Phytoremediation capability of Anabaena sp. for high organic and chromium-loaded wastewater in membrane bioreactors Open Access

The generation and treatment of wastewater is a global problem. Pollution is created due to the discharge of huge amounts of untreated effluent from various sectors. Aquatic environments are directly exposed to these sorts of pollution as they are thought to be the most convenient sinks for disposal (Hiraoka 2021). Therefore, the need to treat and discharge toxic effluent has been in high demand. In this respect, the use of algae a potential microorganism for the remediation of wastewater is nowadays gaining importance. Conventional treatment of wastewater produces water that does not comply with stringent international laws set aside for the quality of discharge water. Microalgae are useful for the removal of phosphorus, nitrogen, toxic heavy metals, etc. (Priyadarshani et al. 2011). Substantial biomass production with simultaneous wastewater remediation has been obtained from microalgae (Cai et al. 2013). According to USEPA (2011) microalgae have been applied for wastewater treatment in ponds for the last 3,000 years (US EPA 2011). Algal treatment of wastewater compared to conventional treatment requires no additional chemicals, is cost-effective, metal removal, lesser generation of sludge, etc. Consortium of Chlorella sp. and Scenedesmus sp. was used for the treatment of domestic wastewater by several authors (Silambarasan et al. 2021; Vaz et al. 2023). The use of Chlorella vulgaris and Scenedesmus quadricauda for the removal of nutrients from wastewater has been studied. C. vulgaris removes more efficiently and S. quadricauda removes BOD and phosphate (Ayodhya & Kshirsagar 2013). Phytoremediation of sewage wastewater was carried out using different microalgae, i.e. Chlorella minutissima, Scendesmus sp. and Nostoc sp. The algae could reduce BOD, COD, NO₃, NH₄, PO₄ , TDS, etc. Chlorella was the best among all the three algae and produced more manure (Sharma & Khan 2013). Five algal species, i.e. Anabaena, Diatoms, Spirogyra, Hyalophacus, and Monoraphidium were studied based on of their ability to reduce pollutants from wastewater. The algae use nitrogen and phosphate and reduce carbon dioxide (Velan & Saravanane 2013). Apart from nutrient pollution, wastewater also contains heavy metals such as chromium, lead, nickel, and zinc that enter the human body and accumulate in the food chain of the ecosystem causing severe health hazards. Treatment technologies for heavy metals, such as reverse osmosis, chemical precipitation ion exchange, and ultrafiltration were used. But these methods are ineffective at lower concentrations and are not cost-effective. The potential use of many living organisms such as algae, fungi, bacteria, yeast, and waste materials is done as biosorbents for the removal of heavy metals. Live organisms are also used for the uptake of metals. Chromium is highly toxic and carcinogenic and imparts hazardous effects on human health. Chromium in its hexavalent state is more toxic than its trivalent state. Chromium is widely used in electroplating industries, tanning industries, the manufacturing of chrome, etc. The ability of algae to take up chromium and accumulate in their cells is studied. Biomass thus produced can be used for the production of biofuel. Algae can bind metal on the surface of their cell wall. Metal-binding sites are present on their cell wall. Their cell is composed of lips, proteins, polysaccharides, etc. which are composed of functional groups such as amino, hydroxyl, and carboxyl groups that act as metal-binding sites. Moreover, algae can use the metal for their growth (Macfie & Welbourn 2000; Dayana et al. 2013; Jyoti & Awasthi 2014). The use of dried algae for chromium uptake was studied using Ulva lactuca with 92% removal efficiency (Gowda 2021). Removal of hexavalent chromium was studied using immobilized Chlorella pyrenoidosa with calcium beads. Removal efficiency was 75 mg/L at pH 3 (El-Sikaily et al. 2007). But very limited literature data are available for the application of live algal cells in metal sequestering (Mohsenpour et al. 2021). Therefore in the present study, Anabaena sp. was studied for the removal of organic matter and chromium from sewage water in a membrane bioreactor (MBR). The biomass produced was studied for the production of biofuel.

