**2. Materials and methods**

#### **2.1. Low-cost biosorbents**

Since cyanide is toxic and Au and Ag being precious metals, non-renewable and finite resource;

The conventional methods adopted for the treatment of MxCN contaminated effluents is alkaline chlorination oxidation process (Ganczarczyk et al 1985). Although this method of treatment can be very efficient in detoxifying free cyanide bearing wastes, it is not effective when challenged with anionic metal species such as MxCN (Eckenfelder 1989). Other methods, such as copper catalyzed hydrogen peroxide oxidation, ozonation, electrolytic decomposition, etc. requires large inputs of energy, cost intensive materials and are rarely used for treatment of MxCN containing wastes. Furthermore, at low concentration, metal-cyanide recovery by conventional means is either not possible and/or very expensive. Thus, there is a big techno‐

Biological treatment systems (i.e. bioremediation) for the detoxification of toxic and hazardous wastes has immense potential of becoming and effective alternative because of their several advantages over conventional methods; and therefore being explored by the researchers all over the world (Patil et al., 2012; Patil and Paknikar, 1999b). However, biological methods like biodegradation / biodetoxification using live microorganisms are subject to toxicity of cyanide and metals. Therefore, removal of precious metal-cyanides species from wastes requires immediate attention of scientists and technologists. The challenge is not limited only to their removal, but also extends to finding competent and inexpensive ways of possible recovery and recycling. It was assumed that if a competent process for removal/recovery could be established, MxCN could be conserved, which in the authors opinion would be an innovative strategy of resource recovery. Since MxCN are anionic chemical species, therefore in principle, a well established physico-chemical methods can be used for removal and recovery of precious metal species. A few physico-chemical methods have been tried for adsorption of Au- and Agcyanide (Niu and Volesky 2001) and MxCN using activated carbon or inorganic chemically active adsorbents (Lee et al 1998). However, the practical utility and cost-effectiveness of these processes are not yet established. Biosorption of metal "cations" have been studied extensively (Paknikar et al 2003). However, very few attempts have been made to adopt this technology for possible removal and recovery of "anions" such as MxCN (Patil 2012; Patil and Paknikar 1999); especially Au- and Ag-cyanide (Niu & Volesky 2001). Literature clearly shows the paucity of references on the removal/recovery of precious Au- and Ag-cyanide using low-cost

It is known that biomass like bacteria, fungi, algae, plants, agricultural biomass and different agro-based industrial waste and byproducts have the ability to bind metals, in some cases selectively, from aqueous solutions (Paknikar *et al.*, 2003). This phenomenon is named as 'metal biosorption' and the biomass responsible for the process are known as 'biosorbents'. Biosorp‐ tion is combination of the processes such as electrostatic interactions, ion exchange, complex‐ ation, formation of ionic bonds, precipitation, nucleation, etc. Biomass surfaces are usually charged. The functional groups like phosphoryl, carboxyl, sulphahydryl and hydroxyl of membrane proteins, lipids and of other cell wall components are responsible for adsorption of metal (both cationic and anionic species). The overall interfaces are a result of complexity of biomass surfaces and chemical/ physical properties of metal ions (Modak and Natarajan,

their complete removal from effluents is the key.

256 Applied Bioremediation - Active and Passive Approaches

logical breach, which needs to be bridged immediately.

waste biomass.

The low-cost biosorbents in the present study were collected from diverse sources (as given below). Some of these biosorbents are reported for the removal of diverse metal species from acqueous solutions (Mohan & Pittman, 2006), while some of them have been employed for the first time.


**2.4. Gold-cyanide (DCAU) and silver-cyanide (DCAG) biosorption studies**

neously to detect the air stripping of cyanide, if any, to confirm biosorption.

mg/l Au and 2.08 mg/l CN-

Cl- , PO4

cations viz. Cu2+, Ni2+, Zn2+, Cd2+, Pb2+, Fe2+, Ag+

**2.5. Adsorption isotherms**

solution (ml); Ci

methanol and acetic anhydride prior to sorption.

