The role of ion charge density and solubility in the biosorption of heavy metals by natural biofilm matrix of polluted freshwater: the cases of Mg(II), Cr(VI), and Cu(II)

ABSTRACT One major cause of aquatic pollution is the accumulation of heavy metal ions. This review is aimed to examine the application of natural biofilm as biosorbent for Mg(II), Cr(VI), and Cu(II), as an eco-friendly, economical, and efficient remediation strategy. Biofilm matrices were collected from different freshwater ecosystems to observe their biosorption properties. The compared EPM values of the different biofilms showed a universal trend. Additionally, the adsorption kinetics of all three ions occurred within 1 minute. The amount of adsorbed Mg(II) was higher than Cu(II), owing to the larger charge density of Mg(II). Interestingly, the b values revealed that Mg(II) was desorbed the quickest among the three ions, which is likely to be influenced by its highest solubility. Thus, both charge density and solubility determined the ions’ biosorption characteristics. Therefore, physicochemical properties of heavy metal pollutants should be understood to achieve an effective bioremediation by natural biofilm.


Introduction
Heavy metal pollution has become one of the most serious environmental problems [1], particularly for freshwater ecosystems.Various innovations have been proposed to tackle aquatic heavy metal pollution, such as precipitation, ion exchange resin, or electrochemical technologies [2][3][4].Some of the developed technologies relied on the process of adsorption as characterized in the study of PEG-maleated rosin polyesters [5].Pesticide pollutant, for example, could be adsorbed by surface post-functionalization of COFs [6].Dye pollutant was removed by adsorption with biomass-derived nitrogen and oxygen co-doped porous carbon [7].Nanocellulose nanocomposite aerogel [8] was effective for adsorption of oil and organic solvent, whereas xanthate adsorption was accomplished by multilayer graphene oxide [9].
Within the last decade, it has been known that some types of biomasses have specific capability to bind and accumulate ions or other molecules from the surrounding aqueous solutions [2].This property was claimed to enable biomass to be an agent for pollutant removal [3].Engineered biomasses were proven to be beneficial for adsorption of heavy metal pollutants, such as the use of aminated lignin [10] and glycine-modified Fe/Znlayered double hydroxides [11] for As(V) adsorption.Similarly, Cd(II) was removed by polyethyleneiminemodified magnetic starch microspheres [12], janus phenol-formaldehyde resin, and periodic mesoporous organic silica nanoadsorbent [13].The latter was also reported to be efficient for MO adsorption.
The principle of adsorption is comparably useful to harvest non-pollutant heavy metals, such as nanofiber membrane carboxyl-functionalized poly(arylene ether nitrile) (CPEN) as a carrier of lanthanide sensor to collect Fe 3+ [14].In a similar manner is the usage of guanidinium-based ionic covalent organic frameworks (iCOFs) to amass uranyl tricarbonate from the ocean [15].
However, despite the advantages, the abovementioned techniques generally require sophisticated technologies, while some are relatively expensive and not all of them are environmentally friendly [16,17].For developing countries that face myriads of environmental problems, inexpensive and simple means may be more helpful.Therefore, endeavors for the development of remediation technologies utilizing native biomasses that are efficient, eco-friendly, and inexpensive have been conducted.
Various bacteria, fungi, yeasts, as well as microalgae and cyanobacteria were examined as candidates of the low-cost biotechnological agents for the treatment of heavy metal pollution [18].They were selected for their versatility and quick adaptation to different environments [18].Streptomyces rimosus [19] and Saccharomyces cerevisiae [20] have been confirmed to adsorb heavy metal pollutants from aqueous solution.The latter bacteria could be engineered as a Ni(II) resistant strain, particularly S. cerevisiae AJ208 [21].This resistance and the resulting ability to adsorb Ni(II) [22] was determined by the availability of complex nutrients, amino acids, and vitamins [23].In addition, techniques such as fixed-bed biofilm reactor (FBBR) employed the ability of microorganisms in the form of biofilm as a biocatalyst [24].Although FBBR did not rely on defined microbial species, it showed quite promising capability as an alternative bioremediation strategy [24].Considering those facts, this review is focused on the utilization of biofilm as sustainable and economical bioremediation tool for aquatic environment.

