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Article

Biological Purification of Heterogenous Car Wash Effluents: Selection of Tolerant Bacteria and Development of Microbial Consortia for Pollutant Biodegradation

by
Katarzyna Starzec
,
Paulina Supel
and
Paweł Kaszycki
*
Faculty of Biotechnology and Horticulture, Department of Plant Biology and Biotechnology, University of Agriculture in Kraków, Al. Mickiewicza 21, 31-120 Kraków, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(18), 8414; https://doi.org/10.3390/su17188414
Submission received: 20 July 2025 / Revised: 6 September 2025 / Accepted: 15 September 2025 / Published: 19 September 2025
(This article belongs to the Special Issue Extremophilic Plants and Microorganisms in Environmental Sustainability)
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Abstract

Car wash wastewaters (CWW) bring growing environmental challenges due to the increasing number of vehicles worldwide and they require novel, optimized and sustainable treatment methods. They are highly heterogenous, typically containing complex mixtures of detergents, waxes, oils, petroleum derivatives, corrosion inhibitors and salts, with the composition depending on installation age, geographic location, season, and weather. This study aimed to select bacteria resistant to variable and potentially toxic CWW, capable of biodegrading organic pollutants. A total of 81 strains isolated from various environmental sites were screened for tolerance to CWW environments by performing growth inhibition tests in 20 real wastewater samples with chemical oxygen demand (COD) ranging from 122 to 2267 mg O2/dm3. Seventeen strain candidates were chosen, identified with molecular proteomics, and further evaluated for biodegradation potential. Based on the most robust isolates, six microbial consortia were developed and examined. Biodegradation experiments were conducted at ambient temperature without active pH control and nutrient supplementation to reflect real conditions occurring in wastewater treatment practice. The best-performing consortium reduced COD by 86% within 7 days. These findings should help improve the treatment of complex CWW by highlighting the potential of thoroughly selected bacteria as effective tools for bioremediation of extremely harsh environments.

1. Introduction

Within recent years there have been a consistent increase in the number of automobiles on the global road network. According to the data provided by the European Automobile Manufacturers’ Association (ACEA), there was an average of 659 motor vehicles per 1000 inhabitants in Europe in 2022 [1]. Despite the observed regional shifts in automotive industry and focus on electrification it is assumed that new vehicle sales will continue to grow with the dynamics of approximately 1.1% per year till 2040 [2]. Earlier predictions indicate the global motorcar number will reach 2 billion by the year 2040 [3]. The water consumption when washing one passenger vehicle is estimated to be approximately 150 L, whereas washing a truck may require 350 to as much as 600 L of water [4,5]. Given the huge number of vehicles worldwide and the extent of car wash operations, the daily water consumption can be estimated as over one million cubic meters [6]. Survey-based research has shown that the average car owner washes their vehicle approximately 11 times per year [7]. Therefore, it is imperative to take appropriate measures to meet the growing global demand for water in the context of increasing scarcity of water resources. At present, a considerable proportion of freshwater suitable for consumption has been contaminated or managed in a manner not conducive to sustainability [8,9,10]. One potential solution to the abovementioned issues is the proper recovery of water from car wash effluents. The problem of car wash wastewater treatment and reuse has been particularly well recognized in Germany and Austria recently [6]; however, it has also attracted considerable attention worldwide, both in terms of basic or model research studies and technological implementations. This growing interest is well documented by a systematic, bibliometric survey of Alazaiza et al. [11] as well as by other thorough review papers [12,13,14,15,16].
Car wash wastewater (CWW) is a complex mixture of numerous pollutants and it can pose severe threat to the environment. It is particularly difficult to treat since both the qualitative and quantitative composition varies significantly and is contingent on several different factors, including the lifespan of the installations, their geographical location, the season, and meteorological conditions [12,15,17,18,19]. The data obtained from 31 cities in 21 countries have confirmed the remarkable wastewater variability [20]. The CWW content is most often dominated by suspended solids, detergents and oils or greases [18,20]. In addition, the presence of slush salt, metal ions as well as many other chemical additives (anticorrosive agents, ammonia, phenolics, waxes, lubricants, dyes, fragrances, volatile organic compounds, etc.) may contribute to the general nuisance of this effluent [21,22,23]. The range of suspended particulate matter was found to be between 68 and 2928 mg/dm3, while the range of oil and grease concentrations was between 12 and 325 mg/dm3 [15]. The greatest discrepancies were identified during the analysis of the organic load, as determined by the chemical oxygen demand (COD). This parameter ranged from 85 to 14,133 mg O2/dm3 [15].
It is evident that many CWW constituents, especially heavy metals and organic pollutants, have the potential to exert detrimental effects on both human health and the environment [11]. Ecotoxicity can often result in the degradation of marine habitats, loss of biodiversity, aquatic eutrophication, accumulation of metals in the food chain, and alterations in ecological balance [24]. The potentially toxic effects of CWW on microorganisms are of particular importance since they can be lethal or inhibitory to activated sludges or bacterial biofilms, and thus hinder or prevent biological treatment.
Considering the aforementioned issues, it is of great importance that the CWW treatment processes be adjusted to extreme values of the changeable parameters. CWW purification and recycling methods have been reviewed by many authors [6,11,12,13,14,16]. They are mostly based on various mechanical, physical and chemical techniques whose effectiveness towards CWW often require elaborate and relatively expensive processes [13,15,20,23] such as electrochemical treatment, membrane ultra- or nanofiltration, reverse osmosis, Fenton reaction or advanced oxidation [12,13,15,18,25].
It should be pointed out here that biological methods, generally regarded as less costly, may also be utilized as an alternative [16]. These methods are based on physiological and biochemical activities of living organisms such as plants used to create model rain gardens (mesocosms) or constructed wetlands [26,27,28], and more often, microorganisms. The latter ones can be employed for CWW bioremediation in several different ways: as specialized biocenoses in biological treatment chambers [29,30,31], floc-forming activated sludges [29], immobilized bacterial strains [32], or complex biofilms developed on bio-carriers [19,33,34] in elaborate bioreactors [17]. Among the most efficient facilities are membrane bioreactors (MBRs) [6,16,29,35,36] where biological treatment is combined with the membrane filtration process. The MBR technology relies on the flow connection of anaerobic, anoxic and aerobic tanks with controlled aeration units and parameter monitoring such as pH and temperature. These systems are designed so as to stimulate bacterial growth under specific conditions occurring in bioreactors [16,35].
While considering various CWW treatment strategies, it is important to note that biological approaches are much less frequently represented in the literature as well in laboratory and environmental practice [11,12,13,14]. Therefore, for the case of microbial remediation, there is a strong need to select robust bacterial strains tolerant to CWW diversity, resistant to potentially toxic substances, and revealing unique catabolic pathways. These bacteria would then be used for development of specialized microbial consortia biochemically capable of metabolizing broad spectrum of contaminants. Such consortia might be used as inocula in bioreactors to launch biological processes and then effectively biodegrade the organic load as well as reduce the level of biogenic elements.
The objective of the present study was to isolate bacterial strains from various environmental habitats including car wash effluents and polluted sites, followed by selecting and testing the most efficient candidates for treatment of several different CWW samples. The most suitable isolates were then chosen to construct variant microbial bacterial consortia to achieve synergy in CWW bioremediation, and thus to enable efficient effluent purification and water reuse.

