Assessment of The Bioremediation Potential of Selected Bacterial Species Isolated from the Textile Printing Wastewater Inoculated with Cow Dung

This study was conducted to isolate and identify bacteria from the wastewater of the textile printing industry inoculated with a mixture of cow dung, jaggery, and urea to assess the bioremediation potential of isolated bacterial species for reducing color and other polluting parameters of the wastewater. Based on colony characteristics, we were able to isolate nine types of bacteria (Pri 1 to 9), capable of thriving in textile printing wastewater. Out of the nine isolates examined, four (Pri 3, Pri 4, Pri 8, and Pri 9) significantly reduced the color and values of other physicochemical parameters of the wastewater. Isolated bacterial cultures were identified using various biochemical tests, antibiotic sensitivity tests, and rRNA sequence analysis. At the end of a 24-h incubation period at room temperature under shaking conditions at 100 rpm on an orbital shaker, isolate Pri 3 was identified as Alcaligenes aquatilis LMG 22996 (T), capable of reducing color by 86.13%, biological oxygen demand (BOD) by 70.44%, chemical oxygen demand (COD) by 80.65%, total dissolved solids (TDS) by 47.31%, total suspended solids (TSS) by 56.56%, and ammoniacal nitrogen by 75.95%; isolate Pri 4 as Priestia aryabhattai B8W22(T), capable of reducing color by 78.35%, BOD by 66.35%, COD by 74.92%, TDS by 34.94%, TSS by 31.66%, and ammoniacal nitrogen by 71.14%; isolate Pri 8 as Citrobacter werkmanii NBRC 105721(T), capable of reducing color by 90.37%, BOD by 82.13%, COD by 85.06%, TDS by 54.83%, TSS by 61.97%, and ammoniacal nitrogen by 80.76%; and isolate Pri 9 as Shewanella chilikensis JC5(T), capable of reducing color by 90.17%, BOD by 84.68%, COD by 83.46%, TDS by 59.13%, TSS by 62.45%, and ammoniacal nitrogen by 90.37%.


INTRODUCTION
Since the existence of mankind, human beings have used colors in their routine lifestyles, which were traditionally derived from natural materials. 1However, with the increase in population and industrial development, these natural materials are being replaced by synthetic colors and dyes.Currently, more than 100,000 types of synthetic dyes are being used across various industrial sectors. 2 Among these sectors, the textile industry stands out as the largest consumer of synthetic dyes and chemicals in its manufacturing process.Textile manufacturing depends on the freshwater requirements, and approximately 20-25% of the used dyes and chemicals return as wastewater from these industries. 3Many of the textile dyes inhibit oxygen transfer in the generated wastewater, thereby increasing both biological and chemical oxygen demands. 4This may lead to toxicity for aquatic life, and continuous exposure to such dyes might pose health risks to humans. 5,6Consequently, proper treatment of industrial wastewater before discharge into the natural environment becomes crucial.Previously, treatment processes relied on chemical and physical methods, 7 which helped in color removal of the dyes to some extent.However, these methods often generate undesirable by-products, such as aromatic amines, which are challenging to maintain, 8 Instead, the biological procedure for the removal of such compounds from the wastewater is cheaper due to the availability of the vast array of microbes in the natural environment and their diverse metabolic pathways that remove or convert complex forms of pollutants to simpler forms. 9,10This can be achieved through biosorption, biodegradation, bioaccumulation, and biostimulation. 11As the bacterial system has diverse metabolic pathways, numerous innovations have been conducted in textile wastewater treatment.Various bacterial genera, including Bacillus sp.ADR, ETL-2012, SF, VUS, UN2, [12][13][14][15][16][17][18][19][20][21][22] Caulobacter subvibrioides 23 ; Enterobacter aerogenes and its spp.SXCR, CV-S1 [24][25][26] ; Klebsiella (ST16.16/034),(facultative) strain VN-31, strain Y3, DA26 11,27,28 ; Moraxella osloensis 29,30 ; Paenibacillus alvei MTCC 10625 and P. azoreducens 7,31 ; Pseudomonas spp.SU-EBT, LBC1, SUK1 [32][33][34] ; Pseudomonas luteola; [35][36][37] Rhodobacter erythropholis MTCC 4688 38 ; and Stenotrophomonas spp.BHUSSp X2, 12,39 have been utilized in the treatment process.
The success of microbial wastewater treatment highly depends on the isolation of microorganisms from the polluted sample, such as textile wastewater 6,26,[40][41][42][43][44] textile effluentcontaminated soil, 19,43,45 and activated sludge. 46n this study, we isolated bacteria from textile printing wastewater inoculated with a mixture of cow dung, jaggery, and urea.The efficiency of different types of bacteria naturally present in manmade polluted samples such as textile printing wastewater, and those present in natural samples such as cow dung that survived in textile printing wastewater was evaluated for their capability to reduce color and values of other physicochemical parameters of the wastewater.