Metal ion pollutants in the aquatic ecosystems
Heavy metal represents elements that have higher density than water and are naturally present in the environment [1].Some of the heavy metal ions such as magnesium, chromium, copper, iron, and zinc are crucial for living organisms' biochemical and physiological activities [1].In other words, they exist as trace elements.Regarding heavy metals in nature, however, improper executions of anthropogenic activities such as mining, agriculture, and industry may cause human exposure of those heavy metals as well as give rise to environmental pollution [25].
The emphasis of this review is given for three heavy metal pollutants in an aquatic environment.They were compared in terms of their profiles of adsorption by biofilm.The first is magnesium ions [Mg(II)] that can be commonly found in the interstitial waters of biofilm [26], thus serving as nutritional element for biofilm.Magnesium also assists cellular function such as cell proliferation and unwinding DNA for transcription, all in the presence of Ca(II) [27].Because of its usefulness for living organisms, Mg(II) toxicity potential is often overlooked despite its relatively large concentrations in wastewater [28].The biota of waters with low ionic content can be extremely sensitive to small additions of Mg(II) at the absence or lack of Ca(II).In this and many other cases, the balance between magnesium and calcium ions determines the toxicology status of Mg(II) [28,29].
Another pollutant metal ion for aquatic environment is Cr(VI), or hexavalent chromium.This structure is the most stable form of the chromium ion [30] and is largely recognized as a waste product from industrial activities such as tannery [31,32].Various health problems can arise due to exposure to hexavalent chromium contamination, such as cancer, respiratory failure, reproduction system disorder, brain damage, and even death [33].Those health risks depend on the oxidation state of the existing chromium [1].The range spans from the low oxidation state of the metal form, hence low toxicity, to the high toxicity of the hexavalent chromium [1].Some biochemical methods have been developed to specifically remove Cr(VI) pollutant, such as ZnNiCr-layered double hydroxides [34] and the utilization of anaerobic granular sludge improved with conductive polyaniline for Cr(VI) bioreduction [10].Both are likely to employ electrostatic attraction as the mechanism of biosorption.Other biosorption mechanisms of Cr(VI) were surface complexation as happened in eucalyptus bark [35] and the apparent mechanism of heterogeneous redox reaction to form Cr(III) ions by Ceratocystis paradoxa MSR2 fungi [36].
Finally, Cu(II) is frequently found as a pollutant in aquatic ecosystems.This ion has been widely used, particularly in the electrical and electroplating industries [37].However, the mishandling of this heavy metal often led to water pollution.Like the oxidation states of Cr(VI), this ion has the transitional ability between Cu(II) and Cu(I) [37].This phenomenon triggers toxicity because the transitions between the oxidized and reduced states promote the generation of superoxide and hydroxyl radicals [37].Cu(II) can form deposits in the skin, brain, pancreas, or liver and may cause various toxicological effects to human body [38].Cu(II) biosorption included several mechanisms, among others are surface biosorption observed in Aspergillus terreus [39], extracellular chelation by chitosan-based hydrogel beads immobilized by silver nanoparticles [40], and biotransformation as happened in phytoplankton Chlamydomonas reinhardtii [41].

Biofilm as potential biosorbent for metal ion pollution
The term 'biosorption' is generally defined as the binding process of ions to the surface of a biomass [42].Biosorption is one of the promising eco-friendly remediation technologies for heavy metal pollution.It involves biosorbent, or biosorption agent, that is derived from biological substances.One of the most important factors for a successful application of this method is the choice of biosorbent, which can be found easily and efficiently, inexpensive, and preferably having high sorption capacities [43,44].
