Exploring indigenous bacteria in wastewater for sustainable bioremediation for red and blue azo dyes decolouration, and chromium (VI) degradation in industrial effluents Open Access

Industrialisation has led to a significant rise in the release of industrial waste into the environment. Industrial waste refers to the byproducts and discarded materials produced during various industrial processes. The pollutants found in industrial waste are diverse. Including heavy metals (such as lead, mercury, chromium, and cadmium), organic and chemical compounds that are toxic to ecosystems and humans, when released into the environment (Shahedi et al. 2020). The industrial sector, undeniably a driving force behind global economic development, serves as the backbone of the modern world by producing the goods and services that sustain our daily lives. However, the remarkable progress achieved by the industrial sector comes with environmental repercussions.

Textile manufacturing wastewater is a complex mixture that contains various aromatic chemicals, colorants, and hazardous metals. Azo dyes are commonly used in the textile industry due to their resistance to chemicals and sunlight and their long-lasting properties (Wojnárovits & Takács 2008). Azo dyes are challenging to break down and can harm living organisms. These dyes contain azo groups (R1 − N = N − R2) and aromatic rings, contributing to their stability and making them difficult to degrade in water (Kishor et al. 2021). Some of these compounds are mutagenic, cytotoxic, and carcinogenic. During the metabolic reduction of azo dyes, aromatic amines like aniline and toluidine and related chemicals such as benzidine are generated. When mammals ingest azo dyes, their intestinal microflora can metabolise these chemicals through the action of azoreductase enzymes, leading to a reduction in the azo bond and nitro groups. Additionally, enzymes in the liver and bacterial skin can also catalyse the reduction of azo bonds and nitro groups, and these derivative compounds have potential carcinogenic effects (Paredes-Quevedo et al. 2021).

Wastewater discharged from the paper and pulp industry poses yet another challenge, with its wastewater containing organic matter, chiefly lignin, chlorinated organic compounds, cyanide, polyphenols, and other aromatic compounds (Munir et al. 2019). It also includes materials like chlorates and transition metal compounds such as chromium. Cr (VI) is mainly generated from anthropogenic activities and is highly toxic to living organisms. Cr (VI) is classified as a group 1 carcinogen by the World Health Organization (WHO) (Sharma et al. 2022). Chromium (VI) can function as an oxidising agent when it comes into direct contact with the skin or can be absorbed through the skin, especially if the skin has any existing damage. If chromium enters the bloodstream through inhalation, the body eliminates it through the kidneys and liver (Pradhan et al. 2017). To avoid the ill effects of Cr (VI) on human health, there is an urgent need to implement strict environmental restrictions to limit the amount of Cr (VI) that can be released into the environment (Sharma et al. 2022).

Various advanced techniques have been employed to address this issue to remove toxic compounds from industrial wastewater. These methods include coagulation/flocculation, ozonation, oxidation, precipitation, ion exchange, reverse osmosis, membrane ultrafiltration, adsorption, electrocoagulation, and electrodialysis (Wojnárovits & Takács 2008; Hassan et al. 2013). However, these processes come with notable drawbacks, such as incomplete pollutant removal, high energy consumption, and the generation of toxic sludge (Crini & Lichtfouse 2019).

Therefore, there is a need to explore sustainable and effective treatment approaches. Bioremediation technology harnesses naturally occurring microorganisms as agents to efficiently break down harmful substances or hazardous industrial wastewater, transforming them into less toxic or nontoxic forms. This is gaining increasing recognition due to its cost-effective and environmentally sustainable approach. Within biological treatment systems, various microorganisms, including fungi, bacteria, and actinomycetes, play a pivotal role in completely degrading organic compounds. Microbial degradation is the predominant process for converting organic molecules into their mineralised states. Additionally, these microorganisms have the capability to sequester, accumulate, and absorb heavy metals within their cellular structures or cell walls. In the bioremediation process, microorganisms utilise these pollutants as sources of nourishment or energy (Harekrushna & Kumar 2012). Our research proposes the use of indigenous bacterial strains for bioremediation from locally present in Rajasthan state, offering an innovative solution that not only reduces toxic pollutants but does so sustainably. The application of local bacterial species represents a significant advancement in addressing the environmental challenges of industrial pollution.