(Q) was calculated using the following equation:

A batch equilibration method was used to determine the sorption of DCAU (0.02 mM i.e. 3.94

Resource Recovery from Industrial Effluents Containing Precious Metal Species Using Low-Cost Biomaterials…

(0.05 to 0.2 g) was contacted with 10 ml solution of DCAU or DCAG of desired pH in a set of 50 ml capacity conical flasks. The flasks were incubated on rotary shaker incubator adjusted to a speed of 150 rpm at 30°C for 1 h. Contents of flasks were filtered using ordinary filter paper and then analysed for residual Au, Ag and cyanide. All experiments were performed in duplicates and repeated twice to confirm the results. Appropriate controls were run simulta‐

Influence of pH on biosorption of DCAU/DCAG was checked in the range of 4-10 with preconditioned biomass. On the basis of maximum DCAU/DCAG uptake values obtained under optimum pH conditions, efficient biosorbents were selected for further studies. DCAU/DCAG loading capacity (μmol DCAU/DCAG bound per gram weight of biosorbent) of each biosorb‐ ent was determined by contacting 0.1 g powdered biomass several times with fresh batches of 10 ml DCAU/DCAG solution till saturation was achieved. To determine optimum biosorbent amount, DCAU/DACG was contacted with varying amounts of biomass powder, ranging from 0.1 to 5% (w/v). Rate of DCAU/DCAG uptake was studied by contacting the biosorbent for a period ranging between 0 to 5 h. Under optimised conditions, effect of various competing

3-, etc. (0.1-1 mM) on biosorption of DCAU/DCAG was also checked. In order to test

the effect of pre-treated biomass on uptake of DCAU/DCAG, the biosorbent were treated for one hour using L-cysteine, boiling water, sodium hydroxide, formaldehyde, acetone, acetate,

To study the impact of initial concentration on adsorption, varying concentration of DCAU/ DCAG was used in the range of 0.01 to 1 mM. In order to obtain sorption data, uptake value

Where, Q is DCAU/DCAG uptake (mmol per gram biomass); V is the volume of DCAU/DCAG

mass of sorbent (g). Based on the 'Q' value obtained adsorption isotherms were plotted

according to Freundlich and Langmuir equations (Freundlich, 1926; Langmuir, 1918):

is the initial concentration (μmol); Cf

) and DCAG (0.1 mM i.e. 10.78 mg/l Ag and 5.2 mg/l CN-

, etc. (0.01-0.1 mM) and anions viz. SO4

Q = V C – C / 1000 m ( ) i f (1)

( ) eq ln Q = ln K + 1/n C Freundlich equation (2)

C / Q = 1 / b Q + C / Q Langmuir equation eq ( ) max eq max ( ) (3)

is the final concentration (μmol); m is

). Biosorbent

259

http://dx.doi.org/10.5772/56965

2-, NO3 - ,

**vii. Reference material:** Activated charcoal was employed as a reference material in order to obtain comparative data.

Biomass samples were collected in polythene bags and transported to laboratory. The samples were washed several times with tap water to remove the dirt and other contaminants, if any, and was then finally washed with deionised water (< 5 μS). Biomass were then subjected for drying at 50°C for 48-72 h to a constant weight and powdered. Dried biomass was pulverized employing electric mixer and sieved; so as to get uniform particle size of ≤ 500 μm (0.5 mm).

## **2.2. Synthesis of stock anionic MxCN solutions**

The stock solutions of Au-cyanide i.e. [Au(CN)2] - (Dicyanoaurate-DCAU) and Ag-cyanide i.e. [Ag(CN)2] - (Dicyanoargentate - DCAG) were prepared stoichiometrically by combining their respective salts with sodium cyanide in the molar proportion of 1:2 (Patil & Paknikar, 2000a; Patil and Paknikar,2000b; Rollinson et al., 1987). Spectral properties were checked and confirmed periodically using UV spectrophotometer. The synthesized DCAU and DCAG solutions were refrigerated at 8-10°C.

#### **2.3. Chemicals and analyses**

Chemicals used for all experiments were of analytical grade (AR). Glassware used were made of borosilicate material. Stock solutions and reagents were prepared in deionized water (< 5 μS) and stored in refrigerator (8-10°C). Gold (Au), silver (Ag), nickel (Ni), copper (Cu), zinc (Zn) and iron (Fe) in the experimental solutions and effluents were analysed using Atomic Absorption Spectrophotometer (Elico, India SL-173). Total cyanide and chemical oxygen demand (COD) content in the solutions were estimated by pyridine-barbituric acid and reflux method, respectively as described in Standard Methods (APHA-AWWA-WEF, 1998). Phos‐ phates (PO4 -3) from effluents were analysed by phenol- disulphonic acid method; sulphates (SO4 -2) were determined by barium chloride method while chlorides (Cl- ) were determined by argentometric method, as per the methods prescribed in Standard Methods (APHA-AWWA-WEF, 1998). Colour and turbidity were recorded by visual observations. pH and electrical conductivity from solutions was measured by their respective meters.