Algal strains of Anabaena sp. were purchased from National Collection of Industrial Microorganism (NCIM), Pune, India. The culture was then aseptically transferred to a culture tank containing Fogg's media (Fogg & Thake 1987) and maintained in a clean atmosphere inside laminar airflow (temperature maintained at ±25 °C), with 12-h photoperiod. The algal species was identified as Anabaena cylindrica with an optical microscope (50× magnification). All the chemicals used for media preparation were purchased from Merck, India. The glass wares used for algal culture and experiments as well as media were properly sterilized before and after each use to minimize contamination.

Since algae require carbon dioxide for growth, wastewater, and control (media) were saturated with carbon dioxide at 50 cc/min from a CO₂ cylinder. The saturation level was detected by a CO₂ hand analyzer.

The initial content of the culture was 25 mg dry weight/L and the initial optical densities at OD₆₅₀ were 0.042. The cell density of alga can be quantified in two basic ways, i.e. as grams of dry or wet weight per litre of the sample or as the number of viable/dead cells per mL. For measuring dry weight, the alga is separated from the solution and dried before weighing which account for dry weight per litre of the sample. To measure the wet weight of algal cells, biomass was detected spectrophotometrically using a UV–Vis spectrophotometer and measured at a wavelength of OD 650. Light absorbance is proportional to the change of cell number for microalgae like Anabaena sp. Fourier transform infrared spectroscopy (FTIR) analysis of dried algal biomass was carried out to understand the functional groups present in the cellular structure.

Wastewater was collected from a nearby sewage pumping station in Kolkata. Immediately after collection, the water was characterized into organic and inorganic matter. Simulated wastewater was prepared by adding hexavalent chromium (5–20 ppm concentration). Algal cells were allowed to grow in wastewater keeping all other culture factors the same.

A large-scale study was carried out in 1 L sterilized glass cylinders covered with cotton plugs. Wastewater was saturated with carbon dioxide and algae (0.5 g/L) were added. Wastewater was drawn at regular intervals for analyzing various parameters and measuring dissolved carbon dioxide. The growth rate was measured at regular intervals by weighing the complete set. Evaporation loss was calculated by setting up an experimental set-up without algae that served as blank. Algae collected after 7 days of culture were subjected to characterization for different biochemical parameters and stress enzymes. These algae-containing effluents were subjected to a microfiltration study.

The membrane support before and after the application was subjected to SEM analysis (Inspect F50, FEI, USA) to understand the surface morphology of the membrane.

Oxidative stress analysis was conducted on algal cells exposed to wastewater and media to compare the induced stress on the cells that might be used for biodiesel production. Algal tissues (0.5 g) were homogenized in a 50 mM cold phosphate buffer (pH 7.0) and centrifuged (Kubota, 6,500, Japan) at 14,000 rpm for 15 min at 4 °C. The supernatant was used for further analysis. For superoxide dismutase (SOD) measurement, the method described by Giannopolitis & Ries (1977) was used and expressed as U/mg protein/min (Giannopolitis & Ries 1977). 1.3 μM riboflavin, 13 mM methionine, 63 μM NBT, and 0.05 M sodium carbonate (pH 10.2) were added to the reaction mixture. Distilled water was added to make the volume up to 3 mL. Samples were properly illuminated and the blank was not exposed to light. After 30 min, samples were measured spectrophotometrically at 560 nm. Beauchamp and Fridovich explained 1 unit of SOD as the amount that inhibits the NBT reduction by 50% (Beauchamp & Fridovich 1971). Catalase (CAT) activity was measured by the disappearance of H₂O₂ when added to enzyme extract and expressed as U/mg protein/min (Aebi 1983). Measurement was carried out spectrophotometrically at 240 nm. The reaction mixture contained phosphate buffer and hydrogen peroxide. Chlorophyll content was measured as per the method described in APHA and measured spectrophotometrically at 664 nm (Greenberg et al. 2005).

Each set of experiments was in three replicates and the standard deviation (S.D.) was determined and expressed as X ± S.D. in GraphPad InStat 3 software (GraphPad, San Diego, CA, USA) by one-way analysis of variance (ANOVA). The means of different treatments were determined using p ≤ 0.05 and p ≤ 0.01 as levels of significance.

