Environmental pollution is a major concern worldwide, especially water pollution, affecting all forms of life (Boretti and Rosa 2019). Industrial and agricultural wastewater are the most abundant and hazardous sources of pollution. Therefore, WWT is a crucial and pivotal step before water discharge into the environment (Abd-Elaty et al. 2022). Cyanide compounds are toxic contaminants in heterogeneous types of industries, such as precious metals mining, coke-ovens, metal refining, pharmaceuticals, foods, biocides, and chemical and plastics production (Luque-Almagro et al. 2016). Sodium salicylate is a co-contaminant associated with some of the industries producing cyanide-contaminated discharges, such as pharmaceuticals, coke-ovens, and chemicals production. Sodium salicylate exerts an added toxicity to these discharges (Borisov et al. 2022). In the present work, KCN was used to evaluate the cyanide-degradation ability of the isolated bacterial and algal species in the presence of sodium salicylate as a co-contaminant. We successfully isolated a bacterial strain from a WWT station, Bacillus licheniformis MMT1, a Gram-positive motile rod with 99.22% similarity to Bacillus licheniformis.

Several studies reported the isolation of cyanide-degrading bacterial strains from various environments. For instance, Kandasamy et al. (2015) reported the coexistence of Bacillus pumilus, Pseudomonas putida, and Bacillus cereus C2, which were enriched in sodium cyanide and glucose-containing liquid media and isolated from a cassava factory wastewater (Kandasamy et al. 2015). Jiang et al. (2022) isolated Aerococcus viridans T1 from electroplating sludge, showing cyanide resistance up to 550 mg L−1 (Jiang et al. 2022). Similarly, Pseudomonas spp. such as Pseudomonas putida and Pseudomonas stutzeri were collected from coke-oven effluent, rich in phenol and cyanide (Singh et al. 2018). Additionally, Pseudomonas parafulva, isolated from polluted gold mine soil, was effectively applied for cyanide degradation (Moradkhani et al. 2018). To enhance degradation efficiency and spectrum, mixed microbial cultures have been employed. For example, Mekuto et al. (2016a) used a mixture of Exiguobacterium acetylicum and Bacillus marisflavi from the Diep River, Cape Town, to achieve co-degradation of cyanide and thiocyanate (Mekuto et al. 2016a). Similarly, mixed cultures of Pseudomonas stutzeri and Bacillus subtilis were employed for optimized cyanide compound degradation (Nwokoro and Dibua 2014).

Microalgae are diverse in terms of sizes, textures, colors, and shapes; therefore, their morphological characteristics can be used for taxonomical identification (Iamsiri et al. 2019). In fact, microscopic identification of microalgae is one of the most widely accepted and commonly used methods for microalgal species identification (Chong et al. 2023). Examination using light microscopy of the microalgal isolate revealed that it belonged to Chlorella species. This identification was corroborated by the TEM examination.

Microalgae has been used in WWT processes as a photosynthetic aeration source (Essam et al. 2014). Prominent algal species in WWT include Chlorella spp., Scenedesmus spp., Oscillatoria spp., and Selenastrum spp.. Particularly, Chlorella species are commonly used in WWT and detoxification processes (Coronado-Reyes et al. 2020). They are also applied in the biodegradation of pollutants symbiotically with biodegrading bacteria in photobioreactors (Mujtaba et al. 2018). In the present study, the Chlorella spp. strain was introduced in the PBR to provide oxygenation via photosynthesis, enabling aerobic degradation of the selected pollutants.

Previously, a microcosm combining Chlorella vulgaris MMI and Pseudomonas MTI efficiently treated simulated wastewater containing phenol and pyridine (Essam et al. 2013). In this system, Pseudomonas MTI was the primary agent responsible for the breakdown of phenol and pyridine, while Chlorella vulgaris contributed to oxygen production via photosynthesis, sustaining the bacterial degradation process (Essam et al. 2013). Similarly, Ryu et al. (2014) investigated thiocyanate removal using a consortium of bacteria (activated sludge) and microalgae in a sequential two-step process. Initially, bacteria facilitated the degradation of thiocyanate into nitrification products such as nitrite and nitrate. Subsequently, microalgae were introduced to assimilate and remove these byproducts, effectively complementing the bacterial degradation (Ryu et al. 2014).

