2021 Volume 12 Issue 2

Pseudomonas sp Isolated from Wastewater and their Interaction with Microalgae


Jessica Pérez-Nava, Fernando Hernández-Aldana, Carmen Martínez-Valenzuela, Antonio Rivera*


During biodegradation processes in Wastewater Treatment Plants (WWTP), bacteria play an important role, and associations of bacteria with microalgae have the potential to remediate wastewater by reducing pollutants. This work aimed to identify bacteria present in wastewater and to analyze their interaction with C. vulgaris to favor their phytoremediation.  The physicochemical and microbiological characterization of the wastewater from the Alseseca-Sur WWTP was carried out, identifying the bacterial isolates, a phytoremediation treatment was performed with C. vulgaris and wastewater as a culture medium, quantifying the proliferation of C. vulgaris in the supplemented culture medium and the wastewater. The physicochemical parameters decreased after phytoremediation, the algae-bacteria system caused a decrease in COD due to its application in symbiosis; Pseudomonas sp. prevailed with 75%; C. vulgaris showed optimal growth when wastewater was used as culture medium, inoculating C. vulgaris in wastewater at 100%, 174 cells/mL were quantified compared to 24 cells/mL of C. vulgaris with bold medium plus drinking water. The phytoremediation process in conjunction with the bacterial load effluent from the Alseseca-Sur WWTP showed a decrease in the values of physicochemical parameters; in addition to increasing the number of cells/mL of C. vulgaris using water from the effluent of the Alseseca-Sur WWTP as a culture medium.

Keywords: Biodegradation, Environmental, Wastewater, Pseudomonas, Microalgae


Biological treatment of wastewater allows for the removal of organic matter and nutrients, enabling its reuse or safe discharge. Bioremediation of wastewater depends in part on the microbial communities present in the bioreactor. Bacteria play an important role in the processes of decomposition and stabilization of organic matter; in wastewater treatment plants the presence of these microorganisms is important due to their participation in the biodegradation process, highlighting their activity in the removal of Biological Oxygen Demand (BOD), coagulation of non-sedimentable colloidal solids and stabilization of organic matter (Barak et al., 2020). These activities carried out by bacteria are intended to complete the cycle of degradation and reproduction from the waste material, which is how man uses these microorganisms to purify wastewater. In Mexico, treatment plants concentrate water from domestic, agricultural and industrial sources, the latter containing a large amount of organic and inorganic substances that are toxic for the microorganisms responsible for stabilizing organic matter, making it appropriate to carry out a pre-treatment for the purification of toxic waste, generating after this treatment an association of bacteria with microalgae to establish an adequate control of all the factors involved in the treatment (Yong et al., 2021). Studies by Chen et al., (2017) have shown that, in natural aquatic environments, microalgae are always associated with bacteria leading to mutual interactions and can be growth promoters, symbionts or simply in coexistence, generating strategies for bioremediation with low action times in the reduction and/or removal of heavy metals present in wastewater used for agricultural irrigation (Matsumoto et al., 2015). Microalgae in laboratory culture are often associated with different bacterial strains that have been co-enriched from natural environments together with algal cells, or that come from the site of contamination. C. vulgaris has a high mitigation potential as it absorbs certain heavy metals through biosorption; in addition to being in direct contact with the microbiota it works together with the bacteria present in the contaminated environment (Salama et al., 2019; Ubando et al., 2021) and C. vulgaris has also been reported to increase the abundance of bacteria that support oxygen production, so a combined application of microalgae and bacteria-based technologies offers a promising remediation approach (Song et al., 2020). Weimer et al., (2020) isolated Pseudomonas putida from wastewater where they used C. vulgaris for tertiary treatment and observed that the bacterial contribution supported the growth and performance of the microalgae in their work within the tertiary phase of treatment; furthermore Leong and Chang (2020) investigated the use of this microalgae in small communities thanks to its low cost and efficiency, describing that it not only removes heavy metals, but also organic matter and inorganic nutrients by working in conjunction with certain bacteria. In wastewater, the presence of heavy metals and xenobiotic compounds is characteristic, generating problems in high concentrations in the development, growth, and reproduction of organisms (Arora, 2020). The joint use of bacteria with C. vulgaris extract or immobilize toxic compounds, leaving the substance unavailable to organisms and preventing them from being affected. It is also important to highlight that bacteria are functionally important in pollutant biotransformation processes and offer resources for their application in bioremediation, as well as achieving various socioeconomic advantages through the use of this process, in particular the environmental advantages of bioenergetics and the economic and social benefits of the economic valorization of wastewater (Ma et al., 2020; Rajhi et al., 2020). This study aimed to identify bacteria present in wastewater and to analyze their interaction with C. vulgaris to promote their phytoremediation.

