Treatment of heavy metals contaminated water: use of B. mojavensis BI2 derived lipopeptide and palm waste flour Open Access

BioS

Biosurfactant

Generally, the presence of certain chemical industries causes many problems of heavy metal pollution. They lead to the toxifying of aquatic and soils environments, plants, animals and humans. So an urgent need for their elimination has developed. Heavy metals are natural metal elements with a density exceeding 5 g/cm³. The most toxic ones are lead, cadmium and mercury. Generally, their presence in soils can be natural and they are often present in the environment as traces such as mercury, lead, cadmium, copper, arsenic, nickel, zinc, cobalt and manganese. Indeed, heavy metals are naturally present in rocks; they are released when they are altered to form the geochemical background (Bouzabata & Djamaa 2015). However, due to multiple human activities, heavy metals concentration increases in the soil and water causing great problems of pollution and ecological toxicity. Moreover, some anthropogenic activities are responsible for increasing metal flows such as pollution from agricultural activities, and industrial releases (Sumiahadi & Acar 2018). They are mainly triggered by discharges from factories (particularly from tanneries, paper mills, chlorine manufacturing plants and metallurgical plants) and mining fields during processing. In addition, the use of certain mercury-containing fungicides or residual sludge from wastewater treatment for agricultural soils could be an important source of contamination with heavy metals. Furthermore, they result from the deposition of atmospheric dust emitted during the incineration of waste or the combustion of motor gasoline containing lead and from the runoff of rainwater on roofs and roads (zinc, copper, lead) (Oves et al. 2013; Sumiahadi & Acar 2018). When the heavy metals are not absorbed by the soil, they could end up in water tables and streams which might be the source of the contamination of many plants. Once in the aquatic environment, metals are distributed among the different compartments: water, suspended solids, sediments and biota. When the contamination of a plant is too great, it affects not only the food properties but also the plant production and fertility (Nagajyoti et al. 2010).

The main processes governing the distribution of heavy metals are dilution, advection, dispersion, sedimentation, adsorption, desorption and bioaccumulation (Paul et al. 2012). Generally, bioaccumulation occurs throughout the food chain. It consists of the concentration of heavy metals in living organisms, due to their high solubility into fats (Oves et al. 2013). As a result, they can achieve very high levels in some aquatic species consumed by humans, such as bivalves and fish (Oves et al. 2013). This ‘bio-accumulation’ explains their very high toxicity (Oves et al. 2013).

The main problem related to heavy metals is their nonbiodegradability, and therefore their endless persistence in the environment (Sumiahadi & Acar 2018). Therefore, being largely widespread, highly toxic and non-biodegradable' an urgent need has developed for their elimination. Numerous physical-chemical methods were developed such as stabilization by physical washing, making them inert, heat treatment by incineration, mobilization, extraction and reduction reaction (Yao et al. 2012). However, they are costly effective and can release toxic by-products that need subsequent treatment for their discharge and elimination. Biological treatment methods involving the use of microorganisms, mainly bacteria and their derived secondary active metabolites, and plants or inert agricultural waste appear as best alternative. Bioprocesses such as biosorption, mobilization, immobilization, volatilization and/or extraction can help to completely or partially eliminate heavy metals. The biosorption process appears to be a best choice due to the rapidity and simplicity of its realization.

The use of bacteria as a support for the biosorption of heavy metals recognizes a particular boom. Interactions between bacterial cells and metals are governed by passive or active mechanisms (Yao et al. 2012; Dixit et al. 2015). They occur at the cell/solution interface and involve mechanisms such as ion exchange, surface complexation or precipitation (Yao et al. 2012; Dixit et al. 2015). With this aim, numerous studies reported the biosorption of heavy metals by bacterial strain (Banik et al. 2013; El Bestawy et al. 2013; Oves et al. 2013; Yang et al. 2016). Moreover, numerous previous studies reported the use of lipopeptides BioS for heavy metals biosorption (Mulligan et al. 2001; Zouboulis et al. 2003; Das et al. 2009, Singh & Cameotra 2013; Zhu et al. 2013; Swapna et al. 2016; Md Badrul et al. 2019; Ayangbenro & Babalola 2020; De Araujo Freire et al. 2020; Ravindran et al. 2020; Zhao et al. 2020; Kalvandi et al. 2022). According to Sarubbo et al. (2015), surfactants or biosurfactants (BioS) can act by solubilization-dissolution; complexation and/or ion exchange increasing therefore the bioavailability of heavy metals (Sarubbo et al. 2015). They can also interact with cellular surfaces, facilitating the adsorption of metals to cellular surfaces or their incorporation into cells (Sarubbo et al. 2015). In addition, BioS can be used as an improver of heavy metals biosorption (Chakraborty & Das 2014; Ayangbenro & Babalola 2018).

