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Microbial biosorption of copper and lead from aqueous systems
The Science
of the Total
Environment
170 (1995)
209-220
Microbial biosorption of copper and lead from aqueous systems Amit A. Pradhan”“, Audrey Il. Levineb bDepartment
aGeotechnical Services, Inc., 10052 Justin Drive, Suite L, Des Moines, Iowa 50322, USA of Civil and Environmental Engineering, Utah State University, Logan, Utah 84322-3876, Received
26 May
1994; accepted
20 December
USA
1994
Abstract Biosorption of metal ions from aqueous systems was evaluated using a culture of acidic soil isolates grown in a completely mixed, aerobic, semi-batch culture reactor. The laboratory scale system was used to test single and bimetallic solutions of copper and lead with sulfates, chlorides, or nitrates. To elucidate the key factors influencing biosorption and to characterize metal uptake by cellular and extra cellular components of the microbial system, a dialysis testing procedure was developed. A direct contact technique was used to determine the rate of metal sorption on cellular surfaces. The effectiveness of biosorption was influenced by pH, initial metal concentrations, and anionic composition. Respirometric tests were carried out to identify potential inhibitory effects of metal accumulation on microbial oxygen uptake rates. Keywords:
Biosorption; Metallic waste;Actinomycetes; Dialysis; Respirometry
schemes have high chemical costs and tend to generate large quantities of highly caustic sludges. The use of biological processes can overcome some of the limitations of physical and chemical treatment and provide a means for cost effective removal of metals [4-61. It is generally believed that microbial metal removal can occur by binding of metals on cell surfaces, immobilization of metal ions by extracellular enzymes, or active transport of metal ions into the cell with intracellular accumulation [6-S]. A viable cell culture is required for active transport, whereas, surface
1. Introduction
Industrial and mining wastes, solid wastes, sewage sludges, and landfill leachates are major sources of metallic contamination [l-31. Chemical and physical treatment processes such as precipitation, catalytic oxidation, and ion exchange columns have been used for the treatment of soluble metallic wastes. However, these treatment
*Corresponding
author.
0048-9697/95/$09.50 0 1995 Elsevier SSDI 0048-9697(95)04709-A
Science
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reserved.
210
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/The
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and extracellular uptake do not require cell viability. To facilitate the development of full scale metal biosorption systems, it is essential to quantify the uptake capabilities of cell surfaces and extracellular materials and to evaluate the degree to which metal ions impair cell viability. Various organisms have been used for the uptake of metals from aqueous systems. Nakajima and Sakaguchi [7] studied metal binding capacities of 83 microorganisms and concluded that actinomycetes show higher capacities to bind metal ions as compared to fungi, yeast, and bacteria. Accumulation of metal ions on biological surfaces results in inhibition of life processes of the cell [lo]. In addition it has been demonstrated that actinomycetes have greater resistance to metal toxicity [ll]. The specific objectives of this study were: (a) to quantify metal binding capacities of cellular and extracellular components of a microbial biosorption system and (b) to evaluate the response of respiratory rates to various concentrations of the test metals. Acidic soil isolates derived from a culture of actinomycetes were used as the test organisms for this study because of their reported metal binding capacities and ability to resist metal toxicity [12]. The test metals used for this study were copper and lead. 2. Background
Microbial treatment reactors contain cellular mass in contact with a liquid medium into which products of microbial metabolism are released. Both cellular and extracellular components of a microbial system are capable of binding metal ions [6-81. Adsorption of metal ions on microbial cells takes place in two stages: (1) instantaneous binding to cellular surfaces and (2) gradual transport and accumulation of metal ions within the cytoplasm [7]. The metal binding capacity of a microbial system varies depending on the metal type. Anions can also influence microbial metal uptake [71. In a previous study, biomass from an acidic soil isolate was demonstrated to be capable of binding copper and lead from bimetallic solutions [13].
of the
Total Environment
170 (1995) 209-220
Maximum binding rates for copper and lead from aqueous solutions occurred at pH 4.0 [13]. To evaluate the capacity of the cellular surfaces and the extracellular material, it is necessary to separate extracellular products from biomass and compare their metal uptake capacities. Semipermeable membranes can function as physical barriers to separate the cellular and extracellular components and thereby provide a means to evaluate uptake capacity on a laboratory scale. Transport of metal ions across a semipermeable membrane is governed by the concentration gradient and the flux can be evaluated as follows [14]: F,=D;A;~
AC
(1)
where, F, =
Net solute flux through the membrane (mole/cm’/s> D, = Diffusion coefficient of the solute (cm’/s> A, = Numerical factor representing certain restrictions imposed by the membrane on the flow of solute AC, = Difference in the concentrations on either sides of the membrane (mole/cm3> AX = Thickness of the membrane (cm> For a specific test system, metal ion flux is directly proportional to the concentration gradient across the membrane. It can also be inferred that the net flux of metal ions through the membrane is zero at equilibrium. Dialysis membranes can be used in this context as an experimental tool. Biosorption coupled with dialysis consists of two stages: (1) diffusion of metal ions through a semipermeable membrane and (2) binding of diffused metal ions by the microbial system. The rate of change of metal concentration, r, in the solution, corresponds to the rate of diffusion and rU is the uptake rate. The rate of diffusion can be calculated as:
cc,- c,> rc= (t,-tl)
(2)
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170 (1995) 209-220
211
where, C, and C, are the concentrations of metal in the solution at times t, and t,, respectively. The uptake rate, r,,, can be computed using equation (3).
cal circuit so that current can be supplied when required.
(B, -B,) r”= (t2-tl)
This study was based on evaluating biosorption of metal ions from single and bimetallic solutions consisting of two metals: copper and lead. All tests were conducted at constant pH (4.0) and temperature (25°C). The biosorbate was derived from the culture of acidic soil isolates. Biosorption from the whole system was compared to the cellular material sorption and non-diffusible microbial metabolism.
where, B, and B, are the concentrations of metals bound to the biological system at times t, and t,, respectively. Mitra et al. [15] demonstrated that the degree of cell inhibition by metallic contamination depends on the type of metal ions. Cadmium was shown to inhibit the log phase of growth. Metal ion valency also influences the degree of toxicity. Cr6+ was shown to be more toxic to microorganisms than Cr3+ [161. Vallee and Ulmer [171 postulated that the binding of metals to thiol on protein molecules and replacement of metals in the enzyme prosthetic group caused inhibition. Bacterial cells can resist toxicity to a certain limit depending on (1) production of enzymes capable of nullifying toxic properties of the metallic contaminants. (2) formation of an impervious layer external to the cell wall, and (3) removal of metals by transport across the cell membrane. Increase in metal concentration beyond the threshold value can result in lysis of microbial cells and loss of viability [18]. Inhibitory effects of metallic contaminants can be judged by monitoring microbial activity directly or indirectly. Respiration rate can be used as an indirect measure of microbial viability. Respirometers measure the rate of oxygen consumption during microbial cell respiration and thereby provide a basis for comparing the growth characteristics of microbial cultures under controlled conditions. Carbon dioxide is generated during microbial respiration. In an electrolytic respirometer, the CO, generated is absorbed by potassium hydroxide solution and is replaced by a stoiciometric quantity of oxygen generated by the electrolysis of acidified water. The current required for electrolysis can be correlated to the volume of oxygen generated. Electrolytic cells are equipped with a break mechanism in their electri-
3. Experimental
only
design
4. Procedures
The experimental study included four stages: (a) culturing acidic soil isolates and preparing test metal solutions; (b) dialysis experiments to evaluate the factors governing metal binding on cellular and non-diffusible extracellular components of the microbial system; cc> direct contact tests to determine the kinetics of metal binding on cellular surfaces, and (d) respirometric tests to evaluate the inhibitory effects of metal accumulation on microbial respiration. The experimental methodology is described below. 4.1. Culture of microbial system
The acidic soil isolates were cultured aerobically at 25°C and a pH range of 6.5-7.5 using a feed containing glycerin and L-arginine as organic substrates and other mineral nutrients. A sodium bicarbonate buffer was used to control pH in the bio-reactor [13]. The composition of feed solution is shown in Table 1 and a diagram of the reactor used for culturing the microorganisms is presented in Fig. 1. The 5.0-l reactor was operated as a completely mixed, suspended growth, semi-batch reactor with a hydraulic retention time of 5 days. To feed the reactor, 1 litre of the reactor fluid was removed daily and was replaced by an equal volume of feed solution. 4.2. Analytical
methods
During the conduct of laboratory experiments, care was taken to eliminate all the possible
212
Ad.