Collection of textile printing wastewater
Wastewater was collected from the effluent treatment plant of the textile printing industry, Western Overseas, situated at 21°43'25.20"N and longitude 70°34'10.27"E, in the village of Jetalsar, Jetpur, Rajkot, Gujarat, India.The sample was collected in a 10-L sterile plastic container and preserved at 4°C in the refrigerator until examination.

Enrichment and isolation of bacteria
Bacteria are present in all ecosystems and are part of all living systems on the Earth. 47ypically, bacteria capable of dye decolorization and degradation are isolated from textile wastewater, textile effluent-contaminated soil, and activated sludge.In this study, we prepared a cocktail of textile printing wastewater, cow dung, jaggery, and urea for isolating bacteria suitable for the bioremediation of pollution caused by the colored effluent.
To obtain a diverse range of bacterial systems, enrichment of bacteria is essential before the isolation process.In 500 L of fresh water, a mixture of 2 kg cow dung, 250 g jaggery, and 100 g urea was prepared and aerated for 48 h using a Nanobubble pump (Century Pump, Udhyog Nagar, Rajkot, India), producing bubbles that can survive in water for up to 60 min or more. 48Subsequently, 250 L of this mixture was combined with 250 L of textile printing wastewater, supplemented with 2 kg cow dung, 250 g jaggery, and 100 g urea.Aeration with the Nanobubble pump was continued for 24 h, and this cycle was repeated for 7 days.This method enriched only those bacteria capable of surviving in textile printing wastewater.The microbially enriched textile printing wastewater was diluted 100 times in sterile distilled water, streaked aseptically onto nutrient agar plates (HiMedia, Mumbai, India) prepared in sterile textile printing wastewater, and incubated at 37°C for 24 h.After incubation, numerous morphologically distinct bacterial colonies were observed on the nutrient agar plates.Nine morphologically different colonies were selected, maintained by subculturing onto nutrient agar slants, and stored in a refrigerator at 4°C.

Study of bacterial growth in textile printing wastewater
For the examination of bacterial growth, nutrient broth (HiMedia, Mumbai, India) was prepared directly in sterile textile printing wastewater.In a 250 mL Erlenmeyer flask, 100 mL of this nutrient broth was added and sterilized in an autoclave.A loopful of the pure culture of all nine selected bacterial isolates was individually inoculated into separate flasks and agitated at 100 rpm in an orbital shaker (Remi, Bharat) at room temperature for 10 h.Aseptically, 10 mL samples were harvested from each flask and collected in 12 mL sterile centrifuge tubes.The tubes containing isolates were centrifuged at 5000 rpm for 10 min using a laboratory centrifuge (Remi, India).The cell-free supernatants were discarded, and pellets of bacterial cells were resuspended in 10 mL distilled water.The absorbance was measured at λ660 nm (Systronics 106, India) to estimate the growth.

Screening of bacterial isolates for their decolorization potential
The ability of all bacterial isolates to remove color from the effluent was assessed.To prepare an inoculum, a loopful of the pure culture of bacteria was transferred from a nutrient agar slant to a 250 mL sterile Erlenmeyer flask containing 100 mL nutrient broth (pH 7.0) prepared in sterile distilled water.These flasks were placed on an orbital shaker (Remi, Ahmedabad, India) at 100 rpm and room temperature for 8 h.Then, 1.0 mL of actively growing inoculum was transferred to another 250 mL sterile Erlenmeyer flask containing 100 mL nutrient broth prepared in the textile printing wastewater itself (pH 7.0).Similar flasks for all nine bacterial isolates were prepared and kept on an orbital shaker at 100 rpm and room temperature.Visual observations of reduction or change in color were recorded at 24 h and 48 h.At the end of this process, four out of nine isolates were identified as the most effective decolorizers of textile printing wastewater and were selected for further testing.