Biomasses are abundant biological materials that are readily available in nature and certainly require less cost to be used or developed as biosorbents.One of the examples of novel biosorbent that can purify wastewater from Cu 2+ , Cd 2+ , Pb 2+ , and Zn 2+ heavy metals was coconut shell-based activated carbon, which can be easily found in agricultural areas [45].Another example of biomass as biosorbent was sodium alginate (SA), which enhances aerogel strength and function during light energy absorption [46].Biomass can also be chemically modified for specific purposes, such as the development of cotton fabric finishing with N-hydroxymethylacrylamide spirophosphate, which enhances the flame resistance property of the fabric [47].
One of the biomasses that had been largely proposed to meet the criteria as a biosorbent is biofilm [48,49].Biofilm is a predominant form, as well as habitat, for microbes in aquatic ecosystems [50,51].Compared to other biomass, the advantage of using biofilm is that it is readily found in virtually all types of ecosystems with a natural ability to absorb ions.Indeed, biofilm is confirmed to play important roles in aquatic environments, such as nutrient cycling and immobilization of dissolved pollutants [52].Considering this fact, biofilm matrices can be very useful as a biosorbent to remove water pollutants in the form of ions, particularly heavy metals [44].
In terms of efficiency, utilization of nanoparticles has an advantage given their increased surface-areato-volume ratio due to their smaller particle size, resulting in greater attractive forces with the target of interaction compared to larger particles, as demonstrated in the mucoadhesive delivery system [53].This advantage was applied in the employment of FeNi nanoparticles in biomass-derived carbon in wastewater treatment [54].
A study of gelatin bio-hybrid nanoparticles stated that the isoelectric point (IEP) of gelatin type A may reach 9.4, significantly higher than gelatin type B that was 4.5-6.0[55].This offers large possibilities when gelatin nanoparticles are applied in association with pH, charged molecules, or ions.However, in terms of bioremediation of heavy metal ions, the use of nanoparticles still warrants further studies.In comparison, biofilm is a natural resource that may need little to no modification for feasible use in pollution treatment.
Despite its potential, only a few studies have focused on natural biofilm matrices.Most of the previous studies utilized laboratory grown biofilms, which were cultivated from a single species of bacteria [56].The application of such engineered biofilms, while useful, may lead to misconception of the characteristics of native biofilm due to the possible differences in the matrices of the laboratoryproduced biofilms and those formed naturally in aquatic ecosystems [57,58].
This review investigated the application of natural biofilm matrices as biosorbents for three heavy metal pollutants.Considering that biofilms from different types of water ecosystems may have their own structures and characteristics, the naturally formed biofilm matrices compared in this review were collected from stone surfaces submerged in different aquatic environments.Upon collection, microscopic examinations confirmed that the main components of these biofilm pellets were microbes and biofilm polymers.The natural biofilms collected for the purpose of this study are then examined to study their properties, possible differences, and potential as biosorbents.

Biofilm from various freshwaters has a uniform electric charge property
The physicochemical interaction between the biofilm polymer charged sites and the ion in the surrounding water is the main impetus for ion adsorption to the biofilm matrix.In other words, adsorption takes place due to the attractive electrostatic interaction between ions and charged sites in the biofilm matrix [45,59].Interestingly, the observed profile of biofilm's electrophoretic mobility (EPM) displayed similar trends over a broad range of conditions.A study about biofilm formed on various surfaces such as stone, stainless steel, and wood submerged in Lake Biwa, Japan, concluded that there was a universal characteristic of the electric charge properties of biofilms, irrespective of the substrate types [59].A recent study also proposed that there were similar electrophoretic mobility values of biofilms of stone surfaces collected after 15, 30, and 60 formation days from a reservoir in East Java, Indonesia [44], suggesting that the electric charge pattern of biofilm does not depend on its maturity.