2. Materials and Methods

2.1. Car Wash Wastewater

A total of 20 car wash wastewater (CWW) samples were collected at various car washes located in southern Poland during different operational phases and seasons. All samples were first analyzed for the content of organic pollutants by determining the chemical oxygen demand (COD) parameter which was used as an indicator of the total amount of organic matter. The measurements were performed based on the standardized potassium dichromate method [37] with an automated, spectrophotometric analytical module Hach-Lange (Hach-Lange GmbH, Düsseldorf, Germany) using commercial LCK cuvette test in combination with a Hach DR 5000 UV-Vis spectrophotometer and a sample digestion kit (Hach Dry LT200 Thermostat, Düsseldorf, Germany). Since the research was focused on analyzing general bacterial biodegradation potential, no individual organic pollutants nor other CWW characteristics were determined throughout most of the CWW utilization and biodegradation experiments, except for the CWW treatment test carried out in the model system employing fixed-bed bioreactor inoculated with a bacterial consortium. In this test, anionic surfactants were quantified using a Hach-Lange module with the appropriate LCK cuvette test kits as described above and total suspended solids (TSS) were assessed gravimetrically by filtration through glass fiber filters, in accordance with PN-EN 872:2007+A1:2007 [38]. The other CWW indicators, namely specific electrical conductivity (SEC), 5-day biochemical oxygen demand (BOD5), and effluent turbidity, were determined following standardized protocols as in Mazur et al. [34].

2.2. Bacterial Screening

Eighty-one bacterial strains were used to select microbial candidates revealing bioremediation potential towards CWW. The strains were isolated from different environmental habitats, especially the sites polluted anthropogenically. Among these were 38 strains tolerant to crude oil processing products or to selected petroleum-derived substances, 10 strains isolated from soils contaminated with pesticides, 6 from activated sludges of wastewater treatment plants (WWTPs), 5 obtained from waters contaminated with mono- and polyaromatic hydrocarbons, 7 derived from brown coal deposits, 3 isolated from heavy metal-contaminated soils, and 3 from soils with an elevated salt content. In addition, eight strains were identified as autochthonous microorganisms present in car wash effluents and one was derived from agricultural soil.
All the isolates were stored in the microbial collection of the Biochemistry Team, Department of Plant Biology and Biotechnology, University of Agriculture in Krakow. In a set of preliminary tests, most of the listed bacteria proved to be able to reveal unique biochemical degradation pathways of selected xenobiotics.
Microbiologically pure strains were typically cultured in 50 cm3 of an enriched SNB liquid medium (Standard Nutrient Broth, Biocorp, Warsaw, Poland) in 300 cm3 Erlenmeyer flasks, rotary-shaken (150 rpm) on orbital laboratory shakers at room temperature with passive aeration. Pathogenic strains were excluded from analyses by growth evaluation on selective media such as Endo LES Lab Agar, Salmonella-Shigella Lab Agar, Pseudomonas CN Lab Agar, or Bacillus cereus Lab Agar (all from Biomaxima, Lublin, Poland).
Bacterial candidates were screened for their tolerance to various CWW environments based on the growth inhibition (survivability) tests carried out in the presence of particular CWW. Each tested bacterial culture was pre-grown for three days on SNB medium as described above to reach the post-logarithmic phase of highly dense population (of the order of 107–108 CFU/cm3). Then, the microbial biomass was washed by centrifugation at 5200× g for 10 min and resuspension of the pellet in Bushnell Haas minimal medium (BH, Biomaxima, Lublin, Poland). After the second round of centrifugation and decantation of the supernatant, the resultant cell pellet was inoculated by resuspending directly into 50 cm3 of a given CWW sample and incubated for 7 days. Next, a 100 mm3 aliquot of the suspension was spread onto bacterial solid agar plate. Growth inhibition was evaluated based on the visual comparison of the number of developed bacterial colonies with the control cultures cultivated at non-toxic conditions (BH). The results were classified into three categories: (1) normal growth observed; no inhibition relative to control conditions; (2) partial inhibition: visible reduction of growth (~50%) compared to the control; (3) total inhibition: lack of visible colonies on plates. Based on the above test, only those bacteria that did not show any growth inhibition (category 1) in all the examined CWW were selected and subjected to further studies.

2.3. Bacterial Identification

The selected microorganisms underwent species identification with the Bruker Biotyper® analyzer, i.e., molecular proteomics automated next generation microbial identification system (Bruker Daltonics GmbH & Co. KG, Bremen, Germany). The method is based on protein profiling with the Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI TOF MS). The proteomic data profiles were for each strain matched with a Bruker data base MBT IVD Library version K (release 2020) and microbial identification accuracy was evaluated as described in detail elsewhere [39,40].