To define, BioS or biological surfactants are secondary active metabolites synthesized by a wide variety of micro-organisms during their growth on hydrophobic or hydrophlilic substrates (Mnif & Ghribi 2015a, 2015b). They are amphiphilic molecules consisting of a polar hydrophilic head and apolar hydrophobic tail. The hydrophilic group consists of amino acids, peptides or polysaccharides (mono or di) and the hydrophobic group consists of saturated or unsaturated fatty acids (Mnif & Ghribi 2015b). Owing to their molecular weight we distinguish high molecular and low molecular weight BioS (Mnif & Ghribi 2015b). Owing to their ionic charges, BioS are classified into anionic, cationic, non-ionic and neutral compounds (Mnif & Ghribi 2015b). Owing to the secretion type, BioS are divided into intracellular, extracellular and those adhered to microbial cells. Owing their biochemical nature, BioS are grouped into 5 groups: glycolipids, lipopeptides, phospholipids, lipopolysaccharides, neutral lipids and polymeric surfactants (Mnif & Ghribi 2015b). Glycolipids and lipopeptides are among the most popular BioS with high structural versatility and various functional activities.

Generally, BioS are well known by their surface activity properties mainly their capacity to reduce the surface and interfacial tension. Moreover, they are characterized by diverse functional properties (emulsification/de-emulsification, dispersing, foaming, viscosity reducers, solubilizing and mobilizing agents, pore forming capacity) and endowed by different biological activities (antimicrobial, hemolytic, antiviral, antioxidant, anticancer and immune-modulator) permitting their use in many domains (Mnif & Ghribi 2015b). Thus, given the undeniable interest of these molecules, their higher biodegradability, biocompatibility, lack of toxicity and higher efficiency towards extreme temperature, pH and salinity, BioS are very advantageous over synthetic emulsifiers and offer great opportunities as ecological and efficient alternatives for chemical surfactants (Kapada & Yagnik 2013; Mnif & Ghribi 2015b). They appear as potential additives in food, cosmetic, pharmaceutical industry and in environment and bioremediation for the enhancement of contaminants removal and for the sequestration of heavy metals (Mnif & Ghribi 2015a).

In this study, we proposed to evaluate a bioremediation process based on the use of B. mojavensis BI2 derived lipopeptides to sequestrate heavy metals. B. mojavensis BI2; newly isolated in our laboratory; was demonstrated to produce different cyclic and linear isoforms of Surfactin and Fengycin lipopeptides. Four heavy metals with higher toxicities were used in this study: copper, cobalt, mercury and lead. It is widely assumed that heavy metals have a toxic effect on plants and their seeds. Indeed, they delay and inhibit or prevent the germination of certain seeds via the generation of oxidative stress and the inhibition of certain enzymatic activities necessary for the mobilization of reserves (Sethy & Ghosh 2013). We evaluated the feasibility of different bioprocess for the sequestration of heavy metals. Therefore, we estimated the removal efficiency of heavy metals by evaluating germination improvement rates of radish seeds irrigated with treated contaminated water against non-treated one. Preliminary studies showed great improvement of germination rates after heavy metals chelation suggesting the abolition of toxicity. Moreover, we studied the biosorption of heavy metals on an inert agricultural waste derived from palm date. Different operational parameters were optimized by the application of a Taguchi design; the waste quantity, the heavy metal concentration, the time of treatment along with the effect of BI2 lipopeptide addition.