Reduction in color and values of other polluting parameters of textile printing wastewater by selected bacterial isolates
Four bacterial isolates were subjected to further testing to assess their efficacy in reducing color and the values of other polluting parameters of wastewater, including BOD, COD, TDS, TSS, and ammoniacal nitrogen.To conduct the experiment, 100 mL of textile printing wastewater was introduced into a 250 mL Erlenmeyer flask, and 0.1 g jaggery was added as a nutrient source.The final pH was adjusted to 7.0, and the flasks were sterilized in an autoclave.A control flask without inoculation served as a reference, while test flasks were inoculated with 1 mL of an actively growing bacterial isolate aseptically.Each bacterial isolate had a dedicated test flask, placed on an orbital shaker at 100 rpm and room temperature.Samples were collected from these flasks at 8 h, 16 h, 24 h, and 48 h and analyzed for color reduction (decolorization), BOD, COD, TDS, TSS, and ammoniacal nitrogen.Decolorization was measured using a UV-visible spectrophotometer (Elico, Model No. SL 159) at λ660 nm, while other parameters were measured following the standard methods for the examination of water and wastewater (APHA). 49ecolorization percent was calculated using equation (1) 4 :

Identification of selected bacteria Morphological examinations
Morphological examination of bacteria involves observing colony characteristics and conducting microscopic examinations of cells.The colony characteristics of bacterial isolates were visually recorded, while cellular characteristics were documented through Gram and spore staining.Additionally, motility was observed through a hanging drop preparation of pure culture suspension.

Biochemical tests
By performing various biochemical tests, bacterial systems can be identified up to the genus level, and sometimes even to the species level. 44These tests were performed following the procedures outlined in Bergey's Manual of Systematic Bacteriology.The identification process was conducted in VITEK-2 (BioMerieux USA), a fully automated microbial identification system.

16S rRNA sequencing
For the identification of bacterial species, 16S rRNA sequencing of the selected bacterial isolates was conducted at the National Center for Microbial Resources (NCMR), Pune, India.

Colony morphology of bacterial isolates
Colony morphology refers to the visible characteristics and appearance of bacterial colonies grown on solidified agar media.This information is valuable for understanding the bacterial species present and their growth patterns. 52The Detailed colony characteristics are mentioned in Table 1.The colonies of isolates Pri 1 to 8 exhibited a circular form, whereas those of isolate Pri 9 displayed an irregular form.Colony sizes ranged from 0.5 mm to 3.0 mm among all isolates.The elevation of colonies varied: isolates Pri 1, Pri 3, Pri 4, Pri 7, Pri 8, and Pri 9 had convex elevations; isolates Pri 2 and Pri 6 were umbonate; and isolate Pri 5 had a flat elevation.The margin of colonies showed filamentous characteristics for isolates Pri 5 and Pri 8, whereas that of other isolates had entire margins.The color of colonies differed: isolates Pri 1, Pri 2, Pri 3, and Pri 7 were white; Pri 9 was milky white; Pri 5 and Pri 8 were grey; Pri 4 was yellow; and Pri 6 was bluish white.The surface of the colonies also varied: Pri 1, Pri 2, Pri 3, and Pri 7 had smooth surfaces; Pri 4 and Pri 9 had smooth and shiny surfaces; Pri 5 and Pri 6 had rough and white surfaces; and Pri 8 had rough and shiny surfaces.Most colonies appeared opaque or slightly opaque, with the exception of isolate Pri 8, which produced transparent colonies.Based on these observed colony characteristics, all nine microbial isolates were primarily identified as bacteria.

Microscopic examination of bacterial isolates
Bacterial cells were examined under a compound light microscope to determine their size, shape, organization, motility, and other structural traits.Gram staining was employed to determine the composition of cell walls, and

Growth curve of bacterial isolates
Bacterial growth curve patterns (Figure 1) revealed that all bacterial isolates were    parameters of textile printing wastewater by bacterial isolates, additional assays are currently being conducted.

Screening of bacterial isolates for their decolorization potential
Through visual observation for color changes after 24 h and 48 h of incubation, four isolates, namely, Pri 3, Pri 4, Pri 8, and Pri 9, were identified as the most effective candidates for decolorizing textile printing wastewater and the details of the same are mentioned in Table 3.These four bacterial isolates successfully transformed the color of the textile printing wastewater from dark brown to light yellow after 48 h of incubation.In contrast, the remaining bacterial isolates showed minimal alteration in the color of the wastewater.

Bioremediation of textile printing wastewater by selected bacterial isolates
Reduction in wastewater color, BOD, COD, TDS, TSS, and ammoniacal nitrogen was assessed to evaluate the bioremediation potential of selected bacterial isolates Pri 3, Pri 4, Pri 8, and Pri 9.
Notably, isolate Pri 8 demonstrated the highest color reduction within 48 h.The results of percent decolorization are illustrated in Figure 2.

