Abstract: The aim of this research is to study the adsorption of mercury from aqueous media by an Acetobacter xylinum biofilm. Effects of pH, adsorption time, initial concentration and the adsorbent dosages on the adsorption of mercury were studied. The influence of pH on the adsorption onto the biofilm was studied in the pH range. The surface charge density varied with pH and the concentration of mercury adsorbed significantly increased from pH 5.0 to maximum levels at pH 8.0. Adsorption equilibrium was established after about 50 min, after which point the level of adsorbed mercury did not significantly change with time. The adsorption equilibrium was also represented with Langmuir and Freundlich adsorption isotherms. The adsorption capacity was 180 mg g-1. Desorption studies were performed with hydrochloric acid. The results suggest that an Acetobacter xylinum biofilm could be used as an effective adsorbent for the removal of mercury.
INTRODUCTION
Mercury is generally considered to be one of the most toxic metals found in the environment. This heavy metal may be introduced into the natural environment in any form and from a variety of sources, at which point it can be converted into more toxic volatile forms (i.e., methyl mercury chloride, ethyl mercury chloride and phenyl mercury chloride) by microorganisms and abiotic processes (Yardim et al., 2003; Choong and Park, 2005). In an aqueous environment, mercury in sediment is subject to methylation, forming the more toxic methylmercury. Once mercury enters the food chain, larger accumulation of mercury compounds takes place in humans and animals (Heyes et al., 2004). The major sources of mercury pollution in the aquatic environment are industries such as chloralkali, paint, pulp and research, oil refining, electrical, rubber processing and fertilizer (Manohar et al., 2002). Mercury causes damage of the central nervous system and chromosomes, impairment of pulmonary function and kidneys, chest pain and dyspnoea. Hence it is essential to remove mercury from wastewaters before its transport and cycling into the environment (Kacar et al., 2002). Various types of technology are available for removing mercury from water and wastewater. These include chemical precipitation, conventional coagulation, lime softening, reverse osmosis, ion-exchange and activated carbon adsorption (Cyr et al., 2002). The use of biological materials for heavy metal removal and recovery technologies has gained high credibility during recent years, due to high-quality performance and low cost of the materials. Developments in the field of environmental biotechnology indicate that bacteria, fungi, yeast and algae and their products can remove heavy metals from aqueous solutions by adsorption (Roux and Fourest, 1997). Cellulose is the most common biopolymer in the world, with an estimated production of 1011 ton/year. Most of this is produced in plant cell walls. The most popular and industrially important isolation of cellulose from plants including separation processes to remove lignin and hemicelluloses. The other way consists of the biosynthesis of cellulose by different types of microorganisms. Several bacteria are in condition to produce cellulose as reported from strains from the genera Acetobacter, Agrobacterium, Pseudomonas, Rhizobium and Sarcina. Acetobacter xylinum is a gram-negative aerobic bacterium that secretes cellulose fibers as part of its normal metabolic activity (Rainer and Luiz, 1998). The cellulose synthesized by Acetobacter xylinum is identical to that made by plants with respect to its molecular structure. However, the secreted polysaccharide is free of lignin, pectin and hemi cellulose, as well as biogenic products, which are associated with plant cellulose (Hong et al., 2001). This cellulose has high crystallinity, high water absorption capacity and mechanical strength in the wet state, ultra-fine network structure and availability in an initial wet state (Astley et al., 2001). Because of these features there is an increasing interest in the development of new fields of application (Klemm et al., 2001). A wide variety of natural products, composed primarily of cellulosic matrix, were investigated by different researchers for their ability to remove various heavy metals from aqueous and nonaqueous solution (Bailey et al., 1999; Chakravarty et al., 2007). These included pine bark, peanut shell, saw dust, cotton, shea butter seed husk and sunflower stalk, among others. A comprehensive list of naturally occurring adsorbents for the removal of heavy metals may be obtained from Bailey et al. (1999). In this study, the possible use of an Acetobacter xylinum biofilm as an adsorbent for mercury ion from aqueous solutions was studied. The effect of pH on the biosorption capacity was characterized by measuring the adsorption isotherms of mercury with the biofilm under pH controlled conditions. The maximum capacity of the biofilm, based on dry weight, was determined by varying the concentration of the mercury in the adsorption medium.