B. mojavensis BI2 (MW130250), a newly isolated strain in our laboratory, was used in this study (Mnif et al. 2021). The strain was demonstrated to produce cyclic and linear isoforms of lipopeptide. It was streaked on a nutrient agar slant and incubated at 37 °C. After 24 h, one loop of cells was dispensed in 50 ml LB medium prepared into a 250 mL shake flask containing 50 mL LB medium: 10.0 g/L peptone, 5.0 g/L yeast extract, 5.0 g/L NaCl. Inoculum culture was cultivated at 37 °C with shaking at 180 rpm for 18 h. For BioS production, four percentage (v/v) inoculums was transferred into a 250 mL shake flasks containing 50 mL of a modified glucose-based medium, which contains: 20.0 g/L glucose, 5.0 g/L yeast extract, 1.5 g/L KH₂PO₄, 0.7 g/L ammonium sulfate, 0.1 g/L KCL, 0.7 g/L MgSO₄, 0.008 g/L FeSO₄, 0.05 g/L CaCl₂, 0.1 g/L KCl and trace elements (Cu, Mn, Zn, Br) (Mnif et al. 2022a) with an initial pH values equal to 7.0. Cultures media were incubated for 24 hours under shaking at 150 rpm.

To prepare a crude lipopeptide extract, we follow the protocol described by Mnif et al. (2021, 2022a, 2022b). Centrifuge the culture broth at 8,000 rpm and 4 °C for 20 minutes. Remove the bacterial pellet and acidify the supernatant with HCl 6N. Incubate overnight at 4 °C to precipitate the most lipopeptide BioS. The next day, centrifuge the precipitated supernatant at 8,000 rpm and 4 °C for 20 minutes. Recover the obtained BioS pellet by suspension in a minimum quantity of distilled water. Adjust pH to 9 with NaOH 1 N to solubilize the lipopeptide and centrifuge again to remove impurities. A second cycle of acid-precipitation-solubilization-neutralization was repeated. The obtained crude lipopeptide was subjected to lyophilization for better conservation. It serves as a sequester of heavy metals and as improver of heavy metals biosorption by waste date palm flour.

Waste date palm was collected in the area of Gabes, dried and crushed. It was provided by a composting company in Nahal-Chenini-Gabes. It was composed of dry or ‘Djerid’ palms, cornafs, lif and damaged fruits. They are collected and used for the production of compost by aerobic fermentation as a method of valorization of agricultural waste in oases. In our present study, we looked for another route of valorization in their use as organic biosorbents. A fine powder obtained after sifting the crushed waste through a 0.2 mm porosity sieve is used for the biosorption tests.

Heavy metal chelation experiments are carried out with artificially contaminated water with copper, cobalt, lead and mercury. The chelation procedure is described by Das et al. (2009). During these experiments, crude lipopeptide preparations at different concentrations are used as bio-chelators. Negative controls without BioS are performed under the same conditions. The whole is incubated overnight in the shaker under a stirring of 150 rpm (except special indication) and then centrifuged at 10,000 rpm for 20 min. The collected supernatant served to evaluate the phytotoxicity of heavy metals using radish seeds. The IG is evaluated against an untreated negative control (as described above).After that, rates of germination improvement were determined as described in the analytical method part.

To optimize heavy metals sequestration, we investigated the effect of increasing concentrations of the selected metals and those of BI2 lipopeptides on the process efficiency. The sequestration efficiency was evaluated in the presence of different heavy metals concentrations ranging from 200 to 1,000 mg/l. Moreover, the sequestration was quantified for increasing BI2 lipopeptide concentrations ranging from 0.025 to 0.1%.

Preliminary studies showed the capacity of date palm waste flour to sequestrate heavy metals. In the first part of this work, we proved the potential use of BI2 lipopeptides as heavy metals sequesters. Hence, to enhance the bio-treatment of heavy metals contaminated water, we proposed to optimize different process parameters to know the date palm waste flour quantity, the heavy metals and B12 lipopeptides concentrations and the incubation duration. Three levels were assigned for each parameter as described in Table 1. The effect of process variables on the treatment efficiency was studied using the L-9 orthogonal array of Taguchi design as presented in Table 2. NemRod W Software was used to perform the design experiments and data analysis (Mnif et al. 2013, 2016). The treatment efficiency was assessed by evaluating the germination improvement of the treated contaminated water after determination of the phytotoxicity as described elsewhere in the analytical methods.