2.4. Bacterial Frequency Analyses

Bacterial abundance was monitored using a modified serial dilution method based on the Koch’s technique [41]. In brief, subsequent geometric dilutions of bacterial culture suspensions were surface-plated onto Petri dishes containing microbial growth media solidified with 2.5% enriched agar (Biocorp, Warsaw, Poland). Colony-forming units (CFUs) were macroscopically evaluated and counted after 3-day incubation at room temperature, and the resultant cell numbers were expressed as CFU per cm3 of the original tested sample.

2.5. Car Wash Wastewater Utilization with Bacterial Monocultures

All the CWW-tolerant bacteria were tested for their ability to utilize organic compounds present in the effluent. This potential was assessed based on analysis of culture growth for 7 days after inoculation with bacterial suspension initially diluted 1000-fold. At the same time the change of the COD parameter was determined as indicative of the biodegradation yield. Both the bacterial population increase and COD removal rate were assumed as parameters proving utilization of CWW components that served as sole sources of carbon and energy for culture proliferation. The monocultures were pre-grown in optimal media and then washed twice as described above. The cell pellets were then inoculated to a given sterile CWW sample so as to obtain the initial biomass density of the order of 104–105 CFU/cm3. After 7-day cultivation, the viability (frequency) of bacteria as well as COD were assessed, then both parameters were compared with the initial values. Uninoculated, sterilized CWW samples served as controls.

2.6. Construction of Bacterial Consortia for CWW Treatment and Biodegradation Tests

Upon choosing the strains that showed the highest bioremediation potential, six variant bacterial consortia were constructed and tested for CWW treatment. They were derived by combining equal volumes of the selected monocultures previously grown to high densities and further cultivating the integrated biocenoses.
All the biodegradation tests were conducted applying three sterilized CWW samples collected at three independent treatment stations to represent considerable differences in the initial load with the organic fraction. The biomass of each consortium (50 cm3 volume) was washed twice as described earlier, centrifuged again and then the pellet inoculated into an equal volume of a given CWW. Fully aerobic conditions were provided by vigorous rotary-shaking the flasks with microorganisms, which enabled efficient passive aeration of the cultures. An independent control experiment involved uninoculated, earlier sterilized wastewaters to rule out any significant contribution of the self-attenuation process.
The analyses of bacterial abundance and COD measurements were carried out at least in triplicates and the results were given as mean values ± standard deviation. The COD data in the CWW samples were subjected to statistical analysis using the ANOVA module of Statistica 13.5 (TIBCO Software Inc., Palo Alto, CA, USA). The significance of differences was assessed employing a Tukey HSD test at an assumed probability level of p < 0.05.

3. Results

3.1. Heterogeneity of the Organic Load

The wastewater samples analyzed in this study were shown to be highly heterogeneous in terms of the organic load and exhibited significant variation of the COD values (Figure 1). The lowest measured COD level was assessed as 122 mg O2/dm3, while the highest one reached 2267 mg O2/dm3, which indicates a wide range of concentrations and possible qualitative differences in organic contamination across the samples. The COD value discrepancies were further confirmed by a more detailed variance analysis that indicated statistical differences between particular samples at p < 0.05 as depicted by different letters over bars in Figure 1. Note that the complexity of the studied CWW samples was not investigated in detail in this work and more information on the composition and quality of these effluents can be found in other studies where similar CWW were used [19,33,42].