AIR D(HAUST
! 1
Pradhan,
A.D. Levine -
/ The Science FEED
i!
of the Total Environment
and membrane surfaces. Acid digestion [19] was used to desorb the metal ions. 4.3. Dialysis
0
0
0
-REACTOR
0
0
FLUID
I ’
0
AIR BUBBLES2 ! 0
-
Fig. 1. Reactor
used for culturing
microorganisms.
sources of contamination. To avoid metallic contamination the glassware was decontaminated using a acid wash containing 5% concentrated nitric acid and was then rinsed using deionized water. To eliminate microbial contamination, the glassware used for storing feed solutions and microbial system was sterilized using an autoclave. Atomic absorption spectroscopy was used to determine the metallic concentrations in the experimental solutions. To establish the mass balance it was necessary to strip the metal ions adhering to glass Table 1 Composition
of the feed solution
for acidic
Component
Concentration
Magnesium sulfate Sodium chloride Calcium chloride LArginine Potassium phosphate Glycerine Sodium bicarbonate
200 200 50 450 450 4.5b 250
“Derived from bConcentration
[lo]. in ml/l.
soil isolate9 (mg/l)
170 (1995) 209-220
experiments
An overview of the design for the dialysis studies is presented in Fig. 2. Tests were conducted on single metal systems with either copper or lead or a bimetallic system containing both metals. The concentrations tested are shown in Table 2. The metal solutions of the desired ionic strength were prepared by dissolving the precalculated quantities of nitrate, chloride, or sulfate salts of copper and lead in deionized water. During the experiments, the solution pH was adjusted using NaOH along with H,SO,, HCl, and HNO, for SO:-, Cl-, and NO; salts, respectively. A flow sheet for the preparation of the dialysis test system is shown in Fig. 3. The cellular and extracellular components of the microbial system were separated using centrifugation followed by vacuum filtration (pore size 1.2 pm). The cellular component was then resuspended in a volume of deionized water equal to the removed extracellular fluid. Parallel tests were conducted on the unseparated microbial system. A control consisting of an equal volume of deionized water was tested to evaluate transport through the membrane and potential losses on the membrane surfaces. For each test, the pH was adjusted to 4.0 and a 50-ml aliquot was poured into individual pieces of prewashed dialysis tubing (Spectra porTM) with a molecular weight cut off of 3500 atomic mass units (amu). Each section of dialysis tubing was immersed in an aerated metal solution until a steady state concentration was reached at about 48 h. The reactor setup for the dialysis experiments is shown in Fig. 4. To minimize evaporation, the reactors were covered with foil during the test. The metal ion concentration in the solution external to the dialysis tubing was monitored at regular intervals. At the end of each test, the contents of the dialysis tubing were acidified and analyzed to determine the chemical oxygen demand (COD), suspended solids (SS), and pH [19l. The concentrations of Cu and Pb were de-
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Science of the Total Environment
213
170 (1995) 209-220
Metal solution I,
I BiitiCCUdpb
Lead
Copper
I
I
*pm
I
20ppm
Fig. 2. Summary
30ppm
of experimental
r 1Oppm
I
20ppm
design for dialysis
termined using flame ionization atomic absorption spectroscopy. Acid digestion was used to free the metal ions adhering to cellular, glass, and membrane surfaces. Rates of diffusion were calculated using Equation 2. The results were expressed as the mean metal uptake capacities and compared with the results of previous studies [7]. 4.4. Kinetic studies To determine the rate of metal uptake by microbial cells, a direct contact technique was used [13]. A 40-ml aliquot of metal solution was pipetted into each of 12 250-ml flasks. Microorganism samples suspended in 10 ml of pH adjusted sodium bicarbonate buffer solution were added to each flask. The reaction vessels were then placed on a shaker table with a lo-cm stroke at 80 strokes per min. At selected time intervals, samples were removed from the shaker table, vacuum filtered using a glass fiber filter, acidified with concentrated nitric acid, and analyzed for metal concentration. The metallic uptake rates were calculated by evaluating the change in metal concentration over time using Equation 3. Mass balance calculations were used to evaluTable Metal
2 concentrations
Type of metal
solution
Bimetallic Cu and Pb Cu in single metal tests Pb in single metal tests
r
I 30ppm
separation
I
I 2CWm
r l%m
of biological
uptake
I 30pPm
of metals.
ate the accuracy of measurements in the various tests. Statistical analyses were carried out to determine the relationship between the total uptake observed in the experiments and the sum of the uptake capacities of the cells and the nondiffusible extracellular material. The metal binding capacities of the two components were subjected to statistical analysis. The metal binding capacities of microbial cells and the extracellular
Cantrifugation ~acumn
and
filtration
deionized
water
To dialysis experiment
tested
using
dialysis
Concentrations 10,20,30 10,20,30 10, 20, 30
tested
(ppm by wt.) To dialysis experiment
Fig. 3. Flow sheet for the separation
of cellular
material.
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/ The Science of the Total Environment
AIR SUPPLY F=-
UYIVV CONTAINER
I I
\
Metal solution \
Fig. 4. Experimental
setup for dialysis
experiments.
metabolites when tested individually were compared with the metal uptake observed for the entire microbial system using the t-test. To determine the accuracy of experimental techniques, the mass balance relationship was established as: MS, + MB, = MS, + MBf + MGf + MDf
(4)
MS, = MB, = MG, = MD, =
209-220
were interfaced with a computer using software developed by Bioscience, Inc. The system was capable of automatically recording data at set time intervals. Respirometric settings used for the study are specified in Table 3. In this study, metal solutions of known ionic strength were prepared by dissolving copper and lead salts in deionized water. The compositions of the metal solutions tested using respirometry are shown in Table 4. For each electrolytic reactor, 100 ml of the microbial aliquot was suspended in 900 ml of metal solution and 50 ml of the completely mixed reaction sample was collected to provide information regarding initial metal concentrations and the organic content. The respirometric data collection was then started in accordance with the experimental settings described in Table 3. For each concentration of the metal solution, the test was conducted in duplicate. Controls were established by testing blank samples consisting of deionized water. A flow sheet for the preparation of the reaction sample in respirometry is shown in Fig. 5. When the oxygen uptake reached a plateau, one of the duplicates was tested for reactivation of microorganisms by adding 50 ml of the feed solution. All the samples were immediately acidified with 5 ml of concentrated nitric acid to prevent further biological activity and were later analyzed to determine metal content, organic content (COD), and suspended solids. 5. Results
Results of the dialysis and respirometric are presented below.
