A METHOD OF PREPARATION OF A MICROBE-DOPED POLYMERIC AEROGEL FOR WASTEWATER TREATMENT

Abstract

The present invention discloses a method of preparation of a microbe-doped polymeric aerogel for wastewater treatment, involving dispersing 3-6 mg of polyvinyl alcohol (PVA) in 100 ml of de-ionized (DI) water, and subsequently adding 50-200 µL of glutaraldehyde to the PVA solution to obtain a glutaraldehyde laced mixture, adding 50-200 µL of hydrochloric acid (HCl) to the glutaraldehyde-PVA solution to obtain a glutaraldehyde-PVA-HCl solution and subsequently stirring the glutaraldehyde-PVA-HCl solution at 90°C to yield a gel, cooling the gel at room temperature for 2 hours, wherein the cooled gel is lyophilized to obtain a sponge like aerogel, doping the obtained aerogel in a nutrient medium, having bacterial cells to obtain a bacteria inoculated aerogel medium, and incubating the bacteria inoculated gel medium in a microbial incubator at 30ºC for 7 days, wherein the bacteria-doped aerogels are collected and lyophilized for storing the bacterial aerogels.

Specification

Description:FIELD OF THE INVENTION
The present invention relates to a method of preparation of a polymeric aerogel. More specifically, the present invention relates to a method of loading of doping of microbes to a polymeric aerogel for wastewater treatment.
BACKGROUND OF THE INVENTION
Environmental concerns are very prevalent and a major source of concern for individuals due to the economy's rapid development. Industrial reagents have a broad range of industrial uses, including food processing, leather, paper, and cosmetics. However, people's living conditions have been significantly impacted by inappropriate use and inadequate monitoring of industrial reagents. Therefore, the elimination of such reagents from industrial effluents has emerged as a significant concern in the realm of water pollution, which has garnered national and international attention.
US10350576B2: Highly porous, lightweight, and sustainable organosilane-coated organic aerogels with ultra-low densities and excellent material properties and methods for preparing them are provided. The aerogels are modified to have a superhydrophobic and superoleophilic surface, thus leading to an extremely high affinity for oils and/or organic solvents. (Abstract)
US10710915B2: Graphene aerogel metallic organic frame composite material loaded with microorganisms as well as preparation method and application thereof in the treatment of azo dye. (Abstract)
CN108928933B: The microbial slow-release treatment method for the organic wastewater is characterized in that a microbial treatment agent prepared by the adsorption-embedding method is adopted, high-concentration organic wastewater can be efficiently treated, and the method has the characteristics of stability, controllability, high purification efficiency and high hydrogen production yield. (Abstract)
Polymer, Volume 205, 28 September 2020, 122879: Polyimide aerogel with controlled porosity: Solvent-induced synergistic pore development during solvent exchange process.
Despite intensive studies on polymer aerogels, the fabrication of polyimide aerogels mostly relies on supercritical drying; however, the development of suitable ambient or vacuum drying methods and a shortened solvent exchange process are important for industrial applications. This study highlights the effect of the solvent type used for the solvent exchange process on the porosity and pore structure of vacuum-dried polyimide aerogels. By combining two different solvents, we could achieve a much higher aerogel porosity than when using either solvent independently. This synergistic effect cannot be simply explained by the surface tension of the exchange solvent but is shown to be related to the solvent-polymer affinity, solvent-induced structural change during the solvent exchange process, and shrinkage behaviour during the solvent exchange and drying processes. The pore structure and porosity are shown to be easily controllable by simply adjusting the solvent ratios. Moreover, the solvent exchange time is much shorter (~10 h) than that in previous reports on polyimide aerogels, which is highly beneficial for industrial applications. The mechanical properties were highly dependent on the porosity and pore structure, and the relative permittivity could be adjusted in a wide range according to the controlled porosity. Together with high thermomechanical stability, various electronic applications are expected.
Moreover, the idea proposed in this study is expected to serve as a useful guideline for the fabrication of various other polymer aerogels with efficient processability. (Abstract)
The prior arts mentioned herein discuss various literature regarding the method of synthesis and the role of polymeric aerogels towards the remediation of wastewater. However, the aerogels mentioned in the prior arts do not cater to the removal of all types of industrial pollutants from wastewater.
Thereby, to overcome the drawbacks, there exists a need in the art to develop a method of preparation of an aerogel complex that would lead to the remediation of a wide range of industrial pollutants from wastewater.
OBJECTS OF THE INVENTION
The principal object of the present invention is to overcome the disadvantages of the prior art.
An object of the present invention is to provide a method of preparation of a microbe-doped polymeric aerogel for wastewater treatment.