Biodiesel and lipid content of algae were measured. Lipid content was measured by extraction in methanol and the biodiesel content was measured by transesterification with alkali (Velan & Saravanane 2013). The algal culture was taken in chloroform solvent (1:2) and set in an orbital shaker for 15 min. It was then heated in a water bath for 1 h at 75–80 °C and then filtered. The solvent extract was esterified with 0.9% NaCl and collected in a separating funnel. Two separate layers were formed where lipid was separated in the lower part and collected in a container (Hamouda et al. 2023).

A batch adsorption study was carried out at varying metal concentrations (0–20 mg/L), pH (3–5), and algal cell density (0–1.23 × 10⁸) at 25 °C ± 2 °C.

Table 2 depicts the values of stress enzymes like superoxide dismutase and catalyze. Since algae were cultured in wastewater and media served as control, an assay of stress enzymes was carried out. Algae generate antioxidant enzymes to cope with reactive oxygen species (ROS) generated in a stressful environment to reduce cellular damage. SOD is the first line of defence that living cell produces to combat cellular damage. It is a metalloprotein that catalyses superoxide dismutation into hydrogen peroxide and oxygen (Rezayian et al. 2019). CAT converts peroxide radicals generated under stress into water and oxygen. The values generated from a 3-day (72 h) experimental study suggest that stress generated within 24 h gradually decreased with time and data were mostly comparable to the control (Mate et al. 1999). Thus algae were able to survive and grow under stress conditions. Chlorophyll content was measured as it is an important parameter that indicates wastewater toxicity. Chlorophyll content was less than control, suggesting stress shock to cells (Table 2).

Lipids are mainly fatty acids and their derivatives. Fatty acids produced by microalgae generally contain combinations of zero to five cis double bonds. Increased lipid content in untreated wastewater might be because under stress conditions algae reduce their cell growth and accumulate available carbon for the synthesis of lipid and other high energy density compounds to combat carbon limiting conditions. This result can be established from the growth rate obtained in this study where, after the 5-day growth, almost attained a steady state. Since wastewater is a nutrient-rich medium, it becomes light and carbon limiting for algae growing in it. But some algae thrive well in the light-limiting condition as well as utilize organic carbon for their growth (Ota et al. 2009; Onyeaka et al. 2021). The lipid content of algae growing in media was 3.7% (±0.03) whereas that of algae growing in wastewater was 5.3% (±0.03). Biodiesel content was on the lesser side than the control (Table 3).

The produced algal biomass can be utilized in many ways. The synthesis of nanoparticles from algal biomass has been widely used because of its smaller size, greater surface-to-volume ratio, and higher reactivity (Cheng et al. 2019). The removal of heavy metals using algae-based adsorption process is studied (Li et al. 2019; Goswami et al. 2022). The produced algal biomass can also be used for the production of biofuel. Studies were conducted using algal biomass where after the adsorption of Pb(II) Chlorella sorokiniana was subjected to biodiesel production. The GC–MS analysis of algae showed that fatty acids and SFA are required for biodiesel production (Nanda et al. 2021). Algal-based treatment of wastewater and heavy metal remediation were done using produced biomass for the generation of biocrude oil or bioenergy-like value-added products (Goswami et al. 2020).

The study shows that algae can efficiently phyto-remediate wastewater-containing hexavalent chromium and organic matter. The growth rate data of algae suggested that the nutrients in wastewater served the growth requirements. 97% removal of chromium was obtained with 93% COD removal. The FTIR analysis proved the presence of polysaccharides that might take part in chromium uptake by algae. An assay of stress enzymes, such as SOD, CAT, and cholorophyll content, showed the generation of ROS in response to toxic wastewater and heavy metal compared to the control in algae within 24–48 h in which the algae were able to combat within 72 h as evident from reduced values of SOD and CAT. Fouling behaviour was observed with 66 LMH flux values. When pore blocking and cake filtration models agreed with fouling data they showed that fouling behaviour was reversible and a membrane can be regenerated by back washing after each run. Thus, a side-stream MBR study using algae showed promise in remediating toxic metal-laden wastewater and treated water could be reused and biomass can be harvested for the production of biofuel.

The financial support from the Department Of Science And Technology, Government of India vide Grant no. DST/WOS-B/WWM-2/2021 (G) dated 17.09.2021 is gratefully acknowledged.

Data cannot be made publicly available; readers should contact the corresponding author for details.

The authors declare there is no conflict.