where, MS, = MB, =
170 (1995)
Initial metal content of the solution Initial metal content of the biological system Final metal content of the solution Final metal content of the biological system Metal quantity attached to glass Metal quantity attached to dialysis tub ing
4.5. Respirometry Electrolytic respirometers
used for this study
studies
5.1. Dialysis experiments Dialysis experiments revealed that cellular and extracellular components were capable of binding Table 3 Respirometer
settings
Parameter
Value
Sample volume Oxygen generation rate Data collection intervals
950 50 15 30 2
ml mg/h min for initial 2 h; min for next 6 h; h for the rest of the time
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Reaction sample 950 ml
1 Fin;; saaple
1
Fig. 5. Flow diagram for the preparation (pH was maintained at 4.0) for respirometry
of reaction studies.
samples
170 (1995) 209-220
215
uptake capacities is shown in Table 5 and is graphically represented in Fig. 6. To compare the results obtained from different tests, the data were normalized based on the sample COD. The experiments revealed that an increase in the initial lead concentration from 10 ppm to 20 ppm resulted in an increase in the metal uptake by the cellular part and the entire microbial system. An increase in the concentration beyond 20 ppm, however, resulted in the apparent desorption of the metal ions from the biosorption system. Similar tests with copper did not indicate such a trend. Statistical analysis of the data revealed that the metal uptake was more efficient by the separated components than the unseparated system at 90% confidence level. In a bimetallic system, the microorganisms appeared to possess greater affinity for lead than copper over the concentration range tested. A comparison of the influence of the anionic composition on the copper uptake is shown in Fig. 7. Sulfate appeared to influence cellular uptake more than chloride and nitrate. 5.2. Dijj‘iuion and uptake rates
metal ions individually as well as combined. In the dialysis experiments, most of the uptake and complexation reached a saturation point within the first few hours. Metal and anionic concentrations influenced the metal binding capacities of cellular and extracellular components. Continuation of the experiments beyond the saturation capacity resulted in the apparent desorption of metal ions from the biological system. The influence of metal concentration on saturation metal Table 4 Concentrations
of metallic
solutions
used in respirometry
Concentrations
Bimetallic
Copper concentration was maintained at 100; lead concentration were 0,50, 100,150 0, 25, 50, 75, 100, 150
Cu in metal Pb in metal
single tests single tests
tion and uptake rate, respectively. These rates, calculated for the various experimental stages, are shown in Table 4. The uptake rate, Y,, was calculated based on data from a previous study [13], whereas, r, was obtained from dialysis experiments data. Based on the data obtained, it can
Uptake
0.6 o4
mmollg COD
0.2
Metal
Type of tests tests
As explained in the background section, rc and r, represent rate of change of metal concentra-
0,50,
tested
100, 125, 150, 175
(ppm by wt.)
.
10 ppm 20 ppm 30 ppm Initial Concentration q Cell Fig. 6. Effect capacity.
q Extracellular of initial
metal
concentration
q Intact System on lead uptake
216
A.A. Pradhan,
Table 5 Copper uptake Cont.
capacities
Anions
@pm)
10
NO3
10 10 10 20 30
so4
Cl NO3 NO3
N/A, Table Rates Time
Not
Levine
/The
Science of the Total Environment
components
Other metal cont.
Uptake cells (mmol/g (A>
N/A N/A
0.033 0.091 0.038 0.189 0.165 0.468
170 (199.5) 209-220
of acidic soil isolates
by COD)
Uptake by extracellular part (mmol/g COD) (B)
Sum of the Cellular and extracellular uptakes (mmol/g COD) cc>
Uptake by microorganisms with extracellular substances (mmol/g COD) CD)
0.116 0.052 0.018 0.090 0.188 0.369
0.149 0.143 0.056 0.279 0.353 0.837
0.053 0.062 0.016 0.044 0.222 0.055
applicable.
6 of diffnsion (mm)
and metal
uptake
at various
stages of the experimenta
Copper
5 30 120 aReported
of the individual
N/A Pb 10 ppm Pb 20 ppm Pb 30 ppm
NO3
A.D.
for
Lead
r, (mmol/min)
r, (mmol/min)
r, (mmol/min)
r, (mmol/min)
1.33 x 10-7 1.00 x 10-r 5.00 x 10-s
2.8268 x lo-’ 1.4650 x 1O-4 1.7500 x 10-5
6.67 x lo-’ 6.67 x lo- * 3.33 x 10-s
2.2844 x 10 - ’ 2.8600 x lo- 4 4.3000 x 10-s
:tallic
test with
10 ppm of each metal
in presence
of nitrate.
0.161
Nitrate
I
0 Cell
Chloride
n Extracellular Fig. 7. Effect
Sulfate
0 Cell + Extracellular
of anions
on copper
uptake.
El Intact System I
AA. Table 7 Comparison
Pradhan,
of mass balance
A.D. Levine
of data
Metal
Bimetallic
tests
cu Pb cu Pb CU Pb cu Pb cu Pb
Cellular
tests component
Extracellular Entire
Science of the Total Environment
component
system
aCalculated by comparing the metal quantity used for starting the experiment.
tested
quantity
observed
No. of observations
Mean
24 12 7 3 7 5 7 5 7 5
11 26 7 50 11 36 9 31 17 25
in the various
be inferred that rU > rC for the initial stages. The difference, however, decreases with time.
Results of the mass balance calculations are shown in Table 7. Although strict controls were observed in all components of the study, a more complete mass balance was obtained for copper than for lead. In general, the extracellular compo-
Experimental Unimetallic 0 wm 50 wm 100 ppm 125 ppm 150 ppm 175 ppm bimetallic
demand conditions
various
experimental
test components
percentage
5.4. Respirometric
results
conditions
Plateau
oxygen
the metal
The results of respirometric tests are provided in Table 8. It is evident that the oxygen uptake
uptakea
(mg/l)
Initial
188.05 70.64 71.58 189.69 163.11 155.34
0.0 5.0 7.5 20.0 20.0 20.0
259.28 22.26 16.81 20.22 175.54 67.71
0.0 50.0 65.0 145.0 90.0 165.0
54.21 17.84 16.22 14.32
0.0 26.0 40.0 20.0
copper
on the averages.
error=
at the end of the tests with
lead
0 PPm 25 ppm 50 wm 75 mm 100 ppm 150 ppm Bimetallic Cu 100 ppm Pb 0 ppm Pb 50 ppm Pb 100 ppm Pb 150 ppm aBased
under
217
nent showed the highest recovery in mass balance calculations whereas, the cellular component showed the least recovery perhaps due to analytical inaccuracies associated with solid phase analysis.