To determine the best operational conditions leading to maximum contaminated water treatment, we studied the effect of increasing BI2 lipopeptides and heavy metals concentrations on germination improvement. Firstly, we evaluated the sequestration of heavy metal with increasing BioS concentrations ranging from 25 to 500 mg/L at constant heavy metal equal to 200 mg/L. The concentration of heavy metal was fixed according to preliminary studies (not presented). Results presented in Table 3 show improved germination rates of radish seeds irrigated with treated water suggesting the sequestration of heavy metals by BioS. Better improvement in the order of 3.8, 2.52, 1.5 and 5 are recorded, respectively, for copper, lead, cobalt and mercury with respective lipopeptides quantities of the order of 200, 300, 200 and 400 mg/L. However, there are improvements in germination rates when treating with low BioS concentrations greater than or equal to the critical micelle concentration (CMC) value; suggesting the possibility of heavy metals chelation like those published by Das et al. (2009a, 2009b) and da Rocha Junior et al. (2018). Optimal improvements are observed for concentration values of 200 mg/L for copper and cobalt, 300 mg/L for lead and 400 mg/L for mercury. Lead and mercury chelation results are similar to those observed by Das et al. (2009) and Santos et al. (2017) showing the enhancement of heavy metal chelation in the presence of increasing BioS concentration above the CMC value.

In the second step of this study, we proposed to assess the sequestration of increasing metal concentrations at constant BI2 lipopeptides maintained at its optimal value obtained previously for each metal (200 mg/L for copper and cobalt, 300 mg/L for lead and 400 mg/L for mercury). We treated contaminated water with increasing heavy metal concentrations corresponding to 200, 300, 400, 500, 750 and 1,000 mg/L. The obtained results showed the attenuation of radish seed germination rates when the contamination with copper was established at concentrations superior to 500 mg/L (showing an optimum germination improvement) (Table 2). However, the improvement in lead chelation is comparable for concentrations ranging from 200 to 750 mg/L with a decrease in the presence of 1,000 mg/L. For cobalt and mercury, we observe an enhancement of germination rates improvement with the increase of their concentrations with an optimum around 750 mg/l. These results show that the BI2 lipopeptide is likely capable to sequestrate high concentrations of heavy metals. They contradict those published by Govarthanan et al. (2017) showing the attenuation of sequestration capabilities with increased metal concentrations (above 100 mg/l). They support the potential use of BI2 lipopeptide in the bioremediation of water contaminated with heavy metals.

Based on the estimation and statistics of the coefficients and to obtain better treatment of heavy metals contaminated water, it is necessary to keep the levels of factors having positive and significant effects on the response at their optimum levels. To better visualize these results, we plotted the evolution of the studied responses according to the levels of the examined factors. All the results show that to have an optimal biosorption of lead, copper, cobalt and mercury, it is necessary to carry it out with 10 g/l waste, 1,000 mg/l metal and 200 mg/l BI2 BioS for 1 hour. Thus, for the 4 metals studied, the increase in processing time does not seem to improve the treatment. One hour treatment is statistically sufficient for a large energy gain. However, the addition of 200 mg/l BioS significantly improves the efficiency of the treatment. Higher BioS concentrations reduce the effectiveness of the treatment. The optimum conditions thus obtained were confirmed in 4 separate experiments, the average of the rates of germination improvement of radish seeds after metal biosorption corresponds to 1,100 times for copper, 140 times for cobalt, 35 times for lead and 1,400 times for mercury. The optimization by the Taguchi method made it possible to improve the chelation potency. In the case of the latter, the effectiveness of the treatment is greatly reduced. BI2 BioS significantly improves treatment with a dose-dependent effect. High concentrations do not appear to have a significant positive effect.

Biosorption corresponds to the use of biological materials including microbial biomass, plant biomass and their secondary active metabolites for the fixation of pollutants by adsorption (Fomina & Gadd 2014). This strategy aims to propose them as an alternative or complement to conventional treatment methods, which are generally costly and generate problems of secondary pollution. BioS, non-toxic and biodegradable bacterial metabolites can offer great potential for use in bioremediation processes. However, they have been widely used to improve bioremediation of soils and aquatic environments (Busi & Rajkumari 2017).