The measurement of the EPM of biofilm polymers was initiated by collecting biofilm fragments from the substrates.As many as 0.03 g of biofilm fragments were placed in an electric field, which works based on the light scattering method.The fragment's movement velocity was measured by electrostatic force under various pH conditions, as described previously [26,60].The result was an estimation of the functional group(s) producing the electric charge in the biofilm, which affects the pattern of interaction between the biofilm and the pollutant metal ions.
Observations of the EPM values in this review were conducted on stone biofilms originating from different rivers, streams, lakes, and drainage ditches.All the stones were submerged at a depth of 50-70 cm under the water surface.It is important to note that those freshwater ecosystems are polluted mainly by the three heavy metals: Mg(II), Cr(VI), and Cu(II).The pH values in those different sites were confirmed to be around neutral.The EPM was measured in a range of experimental pH values, spanning from pH 2.0 to 9.0, to analyze the electric charge properties of the biofilm matrix.
Figure 1 shows that the EPM values of the biofilm from the different freshwater ecosystems have similar trends with almost overlapping lines.There was a negative EPM value around pH neutral, and at pH 2, the EPM value was positive.Those reported patterns are identical with that of the previous studies [26,31,44,59,60].It can be concluded that regardless of the differences in the types of heavy metal pollutants, the freshwater ecosystems, and whether the water was stationary or flowing, the characteristics of the EPM of biofilms were the same.Considering that the substrates [59] and formation time [44] did not affect the EPM, the electric charge properties of native freshwater biofilms are thus universal.Therefore, these biofilms are comparable in terms of their adsorption and desorption capabilities for heavy metal pollutants.
As previously discussed, the negative and positive EPM values indicate the presence of the corresponding charged sites in the biofilm polymers, which is due to the ionization of certain functional groups [58,59,61].The large shift of EPM from positive to negative values at around pH 4.0 strongly indicated the presence of carboxyl groups, where pKa is around 4.0 [62].The positively charged sites may exist due to the amino groups in the biofilm polymers.Meanwhile, at pH 2.0, the negatively charged sites were protonated and became neutrally charged because of the abundance of proton ions.This then engenders the biofilm to have a net positive charge value.
The charge mechanism of biofilm's functional groups can be explained by previous observations that the addition of the biofilm samples to the buffer solution increased the pH of the buffer up to neutral pH range, indicating a decrease in the proton concentrations of the solutions in the acidic conditions [44,59].This phenomenon can be inferred as the activity of protons in the buffer that deionize the negatively charged functional groups of the biofilm polymers.The ionization of the biofilm's negatively charged functional groups was inhibited in the acidic buffer solution [63].Meanwhile, the decrease in pH in the alkaline condition is regarded to be caused by the neutralization of OH − , with H + released after certain functional groups such as the amino group got deprotonated [44,59].
The function of the negatively charged sites of biofilm has been discussed previously [64] and was often indicated as a defense mechanism against antimicrobial environment and increase drug tolerance [65][66][67].This review elaborates and proposes that the overall negative charge of the biofilm polymers may enable the biofilm to attract and adsorb the positively charged heavy metals, for example the Mg(II) and Cu(II) ions.The mechanisms of this process include electrostatic interactions and ion exchange mechanisms that will be further discussed by examining the kinetics and adsorption isotherm.