The motivation for this research arises from the urgent need to address the increasing pollution caused by industrial wastewater in Jaipur, as the textile and pulp & paper industries are key drivers of Rajasthan's economy but are also significant contributors to water contamination, especially in regions such as Sanganer and Jaipur. These effluents contain hazardous substances such as azo dyes and hexavalent chromium (Cr VI), which pose serious threats to local ecosystems and human health. The application of bioremediation techniques using indigenous microbes offers a tailored, cost-effective solution to this local issue, addressing pollution at its source.

The current study aims to assess the bioremediation potential of indigenous microbes for the treatment of influent discharged from two different industries. This research involves the comprehensive analysis of physico-chemical parameters of discharged industrial wastewater, isolation, identification, and characterisation of microbial populations derived from the wastewater of the textile and paper and pulp industries, respectively. The study further investigates the potential for bioreduction of Cr (VI) and decolorisation of azo dye within industrial wastewater, utilising indigenous bacterial isolates. A specific focus was on determining the factors influencing Cr (VI) bioreduction, including inoculum concentration, temperature, and pH. Additionally, the study entails FTIR analysis to elucidate the degradation of azo dye and 16S rRNA sequencing to identify the chromium-degrading bacteria.

The industrial effluent samples were collected from the pulp and paper industry, Sanganer and textile industry (Samuhik Dhulai Kendra, Sanganer, Jaipur, India), by grab sampling method in screw-capped sterilised cans, and the samples were brought to the laboratory on an ice pack in a cooler box and stored at 4 °C before further analyses. The collected samples were further analysed for the required physico-chemical parameters to examine pollution load.

The samples collected from the industries were brought for physico-chemical analysis at the environmental biotechnology laboratory of Dr B. Lal Institute of Biotechnology, Jaipur (India). The Physio-chemical parameters such as pH, temperature, electrical conductivity (EC), total dissolved solids (TDS), and dissolved oxygen (DO) were monitored with the help of the Hanna multiparameter kit as followed in the guidelines (APHA 22nd Edition). biochemical oxygen demand (BOD) was measured by the azide modification method, and chemical oxygen demand (COD) was measured through the closed reflux spectroscopic method (Hach-DR 5000). Nutrients (NO³⁻, NH₃) were also measured according to the methods described by Arora et al. (2016), and APHA 22nd Edition and were measured by the standard method of APHA (2017).

The bacteria effluent sample was enriched by co-incubating in nutrient broth containing 0.01% of each dye (Blue azo dye and red azo dye) at 35 °C for 24 h as shown in Figure 1. Subsequently, 1 mL of each enrichment culture was plated on a molten agar medium. After incubation at 35 °C for 24 h, the resulting bacterial colonies, which exhibited a clear zone around them, were isolated. According to Bergey's Manual, isolated cultures were then identified and classified morphologically and biochemically.

Chromium-degrading bacterial species were isolated by serially diluting the water sample and spreading it on nutrient agar and Luria Bertani (LB) agar plates containing 1% hexavalent chromium with three different inoculum concentrations. The bacterial isolates that demonstrated growth in Cr (VI) presence were chosen for chromium bioreduction. According to Bergey's Manual, isolated cultures were then identified and classified morphologically and biochemically.

The bacterial isolates were characterised based on physiological and biochemical characteristics. Physiological and biochemical characteristics of the isolates were evaluated by Voges–Proskauer, methyl red, indole, catalase, oxidase, glucose fermentation, citrate utilisation, and tests. The isolates were identified up to species based on comparative analysis of the observed characteristics with the standard description of bacterial strains in Bergey's Manual of Determinative Bacteriology.

The nutrient broth medium was inoculated with bacterial cultures and placed in the incubator shaker at 37 °C for 5 days at 200 rpm. A mixed bacterial consortium was prepared by combining equal volumes of culture broths from the nine isolates as shown in Figure 2. The nutrient broth was then prepared and filled into flasks labeled with the name of the organism. The broth culture was thoroughly mixed, and a 0.01% concentration of blue azo dye was added. It was then incubated with all nine strains individually, along with the consortium and control, for 3 days at 37 °C for bioremediation studies.

Biodegradation studies were conducted under optimised conditions, with a pH of 7 and a temperature of 37 °C, in aerobic conditions to assess degradation by the isolated strains. At defined intervals (0th, 1st, 2nd, and 3rd day), the culture was withdrawn and centrifuged at 10,000 rpm and 10 °C for 15 min. The supernatant was examined for absorbance at 530 nm under visible light using a spectrophotometer (ultraviolet-visible reflection (UV–VIS RS) spectrophotometer, LaboMed. Inc.) (Singh et al. 2013).