3.2. Testing CWW Self-Attenuation with Indigenous Microbiota

Autochthonous microbial strains were observed to colonize the collected CWW. In the analyzed samples, the abundance of aerobic bacteria ranged from 1 × 104 to 5 × 106 CFU/cm3. The number of anaerobic bacteria was generally higher, ranging from 1 × 105 to 5 × 107 CFU/cm3. Microscopic fungi were detected only in two samples, with counts of the order of 103–104 CFU/cm3. Bacterial biodiversity was relatively high, with the number of distinct morphotypes ranging from 6 to 13, depending on the sample. Pathogenic strains were also identified. Bacteria from the coliform group including Escherichia coli as well as Salmonella spp. were detected in the majority of the samples. Pseudomonas aeruginosa was the least frequently observed pathogen, yet it was still present in more than half of the samples.
It was therefore of interest to verify whether the indigenous strains would initiate biodegradation of organic pollutants under aeration conditions. In addition, the presence of the mechanism of spontaneous oxidation of some organic compounds could not be excluded upon vigorous shaking that allowed for oxygen penetration. To assess any possible contribution of self-purification processes, an independent test was conducted with 10 selected, non-sterilized and uninoculated CWW samples representing low (no. 7, 17), medium (11, 16, 20), and high (8, 12, 13, 14, 18) initial COD levels.
As shown in Figure 2, the CWW self-attenuation was generally negligible upon a 7-day aerobic incubation of raw effluents. Only in two cases of CWW 14 and 18, where the initial COD levels were the highest (≥2000 mg O2/dm3), an approximately 25% spontaneous decrease of the organic content was observed. However, in all other samples, the changes in organic pollutant concentrations were not statistically significant after incubation and such results justify the necessity of using allochthonous bacteria inoculation to launch efficient biodegradation.
Taking into consideration the high heterogeneity of the investigated wastewaters, a total of 81 strains originating from different environmental sites were used for selection of bacteria tolerant to variable qualitative and quantitative CWW composition. To assess survivability, all bacterial monocultures were incubated in three chosen CWW samples characterized by the COD values of: 246 mg O2/dm3 (sample 6), 1320 mg O2/dm3 (sample 8), and 1500 mg O2/dm3 (sample 10). Given the complex nature of these effluents, variable viabilities of individual strains were observed in the samples collected at different car washes.
All three examined CWW samples were toxic to most of the strains causing strong growth inhibition. Due to the objective of our study, it was essential to select only the strains that demonstrated resilience against all CWW variants. Seventeen strains, including both Gram-positive and Gram-negative bacteria, were found as the most promising candidates, whose survival was not negatively affected by any CWW variant (Table 1). No correlation was observed between the cell wall thickness and the resistance against toxic CWW components (Table 1).
The CWW-tolerant strains were then subjected to testing in order to ascertain their growth as well as organic-compound utilization potential upon 7-day incubation in the representative CWW samples. Three effluents were chosen, which differed in their initial organic pollutant load, namely samples 3, 8, and 18, characterized by COD of 215, 1320, and 2000 mg O2/dm3, respectively. The final biodegradation yields expressed as percentages of COD removal rates are delineated in Table 2. All 17 strains proliferated after inoculation of 1000-times diluted cultures to particular sterilized CWW (the initial density of the inoculated biomass was of the order of 105 CFU/cm3). Depending on the strain, bacterial abundances increased by one to four orders of magnitude at the end of incubation, with the highest value of 8.2 × 108 CFU/cm3 achieved by Cupriavidus necator G4 in CWW 18, the isolate performing relatively well in all tested CWW. Since no additional nutrients were supplemented, the observed biomass growth seemingly resulted from utilization of the accessible carbon sources present in the effluents. However, the effectiveness of a single strain-dependent biodegradation varied considerably for different CWW samples.
For instance, Cellulomonas pakistanensis OW1, Ochrobactrum anthropi ZB1, Bacillus pumilus R2, and Priestia megaterium G7 demonstrated no detectable degradation of pollutants in CWW 3 (COD = 215 mg O2/dm3), yet they attained a 34%, 36%, 29%, and 29%, degradation rate in CWW 8 (COD = 1320 mg O2/dm3), respectively, and an even more notable respective degradation yields of 78%, 71%, 80% and 74% in CWW 18 (COD = 2000 mg O2/dm3). Conversely, Pseudomonas brassicacearum G8 and Pseudomonas koreensis G6, very efficient biodegraders in CWW 3, exhibited substantial loss of degradation efficiency in CWW 8 and CWW 18, characterized by increased COD values. Two strains, Alcaligenes faecalis SC7 and Lysinibacillus fusiformis OC2, exhibited no COD removal activity in the effluent with the highest organic load (CWW 18). For the majority of other bacteria, the degradation levels obtained in CWW 8 and CCW 18 were comparable when the respective COD removal rates observed in the uninoculated control samples were subtracted (Table 2).
Importantly, no single microbial strain was found to exhibit consistently high degradation efficiency across all the tested CWW samples. The variability in pollutant composition and concentration among the samples likely contributed to the differential performance of particular strains, highlighting the pressing need for developing a universal and efficient biodegradation solution for such heterogeneous wastewaters. Therefore, we hypothesized whether the combined complex microbial communities would demonstrate higher capacity to degrade contaminants over a shorter time period. For that reason, six different consortia varying in bacterial components were produced and then tested (Table 3).
Biodegradation potential of the consortia was assessed in three car wash effluents selected to represent intermediate COD levels, that is, samples 11 (COD = 651 mg O2/dm3), 16 (COD = 635 mg O2/dm3), and 19 (COD = 718 mg O2/dm3). The effluents were inoculated with the six consortia (K1–K6) at high initial biomass densities and then treated for 7 days. The results are presented in Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8. Figure 3, Figure 5 and Figure 7 reveal bacterial population dynamics of the consortia during their incubation in the CWW samples 11, 16, and 19, respectively. The charts clearly show that in all cases bacterial abundances remained stable throughout the experiment or even tended to grow (see the population changes observed for K6). These results indicate again effective utilization of some CWW components as sources of carbon and energy for the cells. Figure 4, Figure 6 and Figure 8 document the biodegradation data expressed as changes in COD values during effluent treatment (CWW 11, 16, and 19, respectively).
Despite the fact that the initial COD values were comparable for the three CWW samples examined, notable differences in degradation efficiencies were observed for the variant microbial consortia. Consortium K4, composed of five bacterial strains, proved to be the most effective and achieved a consistent 7-day degradation efficiency of over 80% across all the samples, with the highest yield (86%) observed for CWW 16 (Figure 6). Moreover, this consortium was capable of removal of significant COD fraction (53%) in CWW 11 already within the first day of incubation (Figure 4).
Consortium K5 was also performing relatively well, although showed evident variability in degradation efficiencies depending on the wastewater sample. The lowest degradation rate (52%) was observed for the sample 16 (Figure 6), while the highest yield reached 86% in CWW 19 (Figure 8).
In contrast, consortium K2 exhibited the least effective biodegradation. Astonishingly, the combined five strains that formed this consortium displayed reduced activity compared to individual monocultures (cf. data in Table 2). The final degradation rates were 47% for CWW 16 (Figure 6) and 39% for CWW 19 (Figure 8), but only 2% for the sample 11 (Figure 4).
It is to note that the capacity for degradation of contaminants did not correlate with the number of strains present in bacterial consortia. For example, consortium K3, which contained the highest number of strains (11), exhibited only moderate bioremediation activity (cf. Figure 4, Figure 6 and Figure 8).
In order to verify the applicability of the selected bacteria for removal of pollutants occurring in CWW, the treatment of the CWW sample 16 was carried out with the best-performing consortium K4. The bacteria were inoculated to a laboratory-scale fixed-bed, vertical-flow bioreactor consisting of columns filled with a polyester fiber. The flow rate of 1200 cm3 non-sterilized CWW per day was controlled with a peristaltic pump. The specimens were collected after 7-day treatment. Since the content of heavy metals and biogenic elements in CWW 16 was found not to exceed the permissible levels for discharged wastewaters, the following parameters were assessed: COD, BOD5, concentration of anionic surfactants, turbidity, total suspended solids, and electrical conductivity. The results presented in Table 4 confirm the biodegradation activity of the microbial consortium. While the COD value decreased by 78%, the BOD5 representing the biodegradable organic fraction was reduced by 83% and the removal yield of anionic surfactants was over 91%. The observed high efficiency of turbidity reduction as well as removal of TSS (92% and 96%, respectively) was most probably by the action of the polyester fiber used to fill the columns of fixed-bed reactor. In turn, the observed lack of conductivity change implies the necessity to use an additional physical technique to eliminate salinity.