Materials and Methods

Study Area

The Alseseca-Sur Wastewater Treatment Plant (WWTP) located in the municipality of Puebla, Mexico (18°57'14.9 " N, 98°11'23.5" E), performs advanced primary treatment in the final part of the Alseseca riverbed. The treated water presents a domestic, agricultural and industrial mix, with a maximum treatment in rainy weather of 700 L/s while in the dry season it is 300 L/s.


Weekly visits were made to the Alseseca sur-WWTP during November 2017, March, and November 2018, to know the characteristics of the samples, discarding variations of the components in different sampling periods. Each of the samplings was carried out following the NMX-AA-003-1980 standard, where the sample collection was established consisting of 6 L subsamples every 4 h for 24 h to obtain a composite sample transferred to the laboratory to be stored at 4 °C (DOF, 1992).

Physicochemical Characterization

The physicochemical characterization of the wastewater samples was carried out following Mexican Standards and Water Analysis Techniques, which establish the methodological and permissible parameters for the determination of the water quality in the wastewater samples to be used (DOF, 1997).

Microbiological Quantification

Serial dilutions were made of the wastewater samples and the wastewater treated with C. vulgaris in nutritive broth, taking dilutions from 10-3 to 10-6, spreading 0.1 mL of each of the dilutions in nutrient agar, incubated at 36 °C for 24 h, then the colony-forming units per milliliter (CFU/mL) were counted, performed in triplicate. Bacterial isolation was performed by striated seeding on nutrient agar, incubating at 36 °C for 24 h, selecting various colonies for their morphological characteristics and by Gram staining, placing them again in nutrient broth to be incubated; these isolates were identified with the Chromogenic Compact Dry “Nissui” EC™ medium. DNA extraction was carried out with the Zymo-Research Quick-DNA Fungal / Bacterial kit and it was amplified by Polymerase Chain Reaction (PCR) with universal oligonucleotides of the 16S RM and RB gene for bacteria (5'- AGA GTT TGA TYM TGG CTC AG-3 ') and (5'- GGA CTA CCA GGG TAT CTA ATC C-3') using an MS mini thermal cycler (BioRad®, USA) and the following amplification program: initial denaturation at 95 °C by 5 min, 35 cycles of 92 °C for 1 min, 57 °C for 30 s, and 72 °C for 1 min (Cabra-Cendales et al., 2015). The PCR products were analyzed by 1% agarose gel electrophoresis, stained with Biotium. The sequencing of the PCR products was carried out by the Biomolecular Detection Center of the Benemerita Universidad Autonoma de Puebla, sending vials containing +/- 100 μL of the products of each PCR reaction. The sequences were analyzed for quality using the CLC main Workbench ver. 6.1® 2015 CLC bio, QIAGEN and then annotated using the BLAST program ver. 2.2.27+ comparing them against the NCBI nt database, with a threshold value of 1 × 10-5. These results were filtered with 80% similarity and 80% coverage of the amplified region.

Wastewater Treatment with Chlorella vulgaris

The inoculation of C. vulgaris was carried out directly in the effluent water samples from the Alseseca-Sur WWTP, using the wastewater sample as the culture medium of C. vulgaris in addition to the Bold medium. 10 mL of C. vulgaris were added for each L of residual water, carrying out the inoculation in triplicate. The conditions for the growth of the algae were with continuous aeration at a flow of 19 L/h and exposed to white light at a temperature of 18 °C for 5 days (Magdaleno et al., 2012). At the end of the growth time of C. vulgaris in the wastewater sample, the concentration of 180 cells / mL of C. vulgaris in the wastewater was observed, verifying with this data that the water from the Alseseca-Sur WWTP contains the appropriate characteristics. For the survival and growth of C. vulgaris (stock solution), 10 mL of the stock solution were added for each liter of residual water, making concentrations of 100 (total residual water), 75 (750 mL residual water: 250 mL purified water), 50 (500 mL residual water: 500 mL purified water) and 25% (250 mL residual water: 750 mL purified water) respectively plus a 100% purified water control plus Bold medium as culture medium. At the end of the incubation period, the cell count was performed. The organic matter was separated from the water utilizing centrifugation to determine the physicochemical parameters of the water following the Mexican water analysis standards mentioned above.

The cell count was performed on the total of the samples, estimating the growth by the cell count through the microscope, always considering the Neubauer chamber filling technique (Neubauer Improved 0.1 mm), performing the cell concentration calculations (Ramos & Pizarro, 2012).

Statistical Analysis

The comparison of the CFU number of the wastewater with the wastewater treated with C. vulgaris was performed with the Mann Whitney U test with a significance level of 0.05 (GraphPad InStat Software v2.04a).

Results and Discussion

The physicochemical characterization of the effluent water from the Alseseca-Sur WWTP was determined during the three days following its collection, except for the BOD which was measured once the sample arrived at the laboratory. The Alseseca-Sur WWTP treated with C. vulgaris; the results obtained are shown in Table 1, in comparison with the maximum permissible limits of the corresponding regulation.