The kinetics of heavy metal adsorption occurs rapidly
To uncover the mechanism behind the biosorption of pollutant cations into the biofilm matrix, first, the kinetics of the three heavy metals adsorption into the biofilm were investigated.Cr(VI), in particular, is known to exist as different species at different pH.At pH value of 6.0, Cr(VI) exists as approximately 60% HCrO salt in this study as the experiments were conducted in pH neutral.A 0.5 g of biofilm pellet was resuspended to 50 mL of 25 ppm of MgCl 2 , K 2 CrO 4 , and CuCl 2 , respectively.The samples were taken at various incubation times.It was found that the reactions reach equilibrium within 1 min [60].The time courses of the reactions were extended to see whether there is any change of reaction kinetic after the equilibrium, in which there was no remarkable difference.The biofilm samples were measured with atomic absorption spectroscopy to determine the heavy metal adsorption.Time course adsorption of the metal pollutants into the biofilm matrix is demonstrated in Figure 2. The results informed that the adsorbed Mg(II), Cr(VI), and Cu(II) were relatively constant from 5 min mark until the end of the experiment at 180 min.Thus, it can be said that the biosorption of the three heavy metals by the biofilm matrix was a very rapid process.This type of process is a feature of the physicochemical mechanism that usually drives the biosorption [69].
Figure 2 further demonstrated that adsorption kinetics of Cr(VI) was lower than that of Mg(II) and Cu(II).Due to its unique property, chromium ions exist in aqueous solution in both cationic and anionic forms.Contrary to the Cr(III), the Cr(VI) cannot exist in aqueous solution as a mono atom due to its very high positive charge that would need an exceedingly large amount of energy [70].In such a condition, the Cr ion can only be stabilized by the most electronegative atoms.Thus, in aqueous solutions, Cr(VI) exists as polyatomic ions such as chromate (CrO 4 2-) and dichromate (Cr 2 O 7 2-) [70].
Chromate and dichromate ions have an overall negative charge.Therefore, it explained why Cr(VI) had lower adsorption kinetics at 2.25 mg/g as opposed to 2.5 mg/g of Cu(II) and 3.04 mg/g of Mg(II), as shown in Figure 2.Meanwhile, both Mg(II) and Cu(II) are positively charged, and it was reported that cations have a higher chance of being adsorbed, and retained, within biofilm's interstitial region than anion [32], since biofilm has an overall negative charge at neutral pH as indicated by the EPM results.Despite that, Figure 2 demonstrates that the adsorption kinetics of Mg(II) was higher than Cu(II).Although both of them are divalent metal ions, the ionic radius of Cu(II) is larger than Mg(II).Therefore, the charge density of Mg(II) is higher than Cu(II) [71], as charge density is inversely proportional to the ionic radii.The charge density is significantly influenced by the size of the ion, i.e. the ionic radii, whereas the magnitude of the electrostatic charge of the ion is indicated by the valence number.This confirms that electrostatic attractions that determine biosorption by biofilm depend on the ionic size variations, which is closely related to the charge density of the ions [58].
To sum up, the adsorption of Mg(II), Cr(VI), and Cu(II) into the biofilm may occur through passive uptake process [72].Biofilm does not actively absorb heavy metal ions.Instead, it takes in metal ions by means of entrapment and sorption to specific binding sites within the cellular structure, all independent of the biological metabolism process [72].The same characteristics of time course adsorption were also reported for the case of various ions adsorption to the other types of natural biofilms or biomass [73,74].

Adsorption isotherm parameters revealed both ion charge density and solubility may determine ion desorption
The aqueous solutions of MgCl₂, K 2 CrO 4 , and CuCl₂, each 25 mL, with concentrations of 1.5 to 550 ppm of the respective heavy metals were added to 0.25 g of biofilm pellet.After 15 min, the suspensions were centrifuged at 8,000×g, 4°C for 3 min to separate the pellet and the supernatant.The concentrations of the three heavy metals in their respective supernatants were measured using an Atomic Absorption Spectroscopy Shimadzu AA-6800 (Shimadzu Corporation, Japan).Considering the results of the kinetic of adsorption experiments, the contact time used in the adsorption isotherm investigation for this study was 10 min.
The heavy metals adsorbed into the biofilm were calculated from the differences of the Mg(II), Cr(VI), and Cu(II) concentrations between the supernatant and the control without biofilm.The biosorption characteristics of Mg(II), Cr(VI), and Cu(II) into biofilms were analyzed using a variant of Langmuir equation that can be described as Eq. ( 1).