4. Discussion

Biological wastewater treatment is known as an efficient, sustainable, environment-friendly and economically favorable method for removing pollutants [43]. In order to enhance water purification potential, bio-based technologies are often integrated with other strategies that rely on physical–chemical processes [43]. The combined treatment systems are especially relevant for the case of industrial effluents where the application of biological methods solely may not be efficient enough [25,44]. Such wastewaters usually contain specific, mostly nuisance and often toxic contaminants and therefore require optimized processing. Car wash wastewaters (CWW) are particularly troublesome for their complexity, elevated content of solids and high turbidity, chemical variability, risk of toxicity, as well as changeable load dependent on numerous conditions [14,18,20]. In addition, modern car washes should operate so as to ensure continuous utilization of the recycled water, which requires reduction of the effective time of effluent treatment to approximately 20–24 h. This relatively short hydraulic retention time (HRT) was determined by Włodyka-Bergier et al. [19] and Mazur et al. [34] based on model calculations regarding water consumption and flow during car wash process. As a consequence, the CWW treatment systems must be developed and optimized so as to shorten the purification time while maintaining the best water quality characteristics. Proper CWW treatment is thus a challenging task since it has to deal with multiple parameters simultaneously and it is very difficult to develop a sufficient, self-contained method based on biological component only [13,20,24]. In fact, CWW are not suitable for biological remediation since they typically suffer from markedly lowered BOD/COD ratio [12,13,20]. In most cases this parameter drops below the critical value of 1/3, which causes the organic fraction difficult to metabolize by microbes. The problem of suspended solids, colloids and turbidity might be solved by applying bioflocculant-producing microorganisms in a way analogous to that tested and optimized by Pu et al. [45]. The authors isolated and selected a soil-colonizing fungal strain of Aspergillus niger capable of synthesizing a bioflocculant used for successful treatment of potato starch wastewater. Unfortunately, such an approach has not been reported for CWW, yet.
For the reasons given above, the stand-alone microbiological-based systems have rarely been proposed. Among the successful attempts, although tested on the laboratory-scale only, the work of Khondee et al. [32] is worth noting, in which an internal loop airlift bioreactor containing chitosan-immobilized Sphingobium sp. was applied to remove lubricants present in emulsified CWW. The authors established that the observed high removal rates were due to both sorption mechanism and biodegradation by immobilized bacteria. Also, the systems based on the moving bed bioreactors (MBBR) [46] have been proposed as promising solutions. They were first introduced for CWW purification by Mallick and Chakraborty [31] who reported an interesting study on car service station wastewater bioremediation with a set of connected sequentially-performing bioreactors: an anoxic disc bed reactor and a downstream aerobic MBBR. A kinetic study was conducted to determine the minimum HRT, assess degradation yield of phenol and hydrocarbons, and to optimize biological removal of ammonia and nitrates. The resultant treatment parameters were satisfactory due to strong performance of the bacterial community. From the biomass of both anoxic and aerobic reactors, four strains of Pseudomonas aeruginosa as well as Lysinibacillus sp. and Stenotrophomonas sp. were isolated and then positively tested for xenobiotic degradation and/or ammonia utilization. Later, the MBBR applicability for CWW treatment was researched in a more detail by Włodyka-Bergier et al. [19]. The authors conducted systematic studies on optimizing the quality and performance of microbial biofilm formed on biological carrier fillings and fittings. After establishing best temperature conditions and aeration rigor [42], microbiologically enriched biofilms were tested after bioaugmentation with allochthonous strains [34] (see below). Nevertheless, it was concluded that due to the low biodegradability of CWW, an additional preconditioning step should be included to achieve the desired water qualities within the reduced HRT.
More frequently, biological treatment has been employed as a method complementary to the chemical and physical ones, aiming at enhancing the overall process efficiency [24]. The integration of both methodologies usually yields numerous advantages [15,44]. The predominant function of microorganisms in wastewater treatment lies in biotransformation or biodegradation of the organic fraction as well as removal of biogenic elements [26,47,48]. The introduction of the chemical–physical processes is crucial for elimination of other recalcitrant contaminants and improvement of water quality parameters, namely removal of suspended and dissolved solids, desalinization, reduction of turbidity and decolorization [13,14,15,18]. It was demonstrated that approaches combining the action of microorganisms together with ultrafiltration [17], microfiltration [36], ozonation, or coagulation [35] led to achieving purification rates that exceeded 95%. These results showed substantially improved efficacy compared to using solely physical or chemical methods. Importantly, CWW pretreatment with advanced oxidation processes was shown to enhance biodegradability of CWW organic content by increasing the BOD/COD ratio, which led to shortening HRT [19]. In turn, Hsu et al. [33] underscored the significance of combining diverse treatment technologies and proposed an efficient hybrid BioMF system that incorporated bio-carriers and non-woven membrane filtration. Moazzem et al. [36] drew attention to applicability of the enhanced membrane bioreactor (eMBR) that allowed for obtaining high quality recyclable water upon CWW treatment. In their model system, an anaerobic tank was connected to an anoxic tank and then to an aerobic membrane bioreactor (AMBR) in which the activated sludge was aerated with a diffuser. After membrane filtration process followed by UV disinfection, the purified water could be redirected for car wash purposes.
Note that in most of the cases cited, the authors focused on establishing treatment process parameters rather than on monitoring active microbiota content and performance. Moreover, the microbiological component in the examined systems was typically acquired passively as captured autochthons along with the inflowing wastewater or was adopted after implementation of non-specific activated sludges from municipal or domestic wastewater treatment stations. It is important here to mention the study of Do et al. [30] who presented data on bioreactor inoculation with allochthonous microbial communities and showed their preliminary characteristics. The so-called “seed” microbial sludges were collected from three sources: a municipal WWTP, an airlift MBR for treatment of slaughterhouse wastewater, and from drainages receiving CWW. These mixed bacterial consortia were then independently introduced into the tested MBR bioreactor to treat oily CWW. Although neither bacterial frequencies nor consortial structures were studied, the performance of each community was characterized by determination of the sludge hydrophobicities, oxygen uptake rates and COD removal rates.
Previously, we participated in MBBR testing by implementing allochthonous pre-grown environmental bacteria as inoculants for improving biofilm performance on biochip carriers [34]. The results showed that bioaugmented reactors did not significantly upgrade the treatment parameters compared to the controls (bioreactors colonized spontaneously). However, the observation time was limited only to 20 h of HRT, which was clearly a time too short for complete biodegradation. Moreover, the selection of strains used for inoculation was not based on the screening of their tolerance nor on testing their activity in the CWW environment. In addition, successive batches of the effluent may have differed with regard to both load and content as the samples were collected at car washes at different times.
The aim of the present work was to select robust bacteria upon systematic screening in representative car wash wastewaters of different content and toxicities. Then, the best candidates were chosen to construct variant bacterial consortia and test their potential application in CWW treatment. To the best of our knowledge this is the first reported successful attempt that made it possible to obtain a collection of tolerant and highly specialized strains suitable for CWW bioremediation. Furthermore, the developed versatile microbial consortia are unique for revealing their activity and potential use in different CWW treatment installations under highly variable and often extreme conditions.