5.3. Mass balance calculations
Table 8 Plateau oxygen
170 (1995) 209-220
calculations
Category
Unimetallic
/The
lag time (h)
218
A.A. Pradhan,
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/The
Science of the Total Environment
rate during respiration was higher for nonmetallic solutions than in the presence of metals. Addition of metal ions to the aqueous system caused a lag phase prior to the onset of respiratory activity. The lag phase duration was longer for copper tests than for lead solutions of equal mass concentrations. It can be inferred that an increase in metal concentration resulted in a decrease in the plateau oxygen uptake by the system. Addition of feed to the reaction sample resulted in an increase in oxygen uptake rate. The inhibitory effects of metal accumulation appear to be reversible in this concentration range. 6. Discussion
The observations from these studies and the potential use of biosorption for metallic waste treatment are discussed in the following paragraphs. 6.1. Metal uptake capacities
Saturation capacity exhibited by the microbial system in binding metal ions is a key factor for biological sorption of metals. The saturation capacity can be explained on the basis of cellular biochemistry. Metal binding on cellular surfaces has been shown to be associated with the fnnctional groups such as carboql, hydroxyl, pyruvate, amide, and acetate [20-221. At constant pH, a microbial cell contains a fixed number of binding sites. As the pH increases, some of the aminophosphate groups and lipoproteins of the cellwall are hydrolyzed thus making some of the sites unavailable for metallic binding. In this study, the majority of metal uptake occurred fairly rapidly. The microbial system showed preferential binding capacity for lead. Similar results have been previously reported by Ruzic [23] and Irving and Williams [24]. In presence of more than one metal, the extent of complexation of each metal with the microbial system is related to the metal binding constants. The order of metal enzyme stability is given by Pb > Cu > Ni > Co > Zn > Cd > Fe > Mn > Mg. Thus, it is possible that copper could be displaced by lead. Therefore, a treatment scheme based on biosorption may require optimization
170 (1995) 209-220
depending on the metal content and concentration. In this study, total metal uptake was found to be higher when the cellular and extracellular components were tested individually as compared to the unseparated system. The exact mechanism of interaction and competition between cellular and extracellular components needs to be investigated further. 6.2. Kinetic studies
The metal uptake rate Y, appears to be related to the availability of metal binding sites and the rate of diffusion r, is dependent upon the concentration gradient across the semipermeable membrane as shown by Equation 1. In the initial stages of the experiment, due to the availability of a large number of binding sites, Y, > rc. Consequently, rapid binding of metal ions occurred. As the number of available binding sites decreased, a critical stage was reached when Y, = rc. Allowing contact time beyond this stage resulted in rU < Y, which is represented by the apparent desorption of metal ions. Because the availability of metal binding sites on a cellular surface depends on the pH, it is essential to establish an optimum pH condition for biosorption of metals either individually or in multicomponent mixtures. 6.3. Respirometry
Respiratory inhibition due to metal accumulation was demonstrated in this study. Evidence for metal inhibition was manifested by: (1) a lag phase prior to onset of respiratory activity and (2) a decrease in plateau oxygen uptake capacity of the microbial system with increase in metal concentration. The acidic soil isolates did not show a loss of viability over the range of metal concentrations tested. This capability of the acidic soil isolates may be advantageous in developing a biosorption system to treat metallic wastes, particularly if the biosorption system is to be based on viable organisms. 6.4. Applications
The use of biosorption to remove and concentrate metal ions from solutions has potential application in treatment of metal containing waste streams, such as landfill leachates, mining wastes,
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and industrial and hazardous wastes. This study has provided laboratory data supporting the use of acidic soil isolates in a biosorption system. Optimization of metal uptake, metal recovery, and reactor design is necessary to facilitate the conduct of pilot scale studies and development of design information.
170 (199.5) 209-220
219
(ISWRRI). The authors acknowledge the assistance of James Gaunt, William Graham and Angela Bieldfeldt in the conduct of this project. Amit A. Pradhan is an Environmental Engineer with Geotechnical Services, Des Moines, Iowa and Dr Audrey D. Levine is Associate Professor of Civil and Environmental Engineering at Utah State University, Logan, Utah.
7. Conclusions
The major conclusions from this study are listed below.
References HI
Dialysis experiments provided a means to determine the relative importance of the cellular and extracellular components for uptake of copper and lead from the test microbial system. For both metals tested, a saturation limit was observed for the maximum quantity of metal that could be bound. pH, anion type and concentration, and initial concentrations of the metal solutions all are significant factors in microbial metal uptake using acidic soil isolates. Overall metal uptake capacity of the microbial system is greater when cellular and extracellular components are separated. The presence of copper and lead inhibited microbial respiration. These effects were exhibited as (1) a lag phase in the oxygen uptake curve and (2) a decrease in plateau oxygen uptake potential. The lag phase duration was longer for microbial systems exposed to copper as compared to similar levels of lead. Following the lag phase, respiratory activity could be re-triggered by the addition of feed solution, thus some degree of microbial viability was maintained. Acknowledgements
This project was supported, in part, State Mining and Mineral Resources Institute (ISMMRI) and in part, by State Water Resources Research
by Iowa Research the Iowa Institute
A.D. Levine and L.R. Rear (Eds.), Evaluation of leachate monitoring data from co-disposal, hazardous, and sanitary waste disposal facilities, in Proceedings of the 43rd Annual Purdue Industrial Waste Conference, Lewis, Chelsea, MI, 1989, pp. 173-183. Cation Dl R.E. Marquis, K. Mayzel and D.L. Cartensen, exchange in cell walls of gram-positive bacteria. Can. J. Microbial., 22(7) (1976) 976-982. Geotechnology of Waste t31 I.A. Gweis and R.P. Khera, Management, Butterworth, Guildford, UK, 1990. B.Greene, R.A. McPherson and M.D. [41 D.W. Damall, Alexander, Recovery of heavy metals by algae, in R. Thompson (Ed), Trace Metal Removal from Aqueous Solution, Royal Society of Chemistry, Cambridge, UK, 1986, pp. l-24. and accu151 B. Friedman and P.R. Dugan, Concentration mulation of metallic ions on the bacterium Zooglea, in Developments in Industrial Microbiology, Vol. 9, Chapter 35, (1968) 381-388. and G.G. Geesey, Copper binding 161 M.W. Mittelman characteristics of exopolymers from a freshwater sediment bacterium. Appl. Environ. Microbial., 49(41 (1985) 846-851. and T. Sakaguchi, Selective accumulation [71 A. Nakajima of heavy metals by microorganisms. Appl. Microbial. Biotechnol., 24(l) (1986) 59-64. 181 J.J. Doyle, R.T. Marshall and W.H. Pfender, Effect of cadmium on the growth and uptake of cadmium by microorganisms. Appl. Microbial., 29(4) (197.5) 562-564. Sensitivity of various bacteI91 H. Babich and G. Stotzsky, ria, including actinomycetes and fungi to cadmium and the influence of pH on sensitivity. Appl. Environ. Microbiol., 33(3) (1977) 681-695. in C. Booth [lOI S.T. William and T. Cross, Actinomycetes, (Ed.), Methods in Microbiology, Vol. 4, 1971, 317 pp. of heavy 1111 A.B. Norberg and H. Persson, Accumulation metals by Zooglea ramigera. Biotechnol. Bioeng., 26(3) (1984) 239-246. [121 A.A. Pradhan and A.D. Levine, Role of extracellular components in microbial biosorption of copper and lead. Water Sci. Technol., 26 (9-11) (1992) 2153-2156. 1131 W.S. Graham, Development of Engineering Parameters for the Design of Metal Biosorption Waste Treatment
220
1141
WI
[I61 [171
ml
AA.
Pradhan,
A.D.
Levine
/The
Science
System, Unpublished Master’s Thesis, Iowa State University, Ames, IA, 1991. J.R. Pappenheimer, E.M. Renkin and L.M. Borerro, Filtration, diffusion, and molecular sieving through peripheral capillary membranes - a contributaion to the pore theory of capillary permeability. Am. J. Physiol., 167(l) (19511 13-46. R.S. Mitra, R.H. Gray, B. Chin and I.A. Bernstein, Molecular mechanism of accommodation in Eschepichia coli to toxic levels of Cd*+. J. Bacterial., 121(3) (1975) 1180-1188. R.M. Steritt and J.N. Lester, Interaction of heavy metals with bacteria. Sci. Total Environ., 14 (1980) 5-17. B.L. Vallee and D.D. Ulmer, Biochemical effects of mercury, cadmium and lead. Annu. Rev. Biochem., 41 (1972) 91-128. Z. Vaituzis, J.D. Nelson, L.W. Wan and R.R. Colwell, Effects of mercuric chloride on growth and morphology of selected strains of mercury resistant bacteria. Appl. Microbial., 29(2) (1975) 275-286.
of the
Total Environment
I70 (1995) 209-220
AWWA and WPCF, Standard Methods for the 1191 APHA, Examination of Water and Wastewater, 18th Edition, APHA, AWWA, WPCF, 1991. La A.E. Steiner, D.A. McLaren and C.F. Forster, The nature of activated sludge floes. Water Res., 10(l) (1976) 25-30. Dll S.C. Churms and A.M. Stephens, Studies of molecular weight distribution for hydrolysis products from some KIebsiella capsular polysaccharides. Carbohydr. Res., 35(l) (1974) 73-86.