MATERIALS AND METHODS
Biofilm Production
The Acetobacter xylinum NBRC 13693 used in this study was obtained
from the NITE Biological Resource Center (NBRC), Japan. The stock culture was
grown in SH medium at 28°C under static culture conditions. The SH medium
was composed of 2% (W/V) glucose, 0.5% (W/V) Yeast extract, 0.5% peptone, 0.27%
(W/V) Na2HPO4 and 0.115% (W/V) citric acid (Romling, 2002).
After cultivation, the cellulose sheets were removed and rinsed with distilled
water and divided from bacterial and medium residues by 2% sodium dodecyl sulfate
and 4% NaOH solutions in a boiling water bath. The biofilm was then dried at
70°C.
Biofilm Characterization
The surface morphology of an Acetobacter xylinum biofilm was studied
by scanning microscopy (XL30 Philips model). The specific surface area was determined
using the multiple BET method (Micromeritics, Gemini) with nitrogen gas as the
adsorbate.
Adsorption Experiments
All adsorption experiments were carried out in batch conditions. Mercury
was prepared from a synthetic solution (50 mL) of predetermined concentration,
which was taken separately into 100 mL stoppered conical flasks. The pH of the
working solutions was adjusted to the desired value using HCl and NaOH. The
mercury concentration was analyzed following the cold vapor atomic absorption
method at 253.7 nm (UNICAM, model 929, UK). The calibration curves were prepared
by measuring the absorbance of a series of standard solutions at known mercury
concentrations. A measured quantity of the adsorbent was added to the mercury
solution and shaken in a mechanical shaker for a definite period of time. The
difference in the adsorbate content before and after adsorption represented
the amount of adsorbate adsorbed by the Acetobacter xylinum biofilm.
In order to test the effect of pH on the adsorption capacity, samples were adjusted
at different pH ranging between 2 and 12 using 0.1 M NaOH and HNO3.
The period of contact was varied from 0.0 to 120 min. To describe the adsorption
kinetics of mercury by the biofilm, adsorption isotherms of the Freundlich and
Langmiur models were used.
Desorption Studies
The mercury loaded biofilm was regenerated with 1 M HCl solution on a rotary
shaker at 100 rpm and the regenerated biofilm was reused in the next cycle with
mercury solution from the adsorption experiments.
RESULTS AND DISCUSSION
Acetobacter xylinum Biofilm
The thick, gelatinous membrane formed in static culture conditions. The
structure, not found in plant cellulose, results in high cellulose crystallinity
(60-80%) and enormous mechanical strength (Astley et al., 2001). The
cellulose produced in the form of a gelatinous membrane can be molded into any
shape and size during its synthesis, depending on the cultivation technique
and conditions used. Acetobacter xylinum is an important industrial microorganism
used in the production of bacterial cellulose (Geyer et al., 1994). It
is a simple gram negative bacterium which has an ability to synthesize a large
quantity of high-quality cellulose organized as twisting ribbons of microfibrillar
bundles (Shah and Brown, 2005). During the biosynthesis process, various carbon
compounds of the nutrition medium are utilized by the bacteria, then polymerized
into single, linear β-1-4-glucan chains and finally secreted outside the
cells through a linear row of pores located on their outer membrane. Particularly
impressive is the fact that the size of the microbial cellulose fibrils is about
100 times smaller than that of plant cellulose (Shirai et al., 1997).
This unique nano morphology results in a large surface area that can hold a
large amount of water and at the same time displays great elasticity, high wet
strength and conformability. The small size of the microbial fibrils seems to
be a key factor that determines the microbial cellulose’s remarkable performance
as an effective adsorbent. Unlike cellulose of plant origin, microbial cellulose
is entirely free of lignin and hemicelluloses (Lynd et al., 2002). A
vigorous treatment with strong bases at high temperatures allows the removal
of cells embedded in the cellulose net and it is possible to achieve a high
performance absorbent. The weight loss in the pellicles as a result of alkaline
treatment was found to be around 15-20%, which is attributed to the loss of
protein and nucleic acid contents (Shirai et al., 1997). The specific
surface area of the biofilm determined by the BET method was 650±0.08
m2 g-1.