In order to determine the inherent/actual pH of each powdered unconditioned biomass, the biomass sample and RO water were mixed and serially diluted in the ratio of 1:20, 1:30, 1:40 and 1:50 (w/v) in conical flasks. The contents were stirred vigourously and kept for one hour in stationary conditions and was followed by determining the pH of each dilution ratio. pH value obtained for each dilution was then plotted on graph of "pH against water-to-biomass ratio (v/w)". The straight line obtained after joining all the points was extrapolated backwards so as to intersect with Y-axis (i.e. the pH scale).

### **2.4. Gold-cyanide (DCAU) and silver-cyanide (DCAG) biosorption studies**

A batch equilibration method was used to determine the sorption of DCAU (0.02 mM i.e. 3.94 mg/l Au and 2.08 mg/l CN- ) and DCAG (0.1 mM i.e. 10.78 mg/l Ag and 5.2 mg/l CN- ). Biosorbent (0.05 to 0.2 g) was contacted with 10 ml solution of DCAU or DCAG of desired pH in a set of 50 ml capacity conical flasks. The flasks were incubated on rotary shaker incubator adjusted to a speed of 150 rpm at 30°C for 1 h. Contents of flasks were filtered using ordinary filter paper and then analysed for residual Au, Ag and cyanide. All experiments were performed in duplicates and repeated twice to confirm the results. Appropriate controls were run simulta‐ neously to detect the air stripping of cyanide, if any, to confirm biosorption.

Influence of pH on biosorption of DCAU/DCAG was checked in the range of 4-10 with preconditioned biomass. On the basis of maximum DCAU/DCAG uptake values obtained under optimum pH conditions, efficient biosorbents were selected for further studies. DCAU/DCAG loading capacity (μmol DCAU/DCAG bound per gram weight of biosorbent) of each biosorb‐ ent was determined by contacting 0.1 g powdered biomass several times with fresh batches of 10 ml DCAU/DCAG solution till saturation was achieved. To determine optimum biosorbent amount, DCAU/DACG was contacted with varying amounts of biomass powder, ranging from 0.1 to 5% (w/v). Rate of DCAU/DCAG uptake was studied by contacting the biosorbent for a period ranging between 0 to 5 h. Under optimised conditions, effect of various competing cations viz. Cu2+, Ni2+, Zn2+, Cd2+, Pb2+, Fe2+, Ag+ , etc. (0.01-0.1 mM) and anions viz. SO4 2-, NO3 - , Cl- , PO4 3-, etc. (0.1-1 mM) on biosorption of DCAU/DCAG was also checked. In order to test the effect of pre-treated biomass on uptake of DCAU/DCAG, the biosorbent were treated for one hour using L-cysteine, boiling water, sodium hydroxide, formaldehyde, acetone, acetate, methanol and acetic anhydride prior to sorption.

#### **2.5. Adsorption isotherms**

**v. Algae:** Mixed algae biomass obtained from lake Rankala, Kolhapur

leaves and *Lantana camara*.

258 Applied Bioremediation - Active and Passive Approaches

to obtain comparative data.

**2.2. Synthesis of stock anionic MxCN solutions**

The stock solutions of Au-cyanide i.e. [Au(CN)2]

solutions were refrigerated at 8-10°C.

**2.3. Chemicals and analyses**

[Ag(CN)2]

phates (PO4

(SO4

**vi. Terrestrial and aquatic plant species:***Parthenium* sp., *Eichhornia* root biomass,

**vii. Reference material:** Activated charcoal was employed as a reference material in order

Biomass samples were collected in polythene bags and transported to laboratory. The samples were washed several times with tap water to remove the dirt and other contaminants, if any, and was then finally washed with deionised water (< 5 μS). Biomass were then subjected for drying at 50°C for 48-72 h to a constant weight and powdered. Dried biomass was pulverized employing electric mixer and sieved; so as to get uniform particle size of ≤ 500 μm (0.5 mm).