La
[=I
Dl
S.A. Boyd, L.E. Sommers and D.W. Nelson, Infrared spectra of sewage sludge fractions: evidence for an amide metal binding site. Soil Sci. Sot. Am. J., 43(9) (1979) 893-899. I. Ruzic, Theoretical aspects of direct titration of natural waters and its information yield for trace metal speciation. Anal. Chim. Acta, 140(l) (1982) 99-113. M. Irving and R.J.P. Williams, Order of stability of metal complexes. Nature, 162(8) (1948) 746-747.
of the Total
Environment
170 (1995)
209-220
Microbial biosorption of copper and lead from aqueous systems Amit A. Pradhan”“, Audrey Il. Levineb bDepartment
aGeotechnical Services, Inc., 10052 Justin Drive, Suite L, Des Moines, Iowa 50322, USA of Civil and Environmental Engineering, Utah State University, Logan, Utah 84322-3876, Received
26 May
1994; accepted
20 December
USA
1994
Abstract Biosorption of metal ions from aqueous systems was evaluated using a culture of acidic soil isolates grown in a completely mixed, aerobic, semi-batch culture reactor. The laboratory scale system was used to test single and bimetallic solutions of copper and lead with sulfates, chlorides, or nitrates. To elucidate the key factors influencing biosorption and to characterize metal uptake by cellular and extra cellular components of the microbial system, a dialysis testing procedure was developed. A direct contact technique was used to determine the rate of metal sorption on cellular surfaces. The effectiveness of biosorption was influenced by pH, initial metal concentrations, and anionic composition. Respirometric tests were carried out to identify potential inhibitory effects of metal accumulation on microbial oxygen uptake rates. Keywords:
Biosorption; Metallic waste;Actinomycetes; Dialysis; Respirometry
schemes have high chemical costs and tend to generate large quantities of highly caustic sludges. The use of biological processes can overcome some of the limitations of physical and chemical treatment and provide a means for cost effective removal of metals [4-61. It is generally believed that microbial metal removal can occur by binding of metals on cell surfaces, immobilization of metal ions by extracellular enzymes, or active transport of metal ions into the cell with intracellular accumulation [6-S]. A viable cell culture is required for active transport, whereas, surface
1. Introduction
Industrial and mining wastes, solid wastes, sewage sludges, and landfill leachates are major sources of metallic contamination [l-31. Chemical and physical treatment processes such as precipitation, catalytic oxidation, and ion exchange columns have been used for the treatment of soluble metallic wastes. However, these treatment
*Corresponding
author.
0048-9697/95/$09.50 0 1995 Elsevier SSDI 0048-9697(95)04709-A
Science
BV. Ail rights
reserved.
210
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and extracellular uptake do not require cell viability. To facilitate the development of full scale metal biosorption systems, it is essential to quantify the uptake capabilities of cell surfaces and extracellular materials and to evaluate the degree to which metal ions impair cell viability. Various organisms have been used for the uptake of metals from aqueous systems. Nakajima and Sakaguchi [7] studied metal binding capacities of 83 microorganisms and concluded that actinomycetes show higher capacities to bind metal ions as compared to fungi, yeast, and bacteria. Accumulation of metal ions on biological surfaces results in inhibition of life processes of the cell [lo]. In addition it has been demonstrated that actinomycetes have greater resistance to metal toxicity [ll]. The specific objectives of this study were: (a) to quantify metal binding capacities of cellular and extracellular components of a microbial biosorption system and (b) to evaluate the response of respiratory rates to various concentrations of the test metals. Acidic soil isolates derived from a culture of actinomycetes were used as the test organisms for this study because of their reported metal binding capacities and ability to resist metal toxicity [12]. The test metals used for this study were copper and lead. 2. Background
Microbial treatment reactors contain cellular mass in contact with a liquid medium into which products of microbial metabolism are released. Both cellular and extracellular components of a microbial system are capable of binding metal ions [6-81. Adsorption of metal ions on microbial cells takes place in two stages: (1) instantaneous binding to cellular surfaces and (2) gradual transport and accumulation of metal ions within the cytoplasm [7]. The metal binding capacity of a microbial system varies depending on the metal type. Anions can also influence microbial metal uptake [71. In a previous study, biomass from an acidic soil isolate was demonstrated to be capable of binding copper and lead from bimetallic solutions [13].
of the
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170 (1995) 209-220
Maximum binding rates for copper and lead from aqueous solutions occurred at pH 4.0 [13]. To evaluate the capacity of the cellular surfaces and the extracellular material, it is necessary to separate extracellular products from biomass and compare their metal uptake capacities. Semipermeable membranes can function as physical barriers to separate the cellular and extracellular components and thereby provide a means to evaluate uptake capacity on a laboratory scale. Transport of metal ions across a semipermeable membrane is governed by the concentration gradient and the flux can be evaluated as follows [14]: F,=D;A;~
AC
(1)
where, F, =
Net solute flux through the membrane (mole/cm’/s> D, = Diffusion coefficient of the solute (cm’/s> A, = Numerical factor representing certain restrictions imposed by the membrane on the flow of solute AC, = Difference in the concentrations on either sides of the membrane (mole/cm3> AX = Thickness of the membrane (cm> For a specific test system, metal ion flux is directly proportional to the concentration gradient across the membrane. It can also be inferred that the net flux of metal ions through the membrane is zero at equilibrium. Dialysis membranes can be used in this context as an experimental tool. Biosorption coupled with dialysis consists of two stages: (1) diffusion of metal ions through a semipermeable membrane and (2) binding of diffused metal ions by the microbial system. The rate of change of metal concentration, r, in the solution, corresponds to the rate of diffusion and rU is the uptake rate. The rate of diffusion can be calculated as:
cc,- c,> rc= (t,-tl)
(2)
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211
where, C, and C, are the concentrations of metal in the solution at times t, and t,, respectively. The uptake rate, r,,, can be computed using equation (3).
cal circuit so that current can be supplied when required.
(B, -B,) r”= (t2-tl)
This study was based on evaluating biosorption of metal ions from single and bimetallic solutions consisting of two metals: copper and lead. All tests were conducted at constant pH (4.0) and temperature (25°C). The biosorbate was derived from the culture of acidic soil isolates. Biosorption from the whole system was compared to the cellular material sorption and non-diffusible microbial metabolism.