Normally, microalgae are more sensitive to physical and chemical changes than other microorganisms (Omar 2010). The algal toxicity rank of the tested pollutants was as follows: KCN > Benzonitrile > KSCN > Salycilate. KCN, as a simple salt of hydrocyanic acid, releases free cyanide ions, which are highly toxic due to their ability to inhibit cytochrome c oxidase in the mitochondrial electron transport chain (Park et al. 2017). Benzonitrile, an aromatic nitrile, has reduced toxicity compared to KCN as its cyanide group is covalently bonded to a benzene ring, limiting free cyanide release (Sulistinah and Sunarko 2020). In contrast, the thiocyanate ion has lower toxicity than other cyanide compounds (Mekuto et al. 2016a). Sodium salicylate, an aromatic compound with a hydroxyl group, is non-cyanogenic, and its potential toxicity arises from other mechanisms like membrane destabilization or enzyme inhibition. Nevertheless, salicylates are considered one of the hazardous constituents present in industrial effluents (Borisov et al. 2022). These structural differences influence their behavior and toxicity in biological systems, which are reflected in their degradation profiles during the study.

In the present study, the algal toxicity of the influent feed containing a mixture of the selected pollutants was relatively higher than that of each single pollutant, where the feed inhibited ~ 96% of the algal growth. This may be attributed to the addition of a mixture of chemicals together having a similar mode of action to the feed. This mixture may produce an additive toxic effect that is larger than the effect of each component if applied individually (EC European Commission 2012). However, microalgae continued to grow during the operation of the algal–bacterial PBR, proving the efficient detoxification by the bacterial strain.

To test for the maximum biodegradation capacity, the bacterial strain was allowed to grow in increasing concentrations of each of the four pollutants using MSM as a culture medium with no carbon source. Bacillus licheniformis MMT1 was unable to degrade potassium thiocyanate but it effectively removed all other pollutants tested. The degradation of the higher concentrations of each pollutant showed a period of acclimatization in the degradation curve, where a duration of 12–24 h elapsed before recording a significant decline in concentration of each pollutant. This increase in degradation duration may be interpreted by the increase in the lag time needed by the bacteria to overcome the toxic effect of the pollutants and to activate the machinery of degrading enzymes in the presence of higher pollutant concentrations. Indeed, bacterial activity needs additional time for acclimatization with increasing pollutant loads (Mekuto et al. 2016b).

The adjustment of pH is an important step in cyanide treatment. To avoid volatilization of HCN gas from solution, alkali-tolerant microorganisms, or acclimatization of the degrading microorganism to the alkaline environment may be adopted (Vallenas-Arévalo et al. 2018). Airtight containers may also be used (Akinpelu et al. 2015). In this work, we used airtight Erlenmyer flasks with no shaking, and the pH was adjusted to 7.5–8 in order to minimize HCN loss, maintain the environment hospitable to algal growth in the PBR, and avoid salting out of the chemicals.

The capacity of bacterial degradation of cyanide-containing compounds varies from 0.15 g L−1 to ~ 0.5 g L−1 of cyanide or more. In one study, Aerococcus viridans degraded 84% of 0.2 mg L −1 cyanide within three days and 87% of 0.15 mg L −1 cyanide in 2.3 days. This degradation was accomplished by the addition of glycerol as a carbon source and peptone as a nitrogen source (Jiang et al. 2022). In another study, a Bacillus licheniformis strain degraded 98% of 0.5 g L−1 KCN in 5 days after its adaptation to an alkaline medium (Vallenas-Arévalo et al. 2018). In our work, Bacillus licheniformis MMT1 had the ability to biodegrade up to 1 g L−1 of KCN in nine days. This biodegradation capacity is among the highest reported for cyanide biotreatment (Table 4).

The duration to completely biodegrade the influent load of 1 g L−1 KCN was around 9 days, which was relatively long compared to the time reported in previous studies of cyanide degradation. However, in our work, no pre-treatment steps nor additional external carbon or nitrogen sources were introduced in the degradation process. In addition, only a single cyanide-degrading strain, Bacillus licheniformis MMT1, was inoculated in the PBR, unlike other studies where a consortium of degrading bacteria was used to achieve complete removal of pollutants (Table 4).