Another object of the present invention is to provide a cheaper and hassle-free method of preparation of a microbe-doped polymeric aerogel for wastewater treatment.
The foregoing and other objects, features, and advantages of the present invention will become readily apparent upon further review of the following detailed description of the preferred embodiment as illustrated in the accompanying drawings.
SUMMARY OF THE INVENTION
The process of creating a polymeric aerogel is the subject of the current invention. The method of loading and doping microorganisms onto a polymeric aerogel for wastewater treatment is the specific focus of the current invention.
An aspect of the present invention discloses a method of preparation of a microbe doped polymeric aerogel for wastewater treatment, the method comprising dispersing 3-6 mg of polyvinyl alcohol (PVA) in 100 ml of de-ionized (DI) water, wherein PVA is stirred at 90°C to yield a transparent solution, cooling the PVA solution, to room temperature and subsequently adding 50-200 µL of glutaraldehyde to the PVA solution to obtain a glutaraldehyde laced mixture, wherein the glutaraldehyde laced mixture is stirred for 15 minutes to obtain a glutaraldehyde-PVA solution, adding 50-200 µL of hydrochloric acid (HCl) to the glutaraldehyde-PVA solution to obtain a glutaraldehyde-PVA-HCl solution and subsequently stirring the glutaraldehyde-PVA-HCl solution at 90°C to yield a gel, cooling the gel at room temperature for 2 hours, wherein the cooled gel is lyophilized to obtain an aerogel having a sponge like structure, wherein the sponge like aerogel contains a porous polymeric network, doping the aerogel in a nutrient medium, inoculating bacterial cells on the doped nutrient medium to obtain a bacteria inoculated aerogel medium, and incubating the bacteria inoculated gel medium in a microbial incubator at 30ºC for 7 days, wherein the bacteria-doped aerogels are collected and lyophilized for storing the bacterial aerogels.
In another embodiment of the present invention, the method of determining the adsorption efficacy of the microbe-doped polymeric aerogel with a non-microbial doped aerogel involves, preparing 10mg/mL concentration of pollutants that includes congo red, methylene blue, naphthalene, acenaphthene and phenol, adding 1 mg/mL of microbe doped aerogel into each solution of the pollutants and incubating the microbial aerogel pollutant mixture in an incubator for 5 days and analyzing the sample solution in a UV spectrometer for determining the adsorption efficacy of the aerogels.
While the invention has been described and shown regarding the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. To illustrate the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the present disclosure is not limited to specific instrumentalities disclosed herein. Moreover, those in art will understand that the drawings are not too scale. Wherever possible, elements have been indicated by identical numbers.
Embodiments of the present disclosure will now be described, by way of example only, regarding the following diagrams wherein:
Figure 1: Schematic Diagram for microbe doped aerogel preparation.
Figure 2: Digital photographs of (A) PVA hydrogel, (B) PVA aerogel, and (C) Microbe immobilized PVA aerogel.
Figure 3: X-ray diffraction pattern of microbe doped aerogel.
Figure 4: ATR- FTIR spectra of jarosite microbe doped aerogel.
Figure 5: TGA analysis of microbe doped aerogel.
Figure 6: SEM analysis of microbe doped aerogel.
Figure 7: Optical microscopy of microbe doped aerogel.
Figure 8: EB/AO double staining of microbe doped aerogel showing (A) living, and (B) dead microbial colony.
Figure 9: Removal of different pollutants by bacteria-doped aerogel.
DETAILED DESCRIPTION OF THE INVENTION
The following description includes the preferred best mode of one embodiment of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore, the present description should be seen as illustrative and not limiting. While the invention is susceptible to various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.
In any embodiment described herein, the open-ended terms "comprising," "comprises," and the like (which are synonymous with "including," "having" and "characterized by") may be replaced by the respective partially closed phrases "consisting essentially of," consists essentially of," and the like or the respective closed phrases "consisting of," "consists of, the like.
As used herein, the singular forms "a," "an," and "the" designate both the singular and the plural, unless expressly stated to designate the singular only.
The present invention relates to the method of producing a polymeric aerogel. The present invention focuses specifically on the process of loading and doping microorganisms onto a polymeric aerogel for wastewater treatment.

For several years' different research has been conducted for wastewater treatment by using Nanocomposites, bioremediation, photocatalysis and advanced oxidation. On the other hand, after being developed in the 1930s, aerogel has shown a large spectrum of applications viz., thermal insulation, textiles, biomedical usage, energy storage, sensor development and construction. In this process, the production of bacteria-doped polymeric aerogel is demonstrated using polyvinyl alcohol (PVA) and its application for wastewater treatment. This process can be an economical and sustainable alternative to conventional methods for wastewater treatment.