*Eichhornia* stem biomass, *Eichhornia* leaf biomass, Runners, *Tectona grandis* waste



respective salts with sodium cyanide in the molar proportion of 1:2 (Patil & Paknikar, 2000a; Patil and Paknikar,2000b; Rollinson et al., 1987). Spectral properties were checked and confirmed periodically using UV spectrophotometer. The synthesized DCAU and DCAG

Chemicals used for all experiments were of analytical grade (AR). Glassware used were made of borosilicate material. Stock solutions and reagents were prepared in deionized water (< 5 μS) and stored in refrigerator (8-10°C). Gold (Au), silver (Ag), nickel (Ni), copper (Cu), zinc (Zn) and iron (Fe) in the experimental solutions and effluents were analysed using Atomic Absorption Spectrophotometer (Elico, India SL-173). Total cyanide and chemical oxygen demand (COD) content in the solutions were estimated by pyridine-barbituric acid and reflux method, respectively as described in Standard Methods (APHA-AWWA-WEF, 1998). Phos‐

argentometric method, as per the methods prescribed in Standard Methods (APHA-AWWA-WEF, 1998). Colour and turbidity were recorded by visual observations. pH and electrical

In order to determine the inherent/actual pH of each powdered unconditioned biomass, the biomass sample and RO water were mixed and serially diluted in the ratio of 1:20, 1:30, 1:40 and 1:50 (w/v) in conical flasks. The contents were stirred vigourously and kept for one hour in stationary conditions and was followed by determining the pH of each dilution ratio. pH value obtained for each dilution was then plotted on graph of "pH against water-to-biomass ratio (v/w)". The straight line obtained after joining all the points was extrapolated backwards


conductivity from solutions was measured by their respective meters.

so as to intersect with Y-axis (i.e. the pH scale).


) were determined by

To study the impact of initial concentration on adsorption, varying concentration of DCAU/ DCAG was used in the range of 0.01 to 1 mM. In order to obtain sorption data, uptake value (Q) was calculated using the following equation:

$$\mathbf{Q} = \mathbf{V} \left( \mathbf{C}\_{i} - \mathbf{C}\_{\mathbf{f}} \right) / 1000 \text{ m} \tag{1}$$

Where, Q is DCAU/DCAG uptake (mmol per gram biomass); V is the volume of DCAU/DCAG solution (ml); Ci is the initial concentration (μmol); Cf is the final concentration (μmol); m is mass of sorbent (g). Based on the 'Q' value obtained adsorption isotherms were plotted according to Freundlich and Langmuir equations (Freundlich, 1926; Langmuir, 1918):

$$\ln \mathbf{Q} = \ln \mathbf{K} + \begin{pmatrix} 1/\mathbf{n} \end{pmatrix} \mathbf{C}\_{\text{eq}} \text{ Freundlich equation} \tag{2}$$

$$\mathbf{C\_{eq}} / \mathbf{Q} = \left( \mathbf{l} \, / \, \mathbf{b} \, \mathbf{Q\_{max}} \right) + \left( \mathbf{C\_{eq}} / \, \mathbf{Q\_{max}} \right) \, \text{Langmutuir equation} \tag{3}$$

Where, Ceq is the liquid phase concentration of DCAU/DCAG; b is Langmuir constant; Qmax is maximum DCAU/DCAG uptake; K is constant; n is the number of metal reactive sites and Q is the specific metal uptake.

rate of 40 ml/h using programmable peristaltic pump (Enertech-Victor, India). All connecting silicon tubings used in the experiments were of 0.5 cm outer diameter and 0.3 cm inner diameter. Gold-cyanide effluent was passed through the column no. 1 upto 50 bed volumes, while silver-cyanide effluent was passed upto 34 bed volumes till the breakthrough curve (Sshaped) was obtained. Samples were collected periodically after every two hours and analysed

Resource Recovery from Industrial Effluents Containing Precious Metal Species Using Low-Cost Biomaterials…

Unrecoverable (residual) gold-cyanide and silver-cyanide in the solutions after biosorption treatment (in batch studies) were subjected to biodegradation using "live chemoheterotrophic bacterial consortium". The consortium (comprising of three *Pseudomonas* sp. in a proportion of 1:1:1) capable of degrading free cyanide and thiocyanate as the source of nitrogen was isolated by enrichment culture technique (Patil, 2008) and was available in my laboratory. Biodegradation experiment were conducted under aerobic and optimum conditions of pH

biodegradation process was used as a polishing step to clean the effluent containing traces of