where, B, and B, are the concentrations of metals bound to the biological system at times t, and t,, respectively. Mitra et al. [15] demonstrated that the degree of cell inhibition by metallic contamination depends on the type of metal ions. Cadmium was shown to inhibit the log phase of growth. Metal ion valency also influences the degree of toxicity. Cr6+ was shown to be more toxic to microorganisms than Cr3+ [161. Vallee and Ulmer [171 postulated that the binding of metals to thiol on protein molecules and replacement of metals in the enzyme prosthetic group caused inhibition. Bacterial cells can resist toxicity to a certain limit depending on (1) production of enzymes capable of nullifying toxic properties of the metallic contaminants. (2) formation of an impervious layer external to the cell wall, and (3) removal of metals by transport across the cell membrane. Increase in metal concentration beyond the threshold value can result in lysis of microbial cells and loss of viability [18]. Inhibitory effects of metallic contaminants can be judged by monitoring microbial activity directly or indirectly. Respiration rate can be used as an indirect measure of microbial viability. Respirometers measure the rate of oxygen consumption during microbial cell respiration and thereby provide a basis for comparing the growth characteristics of microbial cultures under controlled conditions. Carbon dioxide is generated during microbial respiration. In an electrolytic respirometer, the CO, generated is absorbed by potassium hydroxide solution and is replaced by a stoiciometric quantity of oxygen generated by the electrolysis of acidified water. The current required for electrolysis can be correlated to the volume of oxygen generated. Electrolytic cells are equipped with a break mechanism in their electri-
3. Experimental
only
design
4. Procedures
The experimental study included four stages: (a) culturing acidic soil isolates and preparing test metal solutions; (b) dialysis experiments to evaluate the factors governing metal binding on cellular and non-diffusible extracellular components of the microbial system; cc> direct contact tests to determine the kinetics of metal binding on cellular surfaces, and (d) respirometric tests to evaluate the inhibitory effects of metal accumulation on microbial respiration. The experimental methodology is described below. 4.1. Culture of microbial system
The acidic soil isolates were cultured aerobically at 25°C and a pH range of 6.5-7.5 using a feed containing glycerin and L-arginine as organic substrates and other mineral nutrients. A sodium bicarbonate buffer was used to control pH in the bio-reactor [13]. The composition of feed solution is shown in Table 1 and a diagram of the reactor used for culturing the microorganisms is presented in Fig. 1. The 5.0-l reactor was operated as a completely mixed, suspended growth, semi-batch reactor with a hydraulic retention time of 5 days. To feed the reactor, 1 litre of the reactor fluid was removed daily and was replaced by an equal volume of feed solution. 4.2. Analytical
methods
During the conduct of laboratory experiments, care was taken to eliminate all the possible
212
Ad.
AIR D(HAUST
! 1
Pradhan,
A.D. Levine -
/ The Science FEED
i!
of the Total Environment
and membrane surfaces. Acid digestion [19] was used to desorb the metal ions. 4.3. Dialysis
0
0
0
-REACTOR
0
0
FLUID
I ’
0
AIR BUBBLES2 ! 0
-
Fig. 1. Reactor
used for culturing
microorganisms.
sources of contamination. To avoid metallic contamination the glassware was decontaminated using a acid wash containing 5% concentrated nitric acid and was then rinsed using deionized water. To eliminate microbial contamination, the glassware used for storing feed solutions and microbial system was sterilized using an autoclave. Atomic absorption spectroscopy was used to determine the metallic concentrations in the experimental solutions. To establish the mass balance it was necessary to strip the metal ions adhering to glass Table 1 Composition
of the feed solution
for acidic
Component
Concentration
Magnesium sulfate Sodium chloride Calcium chloride LArginine Potassium phosphate Glycerine Sodium bicarbonate
200 200 50 450 450 4.5b 250
“Derived from bConcentration
[lo]. in ml/l.
soil isolate9 (mg/l)
170 (1995) 209-220
experiments
An overview of the design for the dialysis studies is presented in Fig. 2. Tests were conducted on single metal systems with either copper or lead or a bimetallic system containing both metals. The concentrations tested are shown in Table 2. The metal solutions of the desired ionic strength were prepared by dissolving the precalculated quantities of nitrate, chloride, or sulfate salts of copper and lead in deionized water. During the experiments, the solution pH was adjusted using NaOH along with H,SO,, HCl, and HNO, for SO:-, Cl-, and NO; salts, respectively. A flow sheet for the preparation of the dialysis test system is shown in Fig. 3. The cellular and extracellular components of the microbial system were separated using centrifugation followed by vacuum filtration (pore size 1.2 pm). The cellular component was then resuspended in a volume of deionized water equal to the removed extracellular fluid. Parallel tests were conducted on the unseparated microbial system. A control consisting of an equal volume of deionized water was tested to evaluate transport through the membrane and potential losses on the membrane surfaces. For each test, the pH was adjusted to 4.0 and a 50-ml aliquot was poured into individual pieces of prewashed dialysis tubing (Spectra porTM) with a molecular weight cut off of 3500 atomic mass units (amu). Each section of dialysis tubing was immersed in an aerated metal solution until a steady state concentration was reached at about 48 h. The reactor setup for the dialysis experiments is shown in Fig. 4. To minimize evaporation, the reactors were covered with foil during the test. The metal ion concentration in the solution external to the dialysis tubing was monitored at regular intervals. At the end of each test, the contents of the dialysis tubing were acidified and analyzed to determine the chemical oxygen demand (COD), suspended solids (SS), and pH [19l. The concentrations of Cu and Pb were de-
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213
170 (1995) 209-220
Metal solution I,
I BiitiCCUdpb
Lead
Copper
I
I
*pm
I
20ppm
Fig. 2. Summary
30ppm
of experimental
r 1Oppm
I
20ppm
design for dialysis
termined using flame ionization atomic absorption spectroscopy. Acid digestion was used to free the metal ions adhering to cellular, glass, and membrane surfaces. Rates of diffusion were calculated using Equation 2. The results were expressed as the mean metal uptake capacities and compared with the results of previous studies [7]. 4.4. Kinetic studies To determine the rate of metal uptake by microbial cells, a direct contact technique was used [13]. A 40-ml aliquot of metal solution was pipetted into each of 12 250-ml flasks. Microorganism samples suspended in 10 ml of pH adjusted sodium bicarbonate buffer solution were added to each flask. The reaction vessels were then placed on a shaker table with a lo-cm stroke at 80 strokes per min. At selected time intervals, samples were removed from the shaker table, vacuum filtered using a glass fiber filter, acidified with concentrated nitric acid, and analyzed for metal concentration. The metallic uptake rates were calculated by evaluating the change in metal concentration over time using Equation 3. Mass balance calculations were used to evaluTable Metal
2 concentrations
Type of metal
solution
Bimetallic Cu and Pb Cu in single metal tests Pb in single metal tests
r
I 30ppm
separation
I
I 2CWm
r l%m
of biological
uptake
I 30pPm
of metals.
ate the accuracy of measurements in the various tests. Statistical analyses were carried out to determine the relationship between the total uptake observed in the experiments and the sum of the uptake capacities of the cells and the nondiffusible extracellular material. The metal binding capacities of the two components were subjected to statistical analysis. The metal binding capacities of microbial cells and the extracellular
Cantrifugation ~acumn
and
filtration
deionized
water
To dialysis experiment
tested
using
dialysis
Concentrations 10,20,30 10,20,30 10, 20, 30
tested
(ppm by wt.) To dialysis experiment
Fig. 3. Flow sheet for the separation
of cellular
material.
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AIR SUPPLY F=-
UYIVV CONTAINER
I I
\
Metal solution \
Fig. 4. Experimental
setup for dialysis
experiments.
metabolites when tested individually were compared with the metal uptake observed for the entire microbial system using the t-test. To determine the accuracy of experimental techniques, the mass balance relationship was established as: MS, + MB, = MS, + MBf + MGf + MDf
(4)
MS, = MB, = MG, = MD, =
209-220
were interfaced with a computer using software developed by Bioscience, Inc. The system was capable of automatically recording data at set time intervals. Respirometric settings used for the study are specified in Table 3. In this study, metal solutions of known ionic strength were prepared by dissolving copper and lead salts in deionized water. The compositions of the metal solutions tested using respirometry are shown in Table 4. For each electrolytic reactor, 100 ml of the microbial aliquot was suspended in 900 ml of metal solution and 50 ml of the completely mixed reaction sample was collected to provide information regarding initial metal concentrations and the organic content. The respirometric data collection was then started in accordance with the experimental settings described in Table 3. For each concentration of the metal solution, the test was conducted in duplicate. Controls were established by testing blank samples consisting of deionized water. A flow sheet for the preparation of the reaction sample in respirometry is shown in Fig. 5. When the oxygen uptake reached a plateau, one of the duplicates was tested for reactivation of microorganisms by adding 50 ml of the feed solution. All the samples were immediately acidified with 5 ml of concentrated nitric acid to prevent further biological activity and were later analyzed to determine metal content, organic content (COD), and suspended solids. 5. Results
Results of the dialysis and respirometric are presented below.