BOD reduction
All bacterial isolates exhibited >50% reduction in BOD of textile printing wastewater within 16 h, except for isolate Pri 4 (<36%).After 24 h and 48 h, BOD reduction increased to 70.44% and 79.52% for Pri 3, 66.35% and 67.47% for Pri 4, 82.13% and 83.14% for Pri 8, and 84.68% and 87.41% for Pri 9, respectively.Notably, isolate Pri 9 achieved the maximum BOD reduction within 48 h.The data on BOD reduction are presented in Figure 3.

COD reduction
All bacterial isolates demonstrated COD reduction of >70% in textile printing wastewater within 16 h.After 24 h and 48 h, COD reduction increased to 80.65% and 88.26% for Pri 3, 74.92% and 75.46% for Pri 4, 85.06% and 87.95% for Pri 8, and 83.46% and 88.57% for Pri 9, respectively.Notably, isolates Pri 3 and Pri 9 achieved the maximum COD reduction within 48 h.The data on COD reduction are presented in Figure 4.

TDS reduction
Al l b acteri al i so l ates exh i b i ted approximately 50% reduction in TDS after 24 h, except for Pri 4 (<33%).After 48 h, TDS reduction further increased to 59.67% for Pri 3, 47.31% for Pri 4, 66.12% for Pri 8, and 68.27% for Pri 9. Notably, isolate Pri 9 achieved the maximum reduction in TDS within 48 h.The data on TDS reduction are presented in Figure 5.

TSS reduction
Approximately 60% reduction in TSS was observed after 24 h by all bacterial isolates, except for isolate Pri 4 (<32%).After 48 h, the reduction increased to 67.22% for Pri 3, 40.89% for Pri 4, 72.55% for Pri 8, and 68.97% for Pri 9. Notably, isolate Pri 8 achieved the maximum reduction in TSS within 48 h.The data on TSS reduction are presented in Figure 6.

Ammoniacal nitrogen reduction
All bacterial isolates demonstrated >75% reduction in ammoniacal nitrogen after 24 h.After 48 h, the reduction increased to 90.37% for Pri 3, 73.54% for Pri 4, 92.68% for Pri 8, and 95.18% for Pri 9. Notably, isolate Pri 9 achieved the maximum reduction in ammoniacal nitrogen within 48 h.The data on ammoniacal nitrogen reduction are presented in Figure 7.

Identification of the selected bacterial isolates using biochemical tests and VITEK 2
We have tested all the isolates against 47 different Sugars for the identification of Bacteria with a biochemical test, and the results we got are mentioned in Table 4.

Antibiotic sensitivity tests of the selected bacterial isolates
We have tested all the isolates against 47 different antibiotics for the selected isolates depending on the biochemical identification test, and the results we got after the incubation period are mentioned in Table 5.
Out of 47 antibiotics tested, isolate Pri 3 exhibited sensitivity to 32; Pri 4 showed sensitivity to 33 and intermediate sensitivity to 1; Pri 8 displayed sensitivity to 26, intermediate sensitivity to 7, and resistance against 1; and isolate Pri 9 demonstrated sensitivity to 35 antibiotics.Thus, the selected bacterial isolates were found to be sensitive to the majority of the antibiotics tested, suggesting vulnerability and positive response to antibiotics in case of infections, even after proper disinfection of the bioremediated textile printing wastewater.

Identification of the selected bacterial isolates by 16S rRNA sequencing
According to the report from NCMR, the results of 16S rRNA sequencing for the selected bacterial isolates are as follows: The Pri 3 isolate matches with the Alcaligenes aquatillis LMG 22996(T) strain with approximately 99.93% similarities, as per the genesequencing database.

TC T TG CGTAG C A AC TA ATG AC A A GGGT TGCGC TCGT TGCGGGAC T TAACCCAA C ATC TC A C G A C A C G A G C TG A C G A C A G C C A TG C A G C A C C TG TG T TC C G G T TC TC T TG C G A G C A C TG C C A A ATC TC T TC G G C AT TC C
The Pri 4 isolate matches with the Priestia aryabhattai B8W22(T) strain with approximately 99.93% similarities, as per the gene-sequencing database.

C C G C A A T G G A C G A A A G T C T G A C G G A G C A A C G C C G C G T G A G T G A T G A A G G C T T T C G G G T C G T A A A A C T C T G T T G T T A G G G A A G A A C A A G TA C G A G A G TA A C TG C T C G T A C C T T G A C G G T A C C T A A C C A G
The Pri 8 isolate matches with the Citrobacter werkmanii NBRC 105721(T) strain with approximately 99.78 % similarities, as per the gene-sequencing database.