Biochemical activity of the selected microbes was determined upon testing biodegradation potential of the organic CWW fraction represented by the chemical oxygen demand (COD). This parameter was chosen to evaluate the CWW quality and to monitor treatment bioprocess since it is generally considered as a simple and reliable measure directly reflecting the degree of organic contamination in waters. It is defined as an amount of the oxygen required to oxidize pollutants. As per the standard method, quantification of the level of organics is most often carried out using potassium dichromate as an oxidizing agent [49,50]. Many monocultures were shown to degrade pollutants only in individual CWW samples and therefore it was concluded that relying solely on single microbial strains was not a suitable strategy for effluent treatment. Development of mixed bacterial communities was a more reasonable option as the construction of universally applicable consortia was considered especially challenging taken the substantial qualitative and quantitative CWW variability. These consortia were composed so as to adapt and actively perform in CWW samples collected from car washes under different operational conditions aiming at capturing a broad range of parameters. It has to be noted, however, that the tested effluents cannot be regarded as representative of all regions worldwide. This is because of regional differences encompassing specific economic, technological, geographical/climatic, seasonal and other factors. A comprehensive screening of CWW obtained from numerous sites carried out by Kuan et al. [15] showed the great variability of all parameters and indicators. Therefore, the developed consortia need additional testing in particular CWW coming from different regions prior to application.
It is generally accepted that microbial consortia are beneficial and superior to the use of single strains. They are especially applicable for pollutant removal from wastewaters, known to expedite biodegradation rates upon cooperation of constituent microorganisms. Most bioprocesses including remediation of complex waste materials or toxic chemical pollutants were demonstrated to be remarkably intensified by the use of metabolically active mixed communities of microorganisms, as opposed to monocultures [51]. This phenomenon can be attributed to the positive interactions occurring within the multispecies consortium, which lead to synergistic enhancement of the overall metabolic activity resulting from the contribution of individual microbial partners [51]. A notable instance of commensalism was observed while studying the relationship between the naphthalene-degrading bacterium Pseudomonas sp. AS1 and the soil bacterium Acinetobacter oleivorans DR1 [52]. The latter strain lacked the naphthalene-degrading enzymes which caused eventual population death in the naphthalene-containing medium. However, when this bacterium was co-cultured with Pseudomonas sp. AS1 in the medium amended with naphthalene, it could survive and grow by utilizing salicylate, a metabolic intermediate produced by Pseudomonas sp. AS1 through naphthalene degradation. Dejonghe et al. [53] demonstrated the importance of adjusting and defining an optimal consortium composition to increase its effectiveness in terms of contaminant bioremediation. The authors tested fourteen distinct bicultures composed upon microorganism pre-selection on the basis of specific criteria, which finally enabled them to degrade petroleum hydrocarbons. While considering the stability of complex microbial communities, also antagonistic interactions were shown to play a pivotal role in maintaining ecological balance [54]. In addition to the above, the influence of environmental factors, such as nutrient concentrations, pH, and the presence of organic pollutants, on the structure and activity of microbial consortia in aquatic environments should be considered. In car wash wastewaters, characterized by variable pollutant levels, especially surfactants, hydrocarbons, and nitrogen compounds, highly dynamic conditions are created that exert strong selective pressure on microbial communities. This pressure can modulate the community composition, favoring taxa with specific metabolic capabilities and stress tolerances. Recently, Pang et al. [55] demonstrated that fluctuations in water quality parameters, particularly nitrogen content, directly affected microbial diversity and functional gene expression. Their study showed that genera such as Pseudomonas, Hydrogenophaga, and Flavobacterium became dominant under elevated N conditions due to expression of genes involved in nitrogen transformation. A similar pattern is expected for car wash effluents, where microbial consortia should restructure in response to pollutant loads, enabling functional resilience and effective biodegradation under changing conditions. However, in the present study the detailed structural dynamics of inoculated consortia was not traced upon CWW treatment since the research was primarily focused on the development of defined microbial communities and on testing their performance.
According to the results of this article, from among the six examined consortia, K4 exhibited the highest overall bioremediation potential by showing satisfactory degradation efficiencies across all wastewater batches. The final degradation yields achieved by the best performing communities K4 and K5 reached 86%, although an extended incubation time was required (4–7 days in the tested batch mode). The collected data on COD reduction does not allow for elucidation of the specific biodegradation mechanisms of organic substances. Xie et al. [56] studied tetracycline-based pollutant removal in a landfill leachate and investigated trends of dissolved organic matter (DOM) by monitoring decomposition of the large molecular structures into medium and small ones and correlating the degree of humification with the COD changes. Such extensive examinations of the DOM composition trends would shed more light on modulation of the activity of microbial consortia as well as bring valuable information on CWW bioremediation trails, yet they require a much more detailed experimental approach.
It is also worth remarking that the selected consortia may not be able to fully mitigate the risk of occurrence of pathogenic microorganisms in real CWW, which makes recirculation of the treated wastewaters difficult. The introduction of a defined and specialized bacterial consortium usually leads to alteration of the microbial equilibrium within the system. This shift may result in partial suppression of pathogenic populations through competitive exclusion and niche displacement. However, the primary objective of bioinoculant consortia is biodegradation of organic pollutants rather than pathogen removal. Therefore, to minimize any risk of pathogenic strain presence, CWW biological treatment facilities are usually equipped with the subsequent units responsible for disinfection (sanitization) of the purified wastewater. This can be achieved by applying membrane filters, ozonation, or ultraviolet irradiation modules [17,36] and is in line with standard wastewater treatment protocols.
The above considerations suggest that the extraordinary biodegradative properties exhibited by thoroughly selected microbes should not be utilized in stand-alone systems, but rather in combined facilities involving physical–chemical processes. These additional elements of purification systems are necessary to support the capacity of biological units by elimination of pathogens as well as enhanced removal of salinity, heavy metals, biogenic substances, and solids, to achieve a shortened HRT and finally make the water reusable.
Among the most prospective solutions, inoculation of aeration chambers, bioreactors, and biocarriers are proposed to enhance the performance of CWW treatment stations. One of such solutions was preliminarily tested on the laboratory scale in a model system based on the fixed-bed bioreactor where the polyester fiber-based columns were colonized with the active bacteria of K4. The treatment system proved efficient in terms of both, removal of the organic fraction including anionic detergents, and reduction of turbidity as well as the content of total suspended solids. We believe that the approach based on bioaugmentation of CWW treatment systems with specialized bacteria will enable development of refined activated sludges or biofilms, thus favoring bioremediation kinetics and elevating the final purification yield. In the next step, further studies are planned to test in detail the specific applicability of chosen consortia to produce potent microbial communities in moving bed bioreactors.

