr 48th hr 72nd hr 96th hr 120th hr 144th hr 2nd week 3rd week 4th week 5th week
Aerobic entrapment
Release from beads 9 x 107 1.2 x 107 3 x 108 4 x 108 1 x 109 1 x 109 2.7 x 109 1 x 109 3 x 108 1.2 x 108
Facultative anaerobe entrapment
Release from beads 1 x 106 1 x 107 3 x 108 5 x 108 9.5 x 108 1 x 109 3.8 x 109 9 x 109 1.6 x 108 2 x 107
Facultative anaerobes + aerobes entrapment
Release from beads 1 x 108 3.4 x 108 1.6 x 108 1.4 x 108 5.4 x 108 2.5 x 109 1 x 109 2.7 x 109 1.8 x 109 2 x 109

7b. Stability after storage
To evaluate the stability and long-term viability of the bacterial cultures entrapped within beads, a systematic stability study was conducted. The microbead-entrapped bacterial cultures were stored under controlled conditions, and at monthly intervals, a representative sample of beads was withdrawn for analysis.

For each time point, the beads were immersed in sterile saline solution to facilitate the release of the entrapped bacteria. To ensure thorough disintegration of the beads and complete release of viable cells, the suspension was subjected to sonication—a process that uses ultrasonic waves to break apart the bead matrix without harming the bacteria. Following sonication, the resulting bacterial suspension was serially diluted to obtain countable concentrations.

The viable bacterial count was then determined using the spread plate method: aliquots of each dilution were plated onto nutrient agar and incubated under suitable conditions for colony development. After incubation, colony-forming units (CFU) were counted, providing a quantitative measure of the total viable bacteria released from the beads at each storage interval.

This process was repeated at each monthly time point, allowing for the monitoring of changes in bacterial viability over the storage period. The resulting data provided insights into the stability of the beads, indicating how well the immobilized bacteria retained their viability and potential functional activity during long-term storage. This information is crucial for assessing the shelf life and practical usability of the microbial beads in biotechnological and environmental applications.

Combinations Count cfu g/beads

0th day 1th month 2nd month 3rd month 4th month 5th month 6nd month
Aerobic strain entrapped microbeads
beads 2 x 109 3 x 109 2 x 108
1 x 109 1 x 109 2 x 108 1 x 108

Facultative anaerobic strain entrapped microbeads
beads 2 x 109 3 x 109 2 x 109
1 x 109 1 x 108 2 x 108 1 x 108

F. Anaerobes + Aerobes in combination
beads 1.8 x 109 9 x 109 6.3 x 109
2 x 109 1.7 x 109 2.4 x 108 1.5 x 108
,CLAIMS:
1. An immobilized biofilm for bioremediation of static and flowing water bodies, wherein the biofilm comprises of:
a. a primary film forming spore bearing bacteria, and
b. a secondary non-spore forming bacteria.

2. The immobilized biofilm as claimed in Claim 1, wherein the biofilm is immobilized on a porous material.

3. The immobilized biofilm as claimed in Claim 2, wherein the porous material is selected from but not limited to polymeric granular gels or polymeric materials, clay aggregates, foam brick pieces, foam sponge, clay brick pieces, alginate beads, sago, seeds of sweet basil.

4. The immobilized biofilm as claimed in Claim 1, wherein
a. Facultative anaerobic microbial population entrapped inside the core of substrate followed by aerobic one on the outer surface of the substrate
b. only aerobic microbial population entrapped in entire substrate, and
c. only facultative anaerobic microbial population entrapped in entire substrate.

5. The immobilized biofilm as claimed in Claim 1, wherein the primary film forming spore bearing bacteria are selected from Bacillus pumilus ATCC 14884, Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 and Bacillus megaterium ATCC 9885, Bacillus subtilis ATCC BS6633

6. The immobilized biofilm as claimed in Claim 1, wherein the secondary non-spore forming bacteria are selected form Pseudomonas spp. and Lactobacillus spp.

7. The immobilized biofilm as claimed in Claim 6, wherein the Lactobacillus spp. is Lactobacillus rhamnosus ATCC 53103.

8. The immobilized biofilm as claimed in Claim 6, wherein the Pseudomonas spp. is Pseudomonas fluorescens NCIM 5226.

9. The immobilized biofilm as claimed in Claim 1, wherein the immobilized biofilm is self-regenerative.

10. The immobilized biofilm as claimed in Claim 9, wherein the immobilized biofilm releases microbial population of live viable microbial cell in range of 101 to 109 cfu/gm.

11. The immobilized biofilm as claimed in Claim 1, wherein the biofilm of the invention reduced COD and BOD upto 99% by 24-120hrs. Under aerobic or anaerobic or anoxic condition.

12. The immobilized biofilm as claimed in Claim 1, wherein the micro beads can be used for liquid waste management, bioremediation of water bodies and as a deodourizer and /or a decolourizer.

13. A process for preparation of the immobilized biofilm as claimed in Claim 1, wherein the process comprises:
a) Preparing of microbial culture;
b) sequential immobilizing each culture of step (a) under aerobic, anaerobic and anoxic conditions;
c) decanting the culture media from the microbeads of step (b), and
d) storing the microbeads of step (c).