Data in Table 2 summarizes the results obtained for DCAU and DCAG sorption under optimal pH conditions. The results showed that optimum sorption in terms of Q (i.e. μmol MxCN sorbed per gram biomass) of 0.02 mM DCAU and 0.1 mM DCAG for most of the waste biomass/ sorbents tested were at pH 4.0 and 6.0, respectively. It was observed that biosorption of DCAU and DCAG was less above pH 7.0 for all the biomass tested. In acidic pH conditions, sorption of DCAU and DCAG increased significantly. The table also shows that other than activated charcoal (chosen as reference material) which showed highest biosorption capacity, biomass of Rice husk (3.65 μmol/g) and *Eichhornia* roots (3.56 μmol/g) were efficient biosorbents for DCAU sorption; while *Eichhornia* roots (4.76 μmol/g) and Tea powder waste (4.73 μmol/g) were efficient biosorbents for DCAG. The overall Q values observed for all the waste sorbents tested for DCAU and DCAG were in the range of 2.69 - 3.65 μmol/g and 2.74 - 4.76 μmol/g, respectively. The observed Q values for efficient biomass were found to be marginally below the Q values obtained for activated charcoal (3.80 - 5.00 μmol/g). As far the optimum pH for sorption was concerned, DCAU uptake was maximal at pH 4.0 for all the biomass tested, while DCAG uptake for majority of the biomass was at pH 5.0 to 6.0. There was no loss of DCAU or

Table 2 also shows the data on pH values of all unconditioned biomass. Other than the reference materials, the lowest pH observed was that of coconut fibers (pH 4.24), while the highest pH was of mixed algae biomass (pH 7.61). pH of unconditioned Rice husk, Tea powder waste and *Eichhornia* root biomass observed were 5.94, 4.94 and 7.01, respectively, while their

cells/ml) and glucose concentration (1 mM). The

http://dx.doi.org/10.5772/56965

261

for Au, Ag and total cyanide content.

**2.9. Biodegradation of residual MxCNs**

(7.0), temperature (30°C), inoculum size (107

**3. Results**

cyanide in order to meet the requirements of statutory agencies.

**3.1. Screening of low-cost waste biomass for DCAU and DCAG sorption**

DCAG in the control flasks without sorbent during the tested time period.

#### **2.6. Adsorption/desorption of DCAU and DCAG**

Samples of 1 g biosorbent loaded with target MxCN was eluted using desorbing agent (1-3 N NaOH) in concentrated form and analysed. Following the elution, biosorbent was washed with DW and then again conditioned to appropriate optimum pH to use in next adsorption/ desorption cycle.

#### **2.7. Biosorption of Au- and Ag-cyanide from industrial wastewaters**

Two types of effluents were procured from silver and gold plating industry. Both effluents were subjected to characterization using Standard methods (APHA-AWWA-WEF, 1998). The proximate analysis of the samples is shown in Table 8 and 9. Batch equilibration method was followed as mentioned earlier. Rice husk (0.1 g) and *Eichhornia* root (0.1 g) biomass was contacted with 10 ml of gold-cyanide and silver-cyanide effluents, respectively. Prior to sorption, the gold- and silver-cyanide effluents were adjusted to desired optimum biosorption pH. All the batch sorption experiments were carried out under optimum conditions as given in Table 1. After contact, the contents of the flasks were filtered and then analysed for residual metal (i.e. gold and silver) and cyanide. Appropriate controls were run simultaneously.


**Table 1.** Optimum conditions used for biosorption experiments

#### **2.8. Continuous biosorption studies using fixed bed column at laboratory level**

Scale-up studies in fixed bed continuous mode at laboratory level for biosorption of gold- and silver-cyanide was carried out in two separate fabricated glass columns of height 44 cm, internal diameter 1.3 cm and filter media height being 30 cm. The total volume of the column was 58.37 cm3 , while the working volume was 39.80 cm3 (figure not shown). Glass column no. 1 was filled with 21 g rick husk biomass pretreated with L-cysteine, while the glass column no. 2 was filled with 24 g *Eicchornia* root biomass also pretreated with L-cysteine. The target effluents were passed through the columns in upward direction in continuous mode at a flow rate of 40 ml/h using programmable peristaltic pump (Enertech-Victor, India). All connecting silicon tubings used in the experiments were of 0.5 cm outer diameter and 0.3 cm inner diameter. Gold-cyanide effluent was passed through the column no. 1 upto 50 bed volumes, while silver-cyanide effluent was passed upto 34 bed volumes till the breakthrough curve (Sshaped) was obtained. Samples were collected periodically after every two hours and analysed for Au, Ag and total cyanide content.