5. Concluding Remarks

The presented systematic research study resulted in obtaining unique bacterial strains tolerant to highly variable, potentially toxic car wash wastewater (CWW) and capable of CWW bioremediation by degradation of organic pollutants. Although single strains may prove applicable for treatment of chosen CWW, they fail to remain viable or active in many different CWW types. Therefore, the strategy of constructing multispecies biocenoses based on the selected strains has been proposed and tested. This resulted in the development of several beneficial microbial consortia able to perform synergistically in various CWW that differed in the chemical content and in the organic load. The consortia designated as the most versatile ones are regarded as well-suited candidates for inoculation of biological units of the CWW treatment stations: activated sludges in aeration chambers or bioreactors of different types. Microbiological treatment of CWW carried out solely may lead to prolonged hydraulic retention time in wastewater treatment plants. Therefore, the bio-based methods have been proposed as components of more complex hybrid systems in which other, physico-chemical treatments play an important role. The results of the study contribute to current trends in CWW treatment, which in general aim at development of universal and efficient technology tailored so as to enable the purified water reuse. The optimized water management and recycling is considered crucial for preserving its natural resources and making the operation of car wash installations sustainable.

Author Contributions

Conceptualization, K.S. and P.K.; methodology, K.S., P.S. and P.K.; validation, K.S. and P.K.; formal analysis, P.S. and P.K.; investigation, K.S., P.S. and P.K.; data curation, K.S. and P.K.; writing—original draft preparation, K.S.; writing—review and editing, K.S. and P.K.; visualization, K.S.; supervision, P.S. and P.K.; project administration, P.K.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Polish National Centre for Research and Development, Smart Growth Operational Programme 2014–2020, Project POIR.01.01.01-00-0636/21 entitled: “Development of technology for an autonomous modular touchless car wash, using dedicated wastewater treatment technology, low-emission washing techniques and renewable energy sources”; Project leader: Marcin Orlik, ECO REDCONST Sp. z o.o.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ACEAEuropean Automobile Manufacturers’ Association
BHBushnell Haas medium
BODBiological oxygen demand
CFUColony-forming unit
CODChemical oxygen demand
CWWCar wash wastewater
DOMDissolved organic matter
HRTHydraulic retention time
MBBRMoving bed biofilm reactor
MBRMembrane bioreactor
NTUNephelometric turbidity units
SECSpecific electrical conductivity
SNBStandard nutrient broth medium
TSSTotal suspended solids
WWTPWastewater treatment plant