The screening test involved the inoculation of autoclaved nutrient broth, spiked with 0.25% Cr (VI), with three different inoculum concentrations (0.1, 0.5, and 1%) of bacterial cultures previously isolated from wastewater influent. The incubation was carried out at 37 °C in aerobic conditions with shaking at 150 rpm for a duration of 10 days, during which the chromium concentrations were periodically determined using the spectrophotometer for all the isolates (Pal & Paul 2004).

The selected isolates, which showed the highest degradation rate, were further selected for the optimization of culture parameters, including temperature and pH, for the paper and pulp industry effluent bioremediation.

The Cr (VI) reduction as well as growth studies of strain were monitored with a different temperature range from 28 to 40 °C. The varying growth of bacterial isolates at different concentrations of Cr (VI) in the Luria broth was measured at O.D. 640 nm. The chromate reduction was measured absorbance at 382 nm for all the bacterial strains at different concentrations of Cr (VI). The bacterial growth was also monitored at different concentrations of pH.

In this study, we conducted an analysis of the physico-chemical parameters of influent samples and effluent samples (after bioremediation treatment) of two industrial sources: the textile industry and the paper and pulp industry. The results obtained from our experiments are summarised in Table 1. The pH values of the influent samples exhibited slight variations, with the textile industry influent having a pH of 4.19 and the paper and pulp industry influent having a pH of 4, but after bioremediation it accounted as pH 6.9 and 7.2 of textile and paper and pulp industry, respectively. The temperature of both influent and effluent samples of both industries was similar, recorded at 31 °C, indicating a uniform thermal environment. Significant differences were observed in the EC measurements between the two industries. The textile influent displayed an EC of 2.9, decreasing by 44% to 1.6 in the effluent. Conversely, the paper and pulp influent exhibited a conductivity of 3.5, decreasing by 60% to 1.4 in the effluent, suggesting variations in the ionic composition of the effluents. Additionally, the DO of textile influent was 2.5 mg/L higher than paper and pulp industry influent, i.e. 1.3 mg/L but after the treatment process, the DO increased by 44 and 74% of both the influents, respectively. The BOD and COD values for the textile influent were 150 and 414 mg/L, respectively, decreasing by 70 and 47% in the effluent. In contrast, the BOD and COD values for the paper and pulp influent were 32.5 and 95.6 mg/L, which decreased by 13 and 5%, respectively. The salinity of the textile industry influent was 5.9 mg/L, which decreased by 53%, i.e. 2.8 mg/L. On the other hand, there was no significant change in salinity observed in the paper and pulp industry influent and effluent, i.e. 3.9 and 3.0 mg/L, respectively. The concentration of nitrogen is evaluated in the form of nitrate, which decreases by 12% in textile industrial influent to effluent and 57% in paper and pulp industrial influent to effluent. The concentration of phosphate decreases by 99% from textile industrial influent to effluent and 94% from paper and pulp industrial influent to effluent as described in Table 1. These physico-chemical parameters provide critical insights into the distinct characteristics of the influent streams from the two industries, which are essential for assessing the feasibility of bioremediation processes.

The microbial diversity analysis was conducted on influent samples from both industries. The results showed that a total of nine microbes were isolated from the textile industry and further identification of these isolates was done by biochemical analysis. In the textile industry, the isolates were designated as follows: T1 – Lactobacillus spp., T2 – Lactobacillus spp., T3 – Corynebacterium spp., T4 – Corynebacterium spp., T5 – Streptococci spp., T6 – Streptococci spp., T7 – Streptococci spp., T8 – Corynebacterium spp., T9 – Corynebacterium spp. Conversely, in the paper and pulp industry, a total of seven microbes were acquired, all categorized under the genus Bacillus and labeled as P1–P7.