In the same framework, heavy metals are inorganic contaminants with high toxic potential. Bibliographic studies reported the bioremediation of heavy metal contaminated water by bacterial biosorption (Banik et al. 2013; El Bestawy et al. 2013; Oves et al. 2013; Yang et al. 2016), using exopolysaccharides (Gupta & Diwan 2017; Kalita & Joshi 2017) and other secondary metabolites (Ayangbenro & Babalola 2018). With this aim, we propose to evaluate the capacity of a lipopeptide BioS to chelate heavy metals. Obtained results show a high potential for the sequestration of mercury, copper, cobalt and lead at BioS concentrations in the range of 300 and 400 mg/l suggesting a high capacity to treat contaminated water. Findings are similar to those published by Govarthanan et al. (2017), Açikel (2011) and Franzetti et al. (2014) reporting the potential use of BioS for the bioremediation of heavy metal contaminated soils and industrial waters. During the bioremediation of heavy metals, surfactants or BioS act by solubilization-dissolution; complexation and ion exchange (Franzetti et al. 2014; Sarubbo et al. 2015).

The date palm Phoenix dactylifera L. is cultivated in the hot arid and semi-arid zones of the northern hemisphere of the Earth and represents the main agricultural resource in the Sahara (Chao & Krueger 2007). It is very abundant in the area of Gabes, located in south-east Tunisia. Thus, a large amount of waste of date palm is generated. They are usually dry palms, cornafs, lif and damaged fruits. The fins, or Djérid, are composed of leaves and pinnate. The petiolate or cornaf base partially engages the trunk and is partially covered by fibrillum or lif (Chao & Krueger 2007). The role of date palm fiber or fibrillum or lif is to give good rigidity to the stems of plants (Chao & Krueger 2007). The stem fibers are obtained from the stems of dicotyledonous plants. The fruit of the palm, the date, is a berry having a single seed commonly called ‘nucleus’. It consists of the epicarp or the skin (it is a thin cellulosic envelope), the mesocarpe more or less fleshy of variable consistency and the endocarp is reduced to a preliminary membrane surrounding the seed (Chao & Krueger 2007). Thus, all of this waste ground into fine powder shows a great potential for biosorption of heavy metals. The addition of 200 mg/l of BI2 Lipopeptide significantly improves the biosorption of heavy metals by this waste.

Generally, agricultural wastes are low cost and widely distributed. They are widely used as a support for the biosorption of heavy metals, including rice bark, peanut shells, sugar cane bagasse, banana and orange peels, date nuclei … (Bharathi & Ramesh 2013; Lakshmipathy et al. 2015; Renu et al. 2017; Dutta et al. 2021). They contain many functional groups such as carbonyl, phenolic, acetamide, alcohol, amide, amine and sulfyril groups with affinities for heavy metals thus forming metal complexes or chelates (Acharya et al. 2018). Various mechanisms are involved in metal biosorption including chemo-sorption, complexation, surface adsorption, pore diffusion and ion exchange (Lakshmipathy et al. 2015; Renu et al. 2017). For the date palm waste flour, it's used for the preparation of active carbon useful in the bioremediation field as adsorbent in water desalination for the removal of heavy metals, dyes, phenolic compounds and pesticides (Faiad et al. 2022). Moreover, palm wastes are used for the production of fructose sweetener, glucose and lactic acid and as a biofuel source (Faiad et al. 2022). In the present study, palm waste flour was applied efficiently as adsorbent of heavy metal for potential involvement in water pollution treatment.

To conclude, B. mojavensis BI2 lipopeptide was evaluated for heavy metals sequestration. An improvement of radish seed germination was obtained after treatment of different concentrations of lead, mercury, cobalt and copper by 200 mg/L lipopeptides. Besides, to valorize agricultural wastes, palm date waste flour was assayed as natural biosorbents in addition to BI2 lipopeptide as improver of heavy metals biosorption. Results show the efficient use of palm waste floor in conjugation with 200 mg/L lipopeptide. The bioprocess parameters were optimized with a Taguchi design. This entire process permit to valorize waste, expand the potential use of lipopeptide BioS and protect the environment from heavy metals toxicity with a low cost manner.

This work has been supported by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology. It is a part of a research project on Biosurfactant Production, Characterization and Application.

Not applicable.

All authors read the final manuscript and approved its submission to Water Science and Technology.

The data sets supporting the conclusions of this article are included in the article.

Funding for this research work was granted by the Ministry of Higher Education and Research of Tunisia.

All authors directly participated in the planning, execution, or analysis of this study. All authors read and approved the final manuscript.

The authors declare that they have no competing interests.

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

The authors declare there is no conflict.