The above equation assumes that a dynamic equilibrium exists between the adsorbed ion (N; mg/g) and the free ion in solution (C; ppm).The Langmuir constant (b) refers to the extent of interaction between adsorbate and adsorbent.A relatively large b value indicates a strong interaction, while a smaller value implies a weak interaction [21].In this equation, the adsorption equilibrium constant b is defined as the ratio of the adsorption and desorption rates.The plot of C/N against C yields a straight line with a slope of 1/N max and a y-axis intercept of 1/(N max )b.Then, the values of N max , which is the maximum amount of adsorbed ion, and b were calculated [3].
The mechanism of ions adsorption to biofilm is determined to be ion exchange mechanism [26,44].That is, the charge density of each ion very likely influences the strength of this interaction.In this case, the ions with a higher charge density can bind more strongly to the opposite charge sites in the biofilm matrix.Previous results indicated that the N max value in biofilm rises along with the increase in the positive charge of a cation, whereas the N max value decreases when there is an increase in the negative charge of an anion [26].Those findings therefore describe that the total ion adsorption to the interstitial region of the biofilm matrix, represented by the N max value, is not only promoted by the ion exchange mechanism but also by the electrostatic interactions [26,58].Additionally, it was discovered that ions with higher valency had a greater value of b than those with a lower valency.In other words, the greater attractive force between the ions and the biofilm or resins seems to result in a greater ability to retain ions [26].
For all the investigated heavy metal ions, the adsorbed concentrations increased along with the longer reaction time.Figure 3 demonstrates that the biofilm matrices from different aquatic ecosystems were able to adsorb the three metal ions within a similar range of concentrations, albeit with different trends.The higher availability of the ions in the surrounding solution in the lower concentration range of the three heavy metal ions seemed to promote the greater accumulation of the ions by the biofilm matrix.The effectiveness of 1.5-550 ppm of Mg(II), Cu(II), and Cr(VI) biosorptions were analyzed.
The biofilm matrix was able to take up ≥50% of Mg(II) and Cu(II) available in the surrounding solution, between the concentrations of 1.5 to 15 ppm.The adsorbed amount of Cr(VI) within that lower concentration range was closer to 50%.However, at higher concentrations, the number of the Cr(VI) taken up by the biofilm matrices was obviously lower than the other two heavy metals, with the Mg(II) adsorbed the most.In general, the results suggest that even though the amount of the adsorbed heavy metals has increased along with the increase of the initial concentration, the effectiveness of the biosorption was higher in the lower concentration range.
The biosorption of the three heavy metals by the biofilm matrix fitted well with the Langmuir Isotherm Model (R 2 = 0.95-0.98).Furthermore, the adsorption of Mg 2+ , Cr(VI), and Cu(II) into the biofilm matrix is likely to occur in the monolayer form.
Since adsorption is shown to occur within a short time frame, the most likely active agent for biosorption is the already-formed EPS of the mature biofilm.The monolayer structure is made up of the target ion and the biofilm charged sites.No other layer is formed by ion-ion interaction, with only charged sites and ions as the functioning monolayer.
The comparison between the Nmax and b values of each heavy metal ion is demonstrated in Table 1.The comparison shows that the Nmax of Mg(II) is the largest, followed by Cu(II) and Cr(VI) with the smallest Nmax value.However, the trend is reversed for b values, whereby Cr(VI) has the highest b value, whereas Cu(II) and Mg(II) follow in the second and third places.The Nmax value of Mg(II) indicated that it gets adsorbed at higher concentrations compared to the other heavy metal ions, while the greater b value of Cr(VI) indicates that the bonding between the ion and the biofilm is stronger than the other two heavy metal ions.