Effect of pH
The effect of pH on the adsorption capacity is characterized by measuring
the mercury adsorption to the biofilm of Acetobacter xylinum under pH
controlled conditions. The maximum mercury adsorption occurred at around neutral
pH. Under acidic conditions the adsorption of mercury by the biofilm was quite
low (Fig. 1). Normally, metal adsorbents exhibit a drastic
decrease in metal affinity at low pH conditions. The occurrence of competition
between protons and metal ions for the same sites should also be considered
particularly at low pH values. There was an increase in mercury adsorption per
unit weight of the biofilm with increasing pH, from pH 3.0 to 5.0. This phenomenon
seems to level off at pH greater than 8.0. The increase in adsorption levels
observed with increasing pH can be explained by the strong relation of adsorption
to the number of surface negative charges, which depends on the dissociation
of functional groups. It can also partly explain the low amounts of metal ions
retained by the adsorbent at pH values below 4, as most functional groups are
expected to dissociate only at neutral pH values.
| Fig. 1: | Adsorption of mercury on the biofilm as a function of pH. Conditions-contact time: 50 min; biofilm mass: 2 g; mercury: 100 mg L-1; temperature: 20°C |
| Fig. 2: | Percentage of mercury ion adsorption on the biofilm as a function of amount of adsorbent. Conditions-contact time: 50 min; mercury: 100 mg L-1; temperature: 20°C |
The effect of pH on the biosorption of Hg (II) was investigated by Chang and Hong (1995). Their results showed that biosorption of Hg (II) by non-living Pseudomonas aeruginosa PU21 was pH dependent and that maximum biosorption was obtained at pH 7.4. Chen et al. (1997) reported that the pH profile of Hg (II) bioaccumulation by recombinant living E. coli remained at the same level within a pH range of 3.0-9.0. Their results demonstrate that, in contrast to biosorbents or ion exchange resins, the Hg (II) bioaccumulation system was resistant to pH over a broad range.
Effect of Adsorption Time
Adsorption equilibrium was established around 50 min, after which the adsorbed
mercury did not significantly change further with time. Several parameters affect
the biosorption rate. These include: structural properties of biosorbent (e.g.,
protein and carbohydrate composition and surface charge density, topography
and surface area), amount of adsorbent, properties of the ions under study,
stirring rate, initial concentration of ionic species and the presence of other
metal ions.
Effect of Adsorbent
The increase of the biofilm leads to the enhancement of percent removal
of mercury (Fig. 2). The result of the present study falls
in line with the findings of Manohar et al. (2002). The capacity of the
biofilm for adsorption of mercury is the same order of magnitude as the heavy
metal ion adsorption capacities of the various biomasses and adsorbents (Kacar
et al., 2002; Saglam et al., 2004). Chen et al. (1997)
used a living E. coli strain for bioaccumulation of mercury, where
the highest bioaccumulation level was about 17.6 mg g-1 dry biomass.
Hafez et al. (1997) reported that the adsorption capacity of Aspergillus
flavus was 46 mg for uranium per g of dry biomass. The adsorption capacity
of Rhizopus arrhizus was 78 mg for Fe (III), 71 mg for Pb (II) and 62
mg for Cd (II) per g of dry biomass (Ozer et al., 1997). Other than microbial
biomass, various sorbent materials have been used for removal of heavy metal
ions from industrial waste, ranging from natural polysaccharide gels to coal
and functionalized synthetic polymers (Namasivayam and Kadirvelu, 1999; Manohar
et al., 2002). In these systems, the metal removal process is based on
a solid-liquid contenting and separation process. Such preparations offer advantages
in terms of mechanical strength and durability, handling and ease of scale up.
