**2.2 Microbial**

 There have been many studies on the effects of metals on soils and water in the natural environment and particularly on the microflora that interact with these metals [59, 60]. Such studies have revealed that soil and natural water streams contain a range of microorganisms with the capability of metal transformation. Such effects can be exploited not only in soil remediation but also in utilising such microorganisms as tools for remediation of wastewaters contaminated with metals. There are currently several categories of interaction that can be observed between microbial populations and metal species as summarised in **Table 6**.


**Table 4.**  *Biochar activation methods.* 


*Advances in Metal Recovery from Wastewaters Using Selected Biosorbent Materials… DOI: http://dx.doi.org/10.5772/intechopen.84335* 

#### **Table 5.**

*Typical metal adsorption levels for unmodified and modified biochars.* 

Fungal and bacterial species that can grow and metabolise in the presence of metal species have been demonstrated to possess a variety of mechanisms of resistance that in many cases have a genetic basis [67–69]. The nature of the microbial species and the mechanism used are often dependent on the metal species present, and several mechanisms have been identified [67]. These include exclusion of the metal species by production of an extracellular matrix that acts as a barrier to entry of the metal. Such material known as extracellular polysaccharide substance (EPS) binds metal species and can have biotechnological applications [2]. Other techniques involve active metal efflux which is often associated with microbial mobile genetic elements [70], sequestration of metals, enzymatic detoxification exemplified by Hg resistance mechanisms and reduction of target sensitivity of the microorganism [67]. Understanding such mechanisms offers insights into the methodologies that have evolved in biological systems over millions of years and may offer new biotechnical approaches that can be exploited for metal remediation.

#### *2.2.1 Microbial adsorption mechanisms*

Microbial EPS material is often associated with the formation of stressresponsive structures on the surface of microbial cells during biofilm formation, and thus its production can be adaptive or protective in nature [2]. EPS can exist in many forms and can be genera specific in its chemical composition. In general


#### **Table 6.**

*Interaction between microbial populations and metal species.* 

 EPS consists of repeating monosaccharide units forming hetero- or homopolymers linked in glycosidic linkage which in turn are linked to a lipid anchor. The complex composition and branching of the EPS with many phosphate, amino sugars and hexuronic acid residues [2, 71] give the EPS structure an anionic charge which can be utilised to protect the microbe from metal toxicity. Key chemical groups on microbial surfaces include carboxyl groups associated with microbial peptidoglycan, phosphate groups on surface material, uronic acid and charged amino acid groups [2].

 There have been many studies of the utility of microbial cells in metal binding [72] although few actual processes have yet been developed. Cyanobacteria and algae have been extensively studied to bind a variety of toxic metals in natural water systems in polluted environments [73–76]. The rationale for their use is that many cyanobacterial and algal species are normal inhabitants of these water courses, and hence their presence would be less environmentally intrusive. Biosorption of a variety of metals has been studied including antimony (Sb), Ni, Cd, Cu, Pb, Co, Mn, As and Zn [2]. A rotating biological reactor containing microbial biofilms has previously demonstrated that metals can be accumulated in a process environment [77], and while this is as yet one of the few processes that have been developed, the potential to utilise microbial biomass is evident. Immobilised photosynthetic bacteria have been utilised to remediate swimming pool muds in Fukushima following the radioactive leak as a result of a Japanese tsunami [78] demonstrating the potential practicality of utilising microbes for metal remediation, in this case radioactive nuclides.

**Table 7** outlines a number of microbes which have been tested for their metal biosorbent capabilities. An uptake of 277.5 mg Pb(II) g<sup>−</sup><sup>1</sup> from aqueous solution at an optimum pH of 6.8 was measured in a study using the exopolysaccharides from *Paenibacillus peoriae* strainTS7 [79]. Another study which used the biosorbent *Anabaena doliolum Ind1* has outlined the presence of a range of surface groups including carboxyl, carbonyl, hydroxyl, amides and sulphate groups as being key binding sites for metals such as Cd(II) [80]. In a study which examined equilibrium and kinetic and thermodynamics of aqueous Al biosorption by *Streptomyces rimosus*  biomass, the presence of methyl, hydroxyl, amine, carboxyl, thiol and phosphate groups was identified as significant binding sites [81], while fatty acid, amide, lipids *Advances in Metal Recovery from Wastewaters Using Selected Biosorbent Materials… DOI: http://dx.doi.org/10.5772/intechopen.84335* 