The P1 and P3 strains, genera of Bacillus, exhibited the most significant chromium bioreduction when exposed to a 1 and 0.5% inoculum concentration, respectively, as illustrated in Table 2. The rate of degradation of Cr (VI) by Bacillus species (P1 and P3 strains) was more pronounced at lower incubation time with respect to the other species. The study from the previous literature also indicated that the commonly used species for chromium reduction is Bacillus sp. (Monica et al. 2011). It can be concluded that the P1 and P3 strains have maximum degradation potential for the chromate metabolism that reduces Cr (VI) to Cr (III) more efficiently with respect to the rest of the isolates. Similar to the results of Biradar et al. (2012), it was found that the rate of chromium reduction increased with the increasing time interval for all the bacterial isolates. In another study conducted by Ge et al. (2013) higher inoculum volume failed to elicit a further increase in Cr (VI) reduction, suggesting that competition for limited nutritional resources restricts bacterial metabolic activity. The conversion of hexavalent chromium into its trivalent form is considered a detoxification mechanism due to the increased stability and reduced toxicity of Cr (III). Recent research has revealed the presence of extracellular chromate reductase activity in these bacteria (Shindhal et al. 2021). Various chromate reductases, including ChrR, YieF, NemA, and LpDH, were identified either in the soluble cytoplasmic fraction or associated with the membrane. These enzymes facilitate the reduction reaction under aerobic, anaerobic, or sometimes both types of conditions (Fernández et al. 2018).

This research establishes a significant connection between water pollution and industrial discharge, highlighting the detrimental effects of industrialization, modernization, and overpopulation on water quality. The research not only addresses a critical local issue of industrial pollution in India but also offers a scalable solution with international relevance. By leveraging the natural bioremediation potential of indigenous microbial species, this study provides a blueprint for tackling industrial wastewater pollution in various global regions, offering a sustainable and adaptable approach to environmental management. The demonstrated potential of indigenous microbes for Cr (VI) bioreduction and dye decolorization aligns closely with practical applications in industrial wastewater management. By enabling the conversion of toxic Cr (VI) to less harmful Cr (III) and reducing the presence of hazardous dyes, these microbial processes offer an effective, scalable alternative for industries. The study identifies indigenous microbial isolates, including Lactobacillus spp., Streptococcus spp., and Corynebacterium spp., which possess notable potential for biodegradation of textile dyes, particularly carcinogenic and toxic azo dyes. Harnessing indigenous microbes for the bioreduction of Cr VI and decolorisation of azo dyes offers an effective, real-world solution for industrial wastewater treatment. Compared to conventional methods like chemical precipitation, microbial bioremediation is cost-effective, sustainable, and reduces secondary pollutants. Industries, particularly those in the textile and paper and pulp sectors, can lower their environmental impact, reduce operational costs, and comply with stricter wastewater regulations. This approach also supports corporate sustainability goals while improving environmental and public health outcomes for communities affected by industrial pollution. The physico-chemical characterization of effluent from the textile, paper, and pulp industries in Jaipur underscores the severity of water pollution, surpassing permissible limits and necessitating urgent remediation. This research also investigates heavy metal contamination, specifically the pollution of water bodies by hexavalent chromium (Cr (VI)), which is prevalent in the paper and pulp industry. Conventional removal methods have been criticized for their inefficiency, high cost, and environmental impact. The bioreduction of Cr (VI) to Cr (III) has emerged as a promising alternative, offering in situ applicability without endangering human, animal, or environmental well-being. This study positions bioremediation as a superior choice, given its eco-friendliness, versatility, and cost-effectiveness compared to conventional methods. The transition from toxic Cr (VI) to nontoxic Cr (III) through biotransformation using indigenous microbes exemplifies the practical application of this eco-friendly approach. In conclusion, this research not only unveils the potential of indigenous microbial strains for dye and heavy metal remediation but also advocates for the immediate adoption of sustainable practices in the face of escalating environmental challenges. The proposed avenues for future research signal a commitment to advancing eco-friendly bioremediation technologies through genetic and protein engineering tools and a detailed understanding of microbial mechanisms.

The present research study is funded by the Department of Science and Technology (DST), Government of Rajasthan (Grant number – 7(3)/V. Pro./R&D/2016/2712).

S. A. (corresponding author) conceptualized the work, investigated the process, provide resources, manuscript writing and editing. S. S. provides the experimental design, protocol standardization, and experimental conduction, rendered support in data analysis. S. V. wrote an original draft, rendered support in data analysis. S. S. provides the experimental design, protocol standardization, and experimental conduction. A. S. provides the experimental design, protocol standardization, and experimental conduction. Y. S. provides the experimental design, protocol standardization, and experimental conduction. R. K. S. provides the experimental design, protocol standardization, experimental conduction. R. M. provides the experimental design, protocol standardization, and experimental conduction.

The study group would like to acknowledge the constant support received from Dr B. Lal Gupta (Director, Dr B. Lal Institute of Biotechnology, Jaipur) and Dr Aparna Datta for inspiring this research and providing daily motivation to work faster.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.