AGT TAG C CG GTG C T TC T TC TG CG AGT A A C G TC A AT TG C TG C G G T TAT TA A C C A C A A CACC T TCC TCC TCGC TGAAAGTAC T T TACAA CCCGAAGGCCTTCTTCATACACGCGGCATGG C TGCATCAGGC T TGCGCCCAT TGTGCAATAT TCCCCACTGCTGCCTCCCGTAGGAGTCTGGAC C G T G T C T C A G T T C C A G T G T G G C T G G T C A T C C T C T C
The Pri 9 isolate has 100% similarities with the Shewanella chilikensis JC5(T) strain, as per the gene-sequencing database.

CONCLUSION
Addressing environmental restoration amidst the current state of pollution poses a significant challenge.Despite decades of research in colored wastewater treatment, finding an effective approach remains elusive.In light of this, the present study explored the utilization of textile printing wastewater inoculated with cow dung to isolate potential bacterial species for the efficient biological treatment of textile printing wastewater.This bioremediation strategy demonstrated the effectiveness of four enriched bacterial species, namely, A. aquatilis LMG 22996(T), P. aryabhattai B8W22(T), C. werkmanii NBRC 105721(T), and S. chilikensis JC5(T), in substantially reducing color, BOD, COD, and other polluting parameters of textile printing wastewater.However, considering their superior capacity to decolorize a diverse range of dyes in natural settings and achieve the complete mineralization of wastewater, C. werkmanii NBRC 105721(T) and S. chilikensis JC5(T) emerged as the most effective biological candidates among the four isolates.To further understand the depth of dye decolorization by these isolates, we are currently studying the biochemical mechanisms involved.Additionally, we are attempting to isolate the genes responsible for dye degradation.We hope that, in the future, cow dung will be recognized as a reservoir of microbial species with high potential for the bioremediation of pollution.

Figure 2 .
Figure 2. Color reduction by selected isolates

Figure 3 .
Figure 3. BOD reduction by selected bacterial isolates

Figure 4 .
Figure 4. COD reduction by selected bacterial isolates

Figure 7 .
Figure 7. Ammoniacal nitrogen reduction by selected bacterial isolates G C A G TC TC C C TA A A G T TC C C G G C AT TA CCCGCTGGCAAGTAAGGATAGGGGT TGCGC TCGT TGCGGGACT TAACCCAACAT T TCACAA CACGAGCTGACGACAGCCATGCAGCACCTGT CTCTCAGT TCCCGAAGGCACACCTGCGTCTC CGCTGGCTTCTGAGGATGTCAAGAGTAGGTA AGGT TCT TCGCGT TGCATCGAAT TAAACCAC ATGCTCCACCGCTTGTGCGGGCCCCCGTCAA T TCAT T TGAGT T T TAACC T TGCGGCCGTAC T CCCCAGGCGGTCTACT TAATGCGT TAGCT TG AGAGCCCAGTGTTCAAGACACCAAACTCCGA GTAGACATCGTTTACGGCGTGGACTACCAGG G TATC TA ATC C TG T T TG C TC C C C A C G C T T TC GTGCCTGAGCGTCAGTCTTTGTCCAGGGGGC CGCC T TCGCCACCGGTAT TCC TCCAGATC TC TACG C AT T TC AC CG C TAC AC C TG G A AT TC TA CCCCCC TC TACAAGAC TC TAGT T TGCCAGT T CGAAATGCGGTTCCCAGGTTGAGCCCGGGGC T T TCACATC TCGC T TAACAAACCGCC TGCGC ACGC T T TACGCCCAGTAAT TCCGAT TAACGC TCGCACCCTCCGTAT TACCGCGGCTGCTGGC ACGGAGTTAGCCGGTGCTTCTTCTGCGAGTA ACGTCACAGC TAGCAGGTAT TAAC TAC TAAC C T T TCC TCC TCGC TGAAAGTGC T

Table 1 .
Colony morphology of bacterial Isolates

Table 2 .
Microscopic examination of bacterial isolates

Table 3 .
Screening of bacterial isolates for their decolorization potential Minor decolorization was observed compared with a reference Brown Light brown 3. Pri 2 Minor decolorization was observed compared with a reference Brown

Table 4 .
Bacterial identification using VITEK-2, the microbial identification system