#### **Table 7.**

*Microbes used for extracellular polysaccharide substance (EPS)-assisted heavy metal removal from selected wastewaters.* 

and protein moieties were identified as significant contributors to the adsorption of Pb(II) on *Providencia vermicola strain SJ2A* [82]. A study to measure the biosorption of Al(III) from waste streams using a *Rhodococcus opacus* strain reported a removal rate of 41.6 mg g<sup>−</sup><sup>1</sup> and indicated that the presence of amine, alkyl, carbonyl and phosphate surface functionalities were significant in its removal [84]. There are several potential advantages of utilising microbial biosorption. They can be classed as environmentally safe and do not generate toxic by-products, while in addition both live and dead cells can be used. On the downside however, mild desorption must be used to recover metals, while the efficiency of biosorption may be low and dependent on sensitive physiochemical parameters. In addition metals may affect the viability of adsorbing strains and limit reuse and cycling [85].

### **2.3 Lignins**

#### *2.3.1 Sources and production of lignin*

 Lignin is a natural biopolymer which makes up typically 15–30% of wood and grass biomass and provides structural rigidity to many plant cell walls. Its structure depends largely on the plant type, age and growth location [86]. Wood-based lignins in particular have been used for adsorption of air pollutants, organics and heavy metals due to their physicochemical properties, low cost, abundant availability and extent of active adsorption sites [87]. Separation and isolation of lignin from cellulose is generally quite difficult due to condensation and oxidation reactions that occur during the separation process which generally consist of either chemical (e.g. alkaline pulping or acid hydrolysis) or mechanical separation processes (e.g. ball milling). In industry, significant quantities of lignin (approximately 70 million tonnes in 2017) are produced as a by-product of the'kraft' paper manufacturing process. In this process woodchips are treated with sodium hydroxide (NaOH) and sodium sulphide (Na2S) to separate lignin from the cellulose fibres of wood which results in a black-coloured lignin-rich liquor. Several efforts to extract lignin from the liquor and use it as an adsorbent of inorganic pollutants, including metals, have been made with varying degrees of success [88, 89].

#### *2.3.2 Unmodified lignin as an adsorbent*

Lignin polymers are hydrophobic in nature containing carboxyl, hydroxyl and phenolic surface groups, which give them an affinity for metal ion adsorption. Their specific surface area is relatively low, typically around 100–200 m2 g<sup>−</sup><sup>1</sup> [90]. Similar to biochars, the absorption research focus to date has been on heavy metals such as Cu, Cd, Cr, Pb and Zn, with typical adsorption rates using unmodified lignin-based adsorbents between 4.2 mg Cu g<sup>−</sup><sup>1</sup> using straw-based lignin and 137 mg Cd g<sup>−</sup><sup>1</sup> using lignin from kraft liquor (**Table 8**). The pH of the solution significantly influences metal uptake as described in the previous section on biochars, with generally higher adsorption rates found at pH 5–6.

Unmodified lignins tend to have relatively low aqueous metal adsorption capacity and in addition have poor selectivity for certain metals [94]. Because of this, modification and activation is frequently carried out on lignins to improve their metal adsorption properties.

#### *2.3.3 Modification of lignins to enhance adsorption*

 Lignins can be chemically modified to improve their physicochemical properties, in particular their adsorption capacity, hydrophobicity and hydrophilicity as well as their overall stability. The modifications are usually carried out with oxygen-, nitrogen- or sulphur-containing functional groups which react mostly with the primary phenolic hydroxyl groups. For metal adsorption, the principal functional groups are divided into three categories [94]. The first category is oxygen-containing functional groups where acid treatment increases the naturally occurring fraction of oxygen groups as well as the hydrophobicity of the lignins. This process can significantly increase the adsorption rates for metals such as Cd and Pb ([95], **Table 9**) provided the pH is also controlled. In general, oxidised lignins have been shown to exhibit stronger adsorption capabilities than unmodified lignins due to the higher amounts of carboxyl groups present [96]. The second category is nitrogen-containing functional groups such as amines and triazoles, which have a high affinity for soluble metals and can be grafted onto the lignin by a Mannich reaction [97]. It has been shown that nitrogen-modified lignin increased the adsorption capacity for Pb(II) by over four times that of the original lignin [98].