Cyanide degradation can be accomplished via several possible enzymatic mechanisms: oxidative, reductive, hydrolytic, and substitution (Oshiki et al. 2019; Alvillo-Rivera et al. 2021). For Bacillus licheniformis MMT1 to effectively degrade the cyanide-containing pollutants (KCN and Benzonitrile), the strain may be producing enzymes such as cyanidase or nitrilase or both. These enzymes catalyze the breakdown of cyanide through hydrolysis. The substitution pathway, which involves the rhodanese enzyme, was ruled out as it requires the presence of thiosulfate in the medium. In addition, thiocyanate concentration did not increase during the operation of the PBR, indicating that no thiocyanate, needed for rhodanese activity, was newly formed. Therefore, it is unlikely that rhodanese contributed to the degradation observed in our work. Bacillus licheniformis MMT1 failed to biodegrade KSCN probably due to the absence of the specific enzymatic systems required for its degradation.

In contrast, the ability of Bacillus licheniformis MMT1to degrade sodium salicylate may involve the production of enzymes such as salicylate hydroxylase, salicylate 5-hydroxylase, or salicylate 1,2-dioxygenase, which are known to metabolize sodium salicylate through hydroxylation or cleavage into various metabolites like catechol, gentisic acid, or 2-oxo-3,5-heptadienedioic acid, respectively (Górny et al. 2019).

For the efficient bacterial removal of a mixture of contaminants, some techniques may be employed, such as the use of more than one biodegrading bacteria, the immobilization of the degrading bacteria, and/or the application of additional pretreatment techniques (e.g., adsorption) (Singh et al. 2016). For instance, to restore the biotreatment efficiency of an established PBR lost upon the addition of ~ 50 mg L −1 cyanide to the feed, two techniques were applied: a 25-fold dilution of the influent and photocatalytic pre-treatment of the wastewater feed (Essam et al. 2014). In our work, the established algal–bacterial microcosm was efficient in removing up to 0.2 g L−1 KCN, 0.1 g L− 1 benzonitrile, and 0.5 g L−1 sodium salicylate from synthetic wastewater in a sustainable manner, without the need for the incorporation of additional biodegrading bacteria, pretreatment steps nor the supplementation of fertilizers such as NaHCO3 to the influent feed.

Biomonitoring is a test for the ability of a microorganism, weed, or plant to survive and/or grow in the presence of treated wastewater (Hybská et al. 2020). Algal growth in the presence of the toxic pollutants was a good indicator of the efficient treatment by the PBR. In algal toxicity testing, the growth of the isolated Chlorella strain was completely inhibited by 0.05 g L− 1 KCN, and the influent feed had 96% inhibition of its growth. However, in the continuous PBR, KCN concentration in the feed reached up to 0.2 g L−1 without harmful effects on algal growth, confirming the efficient detoxification of the pollutants by the system.

In fact, bioassays are essential to assess the effectiveness of bioremediation, as they evaluate the potential of environmental contaminants to interact with living organisms (Mazzeo et al. 2010). Unlike chemical analyses, which focus solely on measuring pollutant concentrations, bioassays provide insights into the bioavailability of substances in environmental samples. Phytotoxicity testing is usually used to assess the impact of various pollutants on seed germination and subsequent growth (Haq and Kalamdhad 2021). Particularly, Lepidium sativum is commonly used in phytotoxicity bioassays due to its sensitivity to contaminants such as heavy metals, petrochemicals, and polycyclic aromatic hydrocarbons (Janecka and Fijalkowski 2008). It is characterized by its rapid growth rate, adaptability to different humidity levels, and high contamination sensitivity. It has been previously applied to evaluate the phytotoxicity of composts (Aslam et al. 2008), sewage sludge (Mañas and De las Heras 2018), and soil saturated with wastewater (Mekki and Sayadi 2017), making it a versatile tool in environmental toxicity assessments. In the present study, the successful germination and stem elongation of the seeds, when incubated in the presence of effluent-treated water, confirmed the complete detoxification of the introduced pollutants in the algal–bacterial photobiorector.

The introduction of symbiotic bacteria to microalgae in biological treatment processses was shown to improve the algal flocculation properties, lipid content, and quality (Cho et al. 2015). The algal–bacterial flocs can be subsequently harvested and used in valuable biotechnological applications. The flocculation efficiency can be affected by the PBR operating conditions such as temperature, pH, and mixing speed (Ernest et al. 2017). Chitosan, as a flocculating agent, can also be used in water treatment since it is environmentally friendly, biodegradable, and readily accessible (Yang et al. 2016). In the present work, natural flocculation of the formed algal–bacterial flocks was satisfactory for the harvest of the effluent-treated wastewater. There was no need for the addition of flocculating agents or changing the operating conditions.