The microbiological quantification showed a decrease in the bacterial load in the phytoremediated sample with C. vulgaris, obtaining for the first dilution 108X106 CFU, while in the sample treated with C. vulgaris a total of 72X106 CFU was quantified for the dilution equivalent; For the 10-6 dilutions of both samples, 108 and 72 CFU were counted respectively, the statistical analysis did not show a significant difference (P>0.05) in the CFU/mL count between the values ​​of the effluent water and the phytoremediation process. The isolates obtained in a nutrient medium presented the colonial characteristics of Pseudomonas sp., (Figure 1a) when performing the Gram stain, they were negative, bacillary-rectum shape and did not develop spores; characteristics like those of Pseudomonas sp. The bacterial cultures seeded in the Chromogenic Compact Dry “Nissui” EC™ medium presented a beige coloration that according to the manufacturer's indications corresponds to Pseudomonas sp., (Figure 1b) to confirm the identification of the isolates, after their amplification by PCR (Figure 2). Table 2 shows the BLAST results, obtaining the identification of the microorganisms (coverage and identity greater than 86%) for 75% of the isolates.

The phytoremediation process using wastewater as a culture medium showed favorable results with a higher percentage of wastewater; where, in 100% water, 174 cells/mL of C. vulgaris, 75% 120 cells/mL, 50% 90 cells/mL and 25% 35 cells/mL of the algae were quantified; unlike the control where 24 cells/mL were quantified.

In several studies where phytoremediation processes are carried out in wastewater, physicochemical parameters that contribute to water pollution are determined, such as arsenic, organic compounds, phosphorus and nitrogen (Gomez-Guzman et al., 2017; Srivastava et al., 2018); however, this determination is only focused on specific pollutants, and it is important to carry out a complete characterization once phytoremediation has been carried out. In this study, a physicochemical characterization was carried out following NOM-001-SEMARNAT-199617, considering all the parameters to determine the variations once the phytoremediation process has been implemented. The microbiological study of the treated samples showed that Pseudomonas sp., in 75% of the isolates from the wastewater from the Alseseca-Sur WWTP, using the effluent water as a culture medium for the growth of C. vulgaris without requiring a simulated medium, thus taking advantage of the characteristics present in the initial sample, in contrast to what has been reported in various studies where phytoremediation processes are carried out with C. vulgaris inoculating certain species of Pseudomonas, Bacillus and bacterial consortia to favor the efficiency of the system in the removal of pollutants present in wastewater (Mu et al., 2020); the data on the increase in the concentration of C. vulgaris with wastewater as a culture medium are similar to those reported in Shen et al., (2017) where they report an increase in the cell density of C. vulgaris in symbiosis with Pseudomonas putida using municipal wastewater as a culture medium, removing ammonium, phosphates and organic compounds from the wastewater. However, in the present work the decrease was in all the physicochemical parameters established in NOM-001-SEMARNAT-199618, except for hardness. This decrease is due to the use of 100% wastewater inoculated with C. vulgaris, increasing the cell density of the algae using the wastewater as a bioreactor and generating adequate phytoremediation, unlike what was reported by Amenorfenyo et al., (2019) where the increase in the concentration of algae increased about the amount of Pseudomonas inoculated in the photoreactors, generating an increase in treatment costs. The reduction of pollutants in phytoremediation wastewater is directly linked to the use of C. vulgaris together with the microbiota present in the wastewater, in particular Pseudomonas.

Table 1. Physicochemical Characterization of the Sample from the Alseseca-Sur WWTP



Initial sample

Sample treated

C. vulgaris






Not specified















































Hardness (CaCO3)

Not specified




Not specified




Not specified



Total solids




Suspended-solids (mL/L)







Figure 1. Colonial growth of Pseudomonas sp., in nutrient agar Panel (a), Pseudomonas sp., identification in Cromogenic medium Compac Dry Nissui EC™ Panel (b).


Figure 2. Agarose gel electrophoresis 1%, lanes 1-3 positive samples to 16S gene, lane 4 positive control, lane 5 molecular weight marker (1 kb).


Table 2. Identification of isolates with BLAST using the 16S gene



Coverage (%)

Identity (%)



P. toyotomiensis





Pseudomonas sp BAB-3709





Pseudomonas sp BAB-3709





S.enterica NCTC10433






The effluent water from the Alseseca-Sur WWTP can be used as a culture medium for the development of C. vulgaris, as it has the necessary characteristics for its proliferation, increasing the number of cells/mL in the wastewater to 100%, thus generating effective phytoremediation of the wastewater, demonstrating the decrease in the values of the physicochemical parameters; without the addition of microorganisms.

Acknowledgments: The first author thanks CONACYT for the scholarship granted (480689) to carry out their doctoral studies. We thank Dra. Cristina Lara Ochoa for the technical support in DNA sequencing. All the authors acknowledge and thank their respective Institutes and Universities.

Conflict of interest: None

Financial support: None

Ethics statement: None


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