For example, Binman et al. (1997) used sulphur-chlorinated jajoba wax
attached to polystyrene beads, for which the maximum inorganic mercury adsorption
capacity was 50 mg g-1. Jyo et al. (1997) prepared phosphoric
acid treated poly (glycidyl methacrylate-co-divinlyl benzene) beads, for which
the mercury adsorption capacity of the sorbent was 40 mg g-1. Unfortunately,
all of the above mentioned sorbents are expensive and require several steps
for preparation. In contrast, the adsorption capacity of the biofilm reached
180 mg g-1 with mercury, a value comparable with the values reported
in the previous studies. It is larger than the adsorption capacity of well known
adsorbents such as coir pith carbon (154 mg g-1) (Namasivayam and
Kadirvelu, 1999), PHC-peanut hull carbon (110 mg g-1) and granular
activated carbon (124 mg g-1) (Namasivayam and Periasamy, 1993).
Ma et al. (1992) report a substantially lower value (0.8 mg g-1)
for a granular activated carbon.
Effect of Hg (II) Concentration
The maximum percentage of mercury was adsorbed at lower initial concentrations.
The percentage mercury adsorption was 91% at a concentration of 80 mg L-1.
A further increase in initial concentration decreased the adsorption percentage
(e.g., an initial concentration of 120 decreased adsorption to 75%). At low
concentrations of mercury, the ratio of sorptive surface area to total metals
ions available is high and thus, there is a greater chance for metal removal
and the removal capacity is higher.
Sorption Isotherms
The experimental data of mercury sorption by biofilms of A. xylinum
were fitted to the Langmuir and Freundlich models. The Langmuir model is applied
to the experimental equilibrium data (Yu et al., 2003).
where, Qe is the amount of mercury adsorbed at equilibrium (mg g-1); Ce is the equilibrium concentration (mg L-1); Q0 is the adsorption capacity (mg g-1) and b is the energy of adsorption (Langmuir constant). The linear plot of the isotherm of the equation is an obligatory condition for the proper application of the theory of monomolecular adsorption. The linear plot of Ce = qe versus Ce shows that the description of the adsorption of mercury on the adsorbent by the Langmuir isotherm model is satisfactory. Q0 and b are determined from the slope and intercept of the plot. Some theoretical parameters in the Langmuir equation were calculated from the experimental data. The linear form of the Freundlich equation (Slejko, 1985) is applied to the results of adsorption.
where, x is the amount of the adsorbed solute (mg), m is the weight of the adsorbent (g), Ce is the equilibrium concentration (mg L-1), 1/n is the slope, showing the variation of the adsorption with concentration and kf is the intercept, showing the adsorption capacity of the adsorbent. Linear plots of log10 (x/m) versus log10Ce show that the adsorption follows the Freundlich isotherm model. The values between 1 and 10 represent beneficial adsorption. The adsorption isotherm of mercury on the biofilm can be included in type L on the Giles classification (AnoopKrishnan and Anirudhan, 2002). Langmuir’s isotherm is based on the assumption that points of valency exist on the surface of the adsorbent and that each of these sites is capable of adsorbing one molecule; thus, the adsorbed layer will be one molecule thick. Furthermore, it is assumed that all the adsorption sites have equal affinities for molecules of adsorbate and that the presence of adsorbed molecules at one site will not affect the adsorption of molecules at an adjacent site. Freundlich’s isotherm is based on the assumption that the adsorbent has a heterogeneous surface composed of different classes of adsorption sites, with adsorption on each class of site following the Langmuir isotherm.
Desorption Experiments
In order to evaluate the reusability of the biofilm, the regeneration and
consecutive adsorption cycles were carried out using 1 M HCl. The results showed
that the adsorption efficiency for the biofilm regenerated with 1 M acid, decreased
to 43% for the first regeneration cycle and to 61% for the second treatment.
CONCLUSION
The results presented here indicate that a biofilm prepared from Acetobacter xylinum is a suitable and effective adsorbent for the removal of mercury from aqueous solutions. The adsorption of mercury on the adsorbent depends on the experimental conditions, particularly on the pH of solution and the metal concentration ion in the medium. The good fit of the adsorption isotherm for mercury was observed for Freundlich and Langmuir models. The calculated values of the Freundlich and Langmuir constants indicated that the mercury adsorption onto the Acetobacter xylinum biofilm was a function of initial mercury concentration.