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Figure 1. Organic load of tested car wash wastewaters. Different letters indicate significant statistical differences at p < 0.05.
Figure 1. Organic load of tested car wash wastewaters. Different letters indicate significant statistical differences at p < 0.05.
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Figure 2. Chemical oxygen demand (COD) value changes over a 7-day incubation period in raw CWW samples containing indigenous microbial strains.
Figure 2. Chemical oxygen demand (COD) value changes over a 7-day incubation period in raw CWW samples containing indigenous microbial strains.
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Figure 3. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in a car wash wastewater (CWW) sample 11 (initial COD = 651 mg O2/dm3).
Figure 3. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in a car wash wastewater (CWW) sample 11 (initial COD = 651 mg O2/dm3).
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Figure 4. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 11 (initial COD = 651 mg O2/dm3) upon treatment with microbial consortia K1–K6 as monitored by changes of chemical oxygen demand (COD).
Figure 4. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 11 (initial COD = 651 mg O2/dm3) upon treatment with microbial consortia K1–K6 as monitored by changes of chemical oxygen demand (COD).
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Figure 5. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in car wash wastewater (CWW) sample 16 (initial COD = 635 mg O2/dm3).
Figure 5. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in car wash wastewater (CWW) sample 16 (initial COD = 635 mg O2/dm3).
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Figure 6. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 16 (initial COD = 635 mg O2/dm3) upon treatment with microbial consortia K1–K6 as monitored by changes of chemical oxygen demand (COD).
Figure 6. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 16 (initial COD = 635 mg O2/dm3) upon treatment with microbial consortia K1–K6 as monitored by changes of chemical oxygen demand (COD).
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Figure 7. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in a car wash wastewater (CWW) sample 19 (initial COD = 718 mg O2/dm3).
Figure 7. Bacterial population dynamics of microbial consortia K1–K6 during 7-day incubation in a car wash wastewater (CWW) sample 19 (initial COD = 718 mg O2/dm3).
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Figure 8. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 19 (initial COD = 718 mg O2/dm3) as monitored by chemical oxygen demand (COD) changes upon treatment of car wash wastewater (CWW) with microbial consortia K1–K6.
Figure 8. Biodegradation of the organic fraction in a car wash wastewater (CWW) sample 19 (initial COD = 718 mg O2/dm3) as monitored by chemical oxygen demand (COD) changes upon treatment of car wash wastewater (CWW) with microbial consortia K1–K6.
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Table 1. Strains that demonstrated highest survival rates in all tested car wash wastewater samples CWW 6, 8, and 10 (COD levels of 246 mg O2/dm3, 1320 mg O2/dm3, and 1500 mg O2/dm3, respectively).
Table 1. Strains that demonstrated highest survival rates in all tested car wash wastewater samples CWW 6, 8, and 10 (COD levels of 246 mg O2/dm3, 1320 mg O2/dm3, and 1500 mg O2/dm3, respectively).
StrainSource of Isolated Strain(s)
Stenotrophomonas maltophilia SC9
Alcaligenes faecalis SC5
Alcaligenes faecalis SC7		Indigenous strains obtained from car wash wastewater
Alcaligenes faecalis OC1
Lysinibacillus fusiformis OC2		Activated sludges of municipal and industrial wastewater treatment plants
Cellulomonas pakistanensis OW1The soil polluted with a natural crude oil leachate
Ochrobactrum anthropi ZB1
Shewanella frigidimarina ZB2		Polluted ground-water environments, the strains capable of biodegradation of selected petroleum substances
Bacillus pumilus R2Water contaminated with mono- and polyaromatic hydrocarbons
Rhodococcus opacus C11Lignite (brown coal) deposits colonized with bacteria
Kocuria rhizophila G3
Cupriavidus necator G4
Sphingomonas yabuuchiae G5
Pseudomonas koreensis G6
Priestia megaterium G7
Pseudomonas brassicacearum G8		Soils anthropogenically contaminated with products of crude oil processing
Microbacterium aerolatum K18The soil contaminated with pesticides
Table 2. Biodegradation yield of organic pollutants by individual strains in tested CWW samples. Numbers show percentage of COD removal upon 7-day incubation.
Table 2. Biodegradation yield of organic pollutants by individual strains in tested CWW samples. Numbers show percentage of COD removal upon 7-day incubation.
CWW Sample Number
3818
COD215 mg O2/dm31320 mg O2/dm32000 mg O2/dm3
Control (uninoculated, sterilized sample)0125
Stenotrophomonas maltophilia SC9284181
Alcaligenes faecalis SC5287146
Alcaligenes faecalis SC751610
Lysinibacillus fusiformis OC215220
Alcaligenes faecalis OC1115377
Cellulomonas pakistanensis OW103478
Shewanella frigidimarina ZB2163879
Ochrobactrum anthropi ZB103671
Bacillus pumilus R202980
Rhodococcus opacus C11423778
Pseudomonas brassicacearum G8951874
Priestia megaterium G702974
Pseudomonas koreensis G6921579
Sphingomonas yabuuchiae G5771977
Cupriavidus necator G4694477
Kocuria rhizophila G3694477
Microbacterium aerolatum K18105576
Table 3. Bacterial strain constituents (marked as x) of microbial consortia K1–K6.
Table 3. Bacterial strain constituents (marked as x) of microbial consortia K1–K6.
Microbial Consortium
K1K2K3K4K5K6
Stenotrophomonas maltophilia SC9x x x
Alcaligenes faecalis SC5 x x x
Alcaligenes faecalis SC7 x x
Lysinibacillus fusiformis OC2x x x
Alcaligenes faecalis OC1 x x
Cellulomonas pakistanensis OW1 x
Shewanella frigidimarina ZB2 xx x
Ochrobactrum anthropi ZB1 xx
Bacillus pumilus R2x x
Rhodococcus opacus C11 xx
Pseudomonas brassicacearum G8x x
Priestia megaterium G7x x x
Pseudomonas koreensis G6 x x x
Sphingomonas yabuuchiae G5x x
Cupriavidus necator G4 x xx
Kocuria rhizophila G3 xx x
Microbacterium aerolatum K18 xxxx
TOTAL NUMBER OF STRAINS6511579
Table 4. Selected parameters determined for car wash wastewater (CWW) sample 16 treated in a fixed-bed bioreactor inoculated with the microbial consortium K4.
Table 4. Selected parameters determined for car wash wastewater (CWW) sample 16 treated in a fixed-bed bioreactor inoculated with the microbial consortium K4.
IndicatorTime (Days)
07
COD (mg O2/dm3)640.0 ± 249.5138.7 ± 7.6
BOD (mg O2/dm3)186.7 ± 41.631.7 ± 14.4
Anionic surfactants (mg/dm3)4.91 ± 1.20.42 ± 0.02
Turbidity (NTU)552.3 ± 174.344.0 ± 7.6
Total suspended solids
(TSS, mg/dm3)		1166.7 ± 442.246.0 ± 28.5
Specific electrical conductivity
(SEC, µS/cm)		2478.0 ± 614.42582.0 ± 275.9
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Starzec, K.; Supel, P.; Kaszycki, P. Biological Purification of Heterogenous Car Wash Effluents: Selection of Tolerant Bacteria and Development of Microbial Consortia for Pollutant Biodegradation. Sustainability 2025, 17, 8414. https://doi.org/10.3390/su17188414

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Starzec K, Supel P, Kaszycki P. Biological Purification of Heterogenous Car Wash Effluents: Selection of Tolerant Bacteria and Development of Microbial Consortia for Pollutant Biodegradation. Sustainability. 2025; 17(18):8414. https://doi.org/10.3390/su17188414

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Starzec, Katarzyna, Paulina Supel, and Paweł Kaszycki. 2025. "Biological Purification of Heterogenous Car Wash Effluents: Selection of Tolerant Bacteria and Development of Microbial Consortia for Pollutant Biodegradation" Sustainability 17, no. 18: 8414. https://doi.org/10.3390/su17188414

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Starzec, K., Supel, P., & Kaszycki, P. (2025). Biological Purification of Heterogenous Car Wash Effluents: Selection of Tolerant Bacteria and Development of Microbial Consortia for Pollutant Biodegradation. Sustainability, 17(18), 8414. https://doi.org/10.3390/su17188414

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