The results for b values implied that although the charge density of Mg(II) is higher than Cu(II) and Cr(VI), it got desorbed easier from the biofilm.It means that the charge density is not the only determining factor of the ion retention by the biosorbent.One possible explanation is that the solubility of each heavy metal may play a role particularly in determining the desorption that determines the b value.The solubility of Mg(II) is the highest among the three heavy metals [75].The second most soluble is Cu(II), followed by Cr(VI) in the last place [75].
The case of absorption of lithium ions might serve as an example of the influence on ion solubility.A large quantity of Li was adsorbed by the biofilm and at the same time K, Ca(II), Na, and Mg(II) were desorbed [76].This ion exchange phenomenon indeed happened to most, if not all, of the previously studied metal ions adsorption and desorption to and from the biofilm [26,31,32,44,[58][59][60]74].Lithium in elemental form is not water-soluble, although it reacts with water.The compound forms are somewhat soluble.This tendency may explain why the more soluble ions such as magnesium were not retained by biofilm, whereas the less soluble ones, like in cases of interaction with ion exchange resins, may not be desorbed as easily [76].In summary, it can be said that the physicochemical properties of a certain heavy metal pollutant will determine the rate of absorption and desorption, hence influencing the effectiveness of biofilm usage in a particular pollution case.

Future directions
As an environmentally friendly and natural biosorbent, the usage of biofilm to tackle pollution in freshwater aquatic ecosystems represents a promising solution for the environmental problem.The use of biofilm is also cost effective compared to the other remediation technologies [26,31,32,44,48,[49][50][51][58][59][60]74].This review analyzed and contrasted three heavy metal pollutants' biosorption characteristics to biofilm matrices collected from various aquatic ecosystems.It is discovered that the negatively charged patches of the natural biofilm matrices play an essential role as binding sites for the Mg(II) and Cu(II) heavy metals, which are positively charged.Although the polyatomic chromate and dichromate ions as the soluble forms of Cr(VI) are likely to possess an overall negative charge, the presence of positively charged patches in the biofilm still helped to adsorb this heavy metal, although with lower adsorption kinetics compared to Mg(II) and Cu(II).This review confirmed that the accumulation of three heavy metals by the biofilm matrix is a physicochemical process promoted by the attractive interaction between the negatively charged sites in the biofilm and the positively charged metal ions.Considering the universal electrical charge properties, biofilm matrices formed naturally in various freshwater environments studied in this review are the promising biosorbent for the biosorption of heavy metal pollutants such as Mg(II), Cu(II), and Cr(VI) in the lotic ecosystems.However, in line with the previous data and results, researchers may consider studying the physicochemical characteristics of each of the heavy metal pollutants that they wished to handle with biofilm as biosorbent.
Future studies should focus on the effect of physicochemical properties of a certain heavy metal ion.Characterization of the metal pollutants, especially regarding ionic size that relate directly to charge density and other properties such as solubility, would provide more insights to comprehend the mechanism of biosorption by biofilm matrix.To confirm the interconnection between the multiple physicochemical conditions of the target heavy metal with their absorption and desorption patterns, it would be beneficial to observe biosorption of heavy metal pollutants that belong to different groups or periods in the periodic table.
The findings and more insights about the effect of ion charge density and solubility will advance our understanding of the mechanism of biosorption with natural biofilm matrix.Endeavors to characterize the process of absorption of various heavy metal ions should also be renewed, followed by detailing the effect of charge density and solubility of the ions towards the ions' retention by biofilm.Determination of the biosorption patterns of common heavy metal pollutants will facilitate the more reliable design of effective bioremediation strategy that is less costly and more environmentally friendly.

Figure 2 .
Figure 2. Time course of Mg 2+ , Cr(VI), and Cu(II) biosorptions by biofilm matrix after various contact times.Experiments were repeated three times, independently.Bars represent the standard errors.

Figure 3 .
Figure 3. Adsorption isotherm of Mg 2+ , Cr(VI), and Cu(II) by the biofilm matrix.Experiments were repeated three times, independently.Bars represent the standard errors.