Preparation of aerogel: To prepare polymeric aerogel, PVA was dispersed in di-ionized (DI) water (5mg/ 100mL) and stirred on a magnetic stirrer at 90 °C until the PVA dissolved completely and the solution became transparent. After that the PVA solution was cooled down to room temperature and 100 microliters of glutaraldehyde was added and again stirred for 15 min. at room temperature. Following this step, 100 microliters of hydrochloric acid (HCl) was added to the mixture and stirred at 90 °C until the liquid sol is converted to gel. The prepared gel is cooled down to room temperature for 2h and lyophilized using a freeze drier. After the lyophilization was complete, a porous polymeric network was developed and stored in a moisture-free condition.

Isolation of microorganisms: The microorganism used in this study was isolated from a soil sample collected from Dhapa, Kolkata, India. The collected soil sample was suspended in sterile water (1g/100ml) and the serial dilution was performed up to 106 concentrations. The suspended soil sample then was used to culture the microbes. Agar plates were prepared, inoculated by the pour plate method and incubated for 24 hours. After the incubation period, another agar plate was prepared, and a single colony was transferred from the incubated plate by the streak plate method. The new plate was again incubated for 24 hours. After 24 hours the cultured colony was isolated from the plate and inoculated into a 100ml liquid nutrient broth medium. The isolated microorganism was identified using the PCR method by Biokart India Pvt Ltd.

Doping of microorganisms: The microorganism used in this invention was Bacillus sp. (GeneBank no.: PP159024) as identified by Biokart India. Sterile nutrient media was utilized to prepare the subculture of that microbe, and the growth curve was observed by using a UV-Vis spectrophotometer. On the other hand, another nutrient media was prepared, and the previously prepared aerogels were merged into the solution. The whole system was sterilized to make it contaminant-free. By observing the bacterial growth curve, the log phase of Bacillus sp. was detected and as they entered that phase, the microorganisms were used to inoculate the nutrient media that contained aerogels. This whole system was incubated for seven days in a BOD shaker incubator at 30 °C for 7 days. After the incubation time, the bacteria-doped aerogels were collected and lyophilized to store them in a sterile condition. Fig. 1 represents the preparation method of bacteria-doped polymeric aerogel. Also, in Fig. 2, the images of PVA hydrogel, aerogel and bacteria-doped aerogel are presented respectively.

The physiochemical characteristics of microbe-doped aerogel were studied by performing XRD, ATR-FTIR, TGA, SEM analysis and optical microscopy. Density, moisture content, oil absorption efficiency, and swelling test of the prepared aerogel were also performed. To study the adsorption efficiency of the microbe doped aerogel, two dyes, namely congo red and methylene blue and three organic pollutants, viz., naphthalene, acenaphthene and phenol are considered as the pollutants of interest. To observe the survival of microorganisms inside the aerogel EB/AO double staining method was used. Microbe-doped aerogel exhibits a distinctive X-ray diffraction (XRD) pattern, which is a result of incident X-rays interacting with its crystal lattice. This interaction produces a discernible diffraction peak at 2?= 19.29° angle with 11.36 % crystallinity of the substance as shown in Figure 3. The uniqueness of this XRD pattern at this precise angle serves as a characteristic signature.

FTIR analysis revealed significant bands at 3261 cm-1 (O-H stretching from intermolecular hydrogen bonds), 2937 cm-1 (C-H from alkyl groups), 2845 cm-1 (C-H from alkyl groups), 1645 cm-1 (C=O group) 1405 cm-1 (CH2 group), 1376 cm-1 (C-H group) 1327 cm-1 (O-H group) 1083 cm-1 (C-O stretching), 916 cm-1 (C-C stretching) and 814 cm-1 (C-H group) in microbe doped aerogel which is mentioned in Figure 4. These findings suggest the presence of functional groups suitable for the adsorption process, highlighting aerogel's potential as an adsorbent in wastewater treatment.

To study the thermal profile of prepared microbe doped aerogel TGA analysis was performed. As shown in Figure 5, no significant weight loss was found. A slight downward curve represents the evaporation of entrapped moisture. From 100 °C a degradation curve was observed signifying the melting of PVA molecules inside the aerogel network. At 210 °C a second peak was observed with rapid degradation. This peak signifies the degradation of the cross-linking of the polymeric network. After this degradation was complete, until 300 °C the carbonation of the sample was observed.