#### **Table 8.**

*Typical metal adsorption capacities of unmodified lignins.* 


*Advances in Metal Recovery from Wastewaters Using Selected Biosorbent Materials… DOI: http://dx.doi.org/10.5772/intechopen.84335* 

#### **Table 9.**

*Typical metal adsorption capacities of modified lignin.* 

The third category is sulphur-containing functional groups which have a strong affinity to metal ions such as Cd, Cu, Pb, Hg and others and are therefore used to enhance the adsorption properties of lignin (**Table 9**).

#### *2.3.4 Activation of lignins to enhance adsorption*

Lignin is one of a number of source materials used to manufacture activated carbons, which are commonly used in the water industry for removal of dissolved organic and inorganic pollutants. Activated carbons normally come it two types, granular activated carbon (GAC) and powdered activated carbon (PAC). GAC is normally used as a tertiary filter in water treatment processes and typically comprises particles of size 0.5–1.5 mm. The operation of a GAC filter is similar to that of a sand filter where GAC can be regenerated once it reaches a particular saturation ratio. PAC on the other hand comprises smaller particles, typically <0.2 mm, which are added to the water as an adsorbent. PAC tends to have very high adsorption rates due to its high specific surface area, but unlike GAC, it is very difficult to regenerate because of the difficulty in recovering the PAC powder from the water. Lignin-derived activated carbons can have very high adsorption rates because of their high micropore volume of up to 1 cm3 g<sup>−</sup><sup>1</sup> and large specific surface area in the range 500–2000 m2 g<sup>−</sup><sup>1</sup> [108, 109]. Preparation of lignin-based activated carbon can be a two-step physical process comprising carbonation and activation or a one-step chemical process. In the two-step physical process, carbonation is achieved by pyrolising the lignin in an inert atmosphere at temperatures in the range 600–900°C,

which results in the formation of a char. Activation then follows by further heating to higher temperatures of 700–1200°C during which the porosity is developed using agents such as CO2, N2, air or steam. This activation step increases the surface area and pore volume by removing internal carbon mass and volatile organic residues. Chemical activation on the other hand normally uses chemical agents such as NaOH and KOH to impregnate the lignin which is then pyrolised at temperatures of 500–900°C in an inert atmosphere. In this process carbonisation and activation occur simultaneously resulting in activated carbon with an open porous structure and high specific surface area [110].

Typical adsorption rates of heavy metals from water, using wood-based activated carbons, range from 5.7 mg Cu g<sup>−</sup><sup>1</sup> using rubberwood sawdust to 255 mg Zn g<sup>−</sup><sup>1</sup> using oakwood (**Table 10**) and are influenced by the pH of the solution. For example, in a study to measure Cu(II) adsorption onto activated carbon-derived from rubberwood sawdust, optimum adsorption was measured at pH 6; however, at pH < 5, uptake decreased because of competition between H<sup>+</sup> protons and free Cu(II) ions to the fixation sites [111]. Similarly, optimum adsorption of Cu, Ni and Zn onto a wood-based activated carbon powder was observed at pH 6, decreasing at pH < 6, while metal precipitation was observed at pH > 6 [112].

Unlike unmodified lignins, modified and activated lignins demonstrate a high adsorption capacity for metals and are considered a promising biotechnology for their adsorption from wastewater. Although abundantly available, one of the difficulties with lignin is its heterogeneity and therefore its ability to consistently produce suitable adsorbents. Lignin quality is very much dependent on its source as well as the processes used to isolate it, and these and other factors very much influence its metal adsorption capacity and selectivity.