where, MS, = MB, =
170 (1995)
Initial metal content of the solution Initial metal content of the biological system Final metal content of the solution Final metal content of the biological system Metal quantity attached to glass Metal quantity attached to dialysis tub ing
4.5. Respirometry Electrolytic respirometers
used for this study
studies
5.1. Dialysis experiments Dialysis experiments revealed that cellular and extracellular components were capable of binding Table 3 Respirometer
settings
Parameter
Value
Sample volume Oxygen generation rate Data collection intervals
950 50 15 30 2
ml mg/h min for initial 2 h; min for next 6 h; h for the rest of the time
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Reaction sample 950 ml
1 Fin;; saaple
1
Fig. 5. Flow diagram for the preparation (pH was maintained at 4.0) for respirometry
of reaction studies.
samples
170 (1995) 209-220
215
uptake capacities is shown in Table 5 and is graphically represented in Fig. 6. To compare the results obtained from different tests, the data were normalized based on the sample COD. The experiments revealed that an increase in the initial lead concentration from 10 ppm to 20 ppm resulted in an increase in the metal uptake by the cellular part and the entire microbial system. An increase in the concentration beyond 20 ppm, however, resulted in the apparent desorption of the metal ions from the biosorption system. Similar tests with copper did not indicate such a trend. Statistical analysis of the data revealed that the metal uptake was more efficient by the separated components than the unseparated system at 90% confidence level. In a bimetallic system, the microorganisms appeared to possess greater affinity for lead than copper over the concentration range tested. A comparison of the influence of the anionic composition on the copper uptake is shown in Fig. 7. Sulfate appeared to influence cellular uptake more than chloride and nitrate. 5.2. Dijj‘iuion and uptake rates
metal ions individually as well as combined. In the dialysis experiments, most of the uptake and complexation reached a saturation point within the first few hours. Metal and anionic concentrations influenced the metal binding capacities of cellular and extracellular components. Continuation of the experiments beyond the saturation capacity resulted in the apparent desorption of metal ions from the biological system. The influence of metal concentration on saturation metal Table 4 Concentrations
of metallic
solutions
used in respirometry
Concentrations
Bimetallic
Copper concentration was maintained at 100; lead concentration were 0,50, 100,150 0, 25, 50, 75, 100, 150
Cu in metal Pb in metal
single tests single tests
tion and uptake rate, respectively. These rates, calculated for the various experimental stages, are shown in Table 4. The uptake rate, Y,, was calculated based on data from a previous study [13], whereas, r, was obtained from dialysis experiments data. Based on the data obtained, it can
Uptake
0.6 o4
mmollg COD
0.2
Metal
Type of tests tests
As explained in the background section, rc and r, represent rate of change of metal concentra-
0,50,
tested
100, 125, 150, 175
(ppm by wt.)
.
10 ppm 20 ppm 30 ppm Initial Concentration q Cell Fig. 6. Effect capacity.
q Extracellular of initial
metal
concentration
q Intact System on lead uptake
216
A.A. Pradhan,
Table 5 Copper uptake Cont.
capacities
Anions
@pm)
10
NO3
10 10 10 20 30
so4
Cl NO3 NO3
N/A, Table Rates Time
Not
Levine
/The
Science of the Total Environment
components
Other metal cont.
Uptake cells (mmol/g (A>
N/A N/A
0.033 0.091 0.038 0.189 0.165 0.468
170 (199.5) 209-220
of acidic soil isolates
by COD)
Uptake by extracellular part (mmol/g COD) (B)
Sum of the Cellular and extracellular uptakes (mmol/g COD) cc>
Uptake by microorganisms with extracellular substances (mmol/g COD) CD)
0.116 0.052 0.018 0.090 0.188 0.369
0.149 0.143 0.056 0.279 0.353 0.837
0.053 0.062 0.016 0.044 0.222 0.055
applicable.
6 of diffnsion (mm)
and metal
uptake
at various
stages of the experimenta
Copper
5 30 120 aReported
of the individual
N/A Pb 10 ppm Pb 20 ppm Pb 30 ppm
NO3
A.D.
for
Lead
r, (mmol/min)
r, (mmol/min)
r, (mmol/min)
r, (mmol/min)
1.33 x 10-7 1.00 x 10-r 5.00 x 10-s
2.8268 x lo-’ 1.4650 x 1O-4 1.7500 x 10-5
6.67 x lo-’ 6.67 x lo- * 3.33 x 10-s
2.2844 x 10 - ’ 2.8600 x lo- 4 4.3000 x 10-s
:tallic
test with
10 ppm of each metal
in presence
of nitrate.
0.161
Nitrate
I
0 Cell
Chloride
n Extracellular Fig. 7. Effect
Sulfate
0 Cell + Extracellular
of anions
on copper
uptake.
El Intact System I
AA. Table 7 Comparison
Pradhan,
of mass balance
A.D. Levine
of data
Metal
Bimetallic
tests
cu Pb cu Pb CU Pb cu Pb cu Pb
Cellular
tests component
Extracellular Entire
Science of the Total Environment
component
system
aCalculated by comparing the metal quantity used for starting the experiment.
tested
quantity
observed
No. of observations
Mean
24 12 7 3 7 5 7 5 7 5
11 26 7 50 11 36 9 31 17 25
in the various
be inferred that rU > rC for the initial stages. The difference, however, decreases with time.
Results of the mass balance calculations are shown in Table 7. Although strict controls were observed in all components of the study, a more complete mass balance was obtained for copper than for lead. In general, the extracellular compo-
Experimental Unimetallic 0 wm 50 wm 100 ppm 125 ppm 150 ppm 175 ppm bimetallic
demand conditions
various
experimental
test components
percentage
5.4. Respirometric
results
conditions
Plateau
oxygen
the metal
The results of respirometric tests are provided in Table 8. It is evident that the oxygen uptake
uptakea
(mg/l)
Initial
188.05 70.64 71.58 189.69 163.11 155.34
0.0 5.0 7.5 20.0 20.0 20.0
259.28 22.26 16.81 20.22 175.54 67.71
0.0 50.0 65.0 145.0 90.0 165.0
54.21 17.84 16.22 14.32
0.0 26.0 40.0 20.0
copper
on the averages.
error=
at the end of the tests with
lead
0 PPm 25 ppm 50 wm 75 mm 100 ppm 150 ppm Bimetallic Cu 100 ppm Pb 0 ppm Pb 50 ppm Pb 100 ppm Pb 150 ppm aBased
under
217
nent showed the highest recovery in mass balance calculations whereas, the cellular component showed the least recovery perhaps due to analytical inaccuracies associated with solid phase analysis.