Figure 6 shows the surface morphology of microbe doped aerogel. From the figure it was observed that the aerogel consists of an uneven surface that is favourable for adsorption. The presence of bacteria inside the aerogel contributes to bioremediation. The same result was observed from optical microscopy as shown in figure 7. The presence of microorganisms was also observed in this analysis and encircled in that image.

From the physical parameter analysis, it was observed that the density, moisture content and swelling capacity were 0.104 g/cm3, 16.04% and 74.91% respectively. From the oil absorption study, it was observed that 1gm of the prepared aerogels can observe 10ml/min. From the EB/AO double staining method, a dominance of green microbial colony was observed signifying the survival of microorganisms inside the aerogel matrix as shown in Figure 8. This observation supports the main aim of this work which is to perform adsorption and bioremediation simultaneously for effective wastewater treatment.

The adsorption efficiency of the microbe doped aerogel was studied by using naphthalene, phenol, acenaphthene, congo red and methylene blue. Each solution with 10 mg/L concentration was used for the adsorption study. Microbe-doped aerogel with a 1g/L dose was added to the solution and treated in a BOD shaker incubator for five days. After the treatment, the sample solution was collected and analysed by a UV-Viz spectrophotometer (Thermo Scientific Orion Automate 8000). The removal % was calculated by using equation (1).

Removal %= ((Initial pollutant concentration-Final pollutant concentration)/ (Initial pollutant concentration)) *100……… (1)

It was found that the highest adsorption was 98.25%, 95.46%, 93.9%, 95.2%, and 93.37% respectively. Another adsorption experiment was performed by normal PVA aerogel, and the adsorption efficiency was found to be 74.29%, 67.15%, 72.17%, 87.29%, and 85.24% respectively for the mentioned pollutants. Both the results were compared as shown in Figure 9, clearly stating that the microbe-doped aerogels have a higher capacity for the adsorption of pollutants.

Novel features of the present invention
• Cost-effective production of aerogel without solvent substitution.
• Production of microbe-doped aerogel.
• Application of microbe-immobilized aerogel for organic pollutant and dye removal from wastewater.
, Claims:We Claim:
1. A method of preparation of a microbe doped polymeric aerogel for wastewater treatment, the method comprising:
(a) dispersing 3-6 mg of polyvinyl alcohol (PVA) in 100 ml of de-ionized (DI) water, wherein PVA is stirred at 90°C to yield a transparent solution;
(b) cooling the PVA solution, to room temperature and subsequently adding 50-200 µL of glutaraldehyde to the PVA solution to obtain a glutaraldehyde laced mixture, wherein the glutaraldehyde laced mixture is stirred for 15 minutes to obtain a glutaraldehyde-PVA solution;
(c) adding 50-200 µL of hydrochloric acid (HCl) to the glutaraldehyde-PVA solution to obtain a glutaraldehyde-PVA-HCl solution and subsequently stirring the glutaraldehyde-PVA-HCl solution at 90°C to yield a gel;
(d) cooling the gel at room temperature for 2 hours, wherein the cooled gel is lyophilized to obtain an aerogel having a sponge-like structure, wherein the aerogel contains a porous polymeric network;
(e ) doping the powdered aerogel in a nutrient medium;
(f) inoculating bacterial cells on the doped nutrient medium to obtain a bacteria-inoculated aerogel medium; and
(g) incubating the bacteria-inoculated gel medium in a microbial incubator at 30ºC for 7 days, wherein the bacteria-doped aerogels are collected and lyophilized for storing the bacterial aerogels.
2. The method as claimed in claim 1, wherein 5 mg of PVA is dissolved in 100 ml of DI water.
3. The method as claimed in claim 1, wherein 100 µL of glutaraldehyde is dissolved in PVA solution.
4. The method as claimed in claim 1, wherein 100 µL of HCl is added in the glutaraldehyde-PVA solution.
5. The method as claimed in claim 1, wherein Bacillus sp. is used to inoculate the aerogel-doped nutrient medium.
6. The method as claimed in claim 1, wherein the method of determining the adsorption efficacy of the microbe doped polymeric aerogel with a non-microbial doped aerogel comprising the steps of: (a) preparing 10mg/mL concentration of pollutants that includes congo red, methylene blue, naphthalene, acenaphthene and phenol; (b) adding 1 mg/mL of microbe doped aerogel into each solution of the pollutants and incubating the microbial aerogel pollutant mixture in an incubator for 5 days; and (c ) analyzing the sample solution in a UV spectrometer for determining the adsorption efficacy of the aerogels.
7. The method as claimed in claim 1, wherein viability of the microbes doped within the polymeric aerogel has been evaluated by Ethidium bromide-Acridine orange staining protocol.

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