5.3. Mass balance calculations
Table 8 Plateau oxygen
170 (1995) 209-220
calculations
Category
Unimetallic
/The
lag time (h)
218
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Science of the Total Environment
rate during respiration was higher for nonmetallic solutions than in the presence of metals. Addition of metal ions to the aqueous system caused a lag phase prior to the onset of respiratory activity. The lag phase duration was longer for copper tests than for lead solutions of equal mass concentrations. It can be inferred that an increase in metal concentration resulted in a decrease in the plateau oxygen uptake by the system. Addition of feed to the reaction sample resulted in an increase in oxygen uptake rate. The inhibitory effects of metal accumulation appear to be reversible in this concentration range. 6. Discussion
The observations from these studies and the potential use of biosorption for metallic waste treatment are discussed in the following paragraphs. 6.1. Metal uptake capacities
Saturation capacity exhibited by the microbial system in binding metal ions is a key factor for biological sorption of metals. The saturation capacity can be explained on the basis of cellular biochemistry. Metal binding on cellular surfaces has been shown to be associated with the fnnctional groups such as carboql, hydroxyl, pyruvate, amide, and acetate [20-221. At constant pH, a microbial cell contains a fixed number of binding sites. As the pH increases, some of the aminophosphate groups and lipoproteins of the cellwall are hydrolyzed thus making some of the sites unavailable for metallic binding. In this study, the majority of metal uptake occurred fairly rapidly. The microbial system showed preferential binding capacity for lead. Similar results have been previously reported by Ruzic [23] and Irving and Williams [24]. In presence of more than one metal, the extent of complexation of each metal with the microbial system is related to the metal binding constants. The order of metal enzyme stability is given by Pb > Cu > Ni > Co > Zn > Cd > Fe > Mn > Mg. Thus, it is possible that copper could be displaced by lead. Therefore, a treatment scheme based on biosorption may require optimization
170 (1995) 209-220
depending on the metal content and concentration. In this study, total metal uptake was found to be higher when the cellular and extracellular components were tested individually as compared to the unseparated system. The exact mechanism of interaction and competition between cellular and extracellular components needs to be investigated further. 6.2. Kinetic studies
The metal uptake rate Y, appears to be related to the availability of metal binding sites and the rate of diffusion r, is dependent upon the concentration gradient across the semipermeable membrane as shown by Equation 1. In the initial stages of the experiment, due to the availability of a large number of binding sites, Y, > rc. Consequently, rapid binding of metal ions occurred. As the number of available binding sites decreased, a critical stage was reached when Y, = rc. Allowing contact time beyond this stage resulted in rU < Y, which is represented by the apparent desorption of metal ions. Because the availability of metal binding sites on a cellular surface depends on the pH, it is essential to establish an optimum pH condition for biosorption of metals either individually or in multicomponent mixtures. 6.3. Respirometry
Respiratory inhibition due to metal accumulation was demonstrated in this study. Evidence for metal inhibition was manifested by: (1) a lag phase prior to onset of respiratory activity and (2) a decrease in plateau oxygen uptake capacity of the microbial system with increase in metal concentration. The acidic soil isolates did not show a loss of viability over the range of metal concentrations tested. This capability of the acidic soil isolates may be advantageous in developing a biosorption system to treat metallic wastes, particularly if the biosorption system is to be based on viable organisms. 6.4. Applications
The use of biosorption to remove and concentrate metal ions from solutions has potential application in treatment of metal containing waste streams, such as landfill leachates, mining wastes,
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and industrial and hazardous wastes. This study has provided laboratory data supporting the use of acidic soil isolates in a biosorption system. Optimization of metal uptake, metal recovery, and reactor design is necessary to facilitate the conduct of pilot scale studies and development of design information.
170 (199.5) 209-220
219
(ISWRRI). The authors acknowledge the assistance of James Gaunt, William Graham and Angela Bieldfeldt in the conduct of this project. Amit A. Pradhan is an Environmental Engineer with Geotechnical Services, Des Moines, Iowa and Dr Audrey D. Levine is Associate Professor of Civil and Environmental Engineering at Utah State University, Logan, Utah.
7. Conclusions
The major conclusions from this study are listed below.
References HI
Dialysis experiments provided a means to determine the relative importance of the cellular and extracellular components for uptake of copper and lead from the test microbial system. For both metals tested, a saturation limit was observed for the maximum quantity of metal that could be bound. pH, anion type and concentration, and initial concentrations of the metal solutions all are significant factors in microbial metal uptake using acidic soil isolates. Overall metal uptake capacity of the microbial system is greater when cellular and extracellular components are separated. The presence of copper and lead inhibited microbial respiration. These effects were exhibited as (1) a lag phase in the oxygen uptake curve and (2) a decrease in plateau oxygen uptake potential. The lag phase duration was longer for microbial systems exposed to copper as compared to similar levels of lead. Following the lag phase, respiratory activity could be re-triggered by the addition of feed solution, thus some degree of microbial viability was maintained. Acknowledgements
This project was supported, in part, State Mining and Mineral Resources Institute (ISMMRI) and in part, by State Water Resources Research
by Iowa Research the Iowa Institute
A.D. Levine and L.R. Rear (Eds.), Evaluation of leachate monitoring data from co-disposal, hazardous, and sanitary waste disposal facilities, in Proceedings of the 43rd Annual Purdue Industrial Waste Conference, Lewis, Chelsea, MI, 1989, pp. 173-183. Cation Dl R.E. Marquis, K. Mayzel and D.L. Cartensen, exchange in cell walls of gram-positive bacteria. Can. J. Microbial., 22(7) (1976) 976-982. Geotechnology of Waste t31 I.A. Gweis and R.P. Khera, Management, Butterworth, Guildford, UK, 1990. B.Greene, R.A. McPherson and M.D. [41 D.W. Damall, Alexander, Recovery of heavy metals by algae, in R. Thompson (Ed), Trace Metal Removal from Aqueous Solution, Royal Society of Chemistry, Cambridge, UK, 1986, pp. l-24. and accu151 B. Friedman and P.R. Dugan, Concentration mulation of metallic ions on the bacterium Zooglea, in Developments in Industrial Microbiology, Vol. 9, Chapter 35, (1968) 381-388. and G.G. Geesey, Copper binding 161 M.W. Mittelman characteristics of exopolymers from a freshwater sediment bacterium. Appl. Environ. Microbial., 49(41 (1985) 846-851. and T. Sakaguchi, Selective accumulation [71 A. Nakajima of heavy metals by microorganisms. Appl. Microbial. Biotechnol., 24(l) (1986) 59-64. 181 J.J. Doyle, R.T. Marshall and W.H. Pfender, Effect of cadmium on the growth and uptake of cadmium by microorganisms. Appl. Microbial., 29(4) (197.5) 562-564. Sensitivity of various bacteI91 H. Babich and G. Stotzsky, ria, including actinomycetes and fungi to cadmium and the influence of pH on sensitivity. Appl. Environ. Microbiol., 33(3) (1977) 681-695. in C. Booth [lOI S.T. William and T. Cross, Actinomycetes, (Ed.), Methods in Microbiology, Vol. 4, 1971, 317 pp. of heavy 1111 A.B. Norberg and H. Persson, Accumulation metals by Zooglea ramigera. Biotechnol. Bioeng., 26(3) (1984) 239-246. [121 A.A. Pradhan and A.D. Levine, Role of extracellular components in microbial biosorption of copper and lead. Water Sci. Technol., 26 (9-11) (1992) 2153-2156. 1131 W.S. Graham, Development of Engineering Parameters for the Design of Metal Biosorption Waste Treatment
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WI
[I61 [171
ml
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/The
Science
System, Unpublished Master’s Thesis, Iowa State University, Ames, IA, 1991. J.R. Pappenheimer, E.M. Renkin and L.M. Borerro, Filtration, diffusion, and molecular sieving through peripheral capillary membranes - a contributaion to the pore theory of capillary permeability. Am. J. Physiol., 167(l) (19511 13-46. R.S. Mitra, R.H. Gray, B. Chin and I.A. Bernstein, Molecular mechanism of accommodation in Eschepichia coli to toxic levels of Cd*+. J. Bacterial., 121(3) (1975) 1180-1188. R.M. Steritt and J.N. Lester, Interaction of heavy metals with bacteria. Sci. Total Environ., 14 (1980) 5-17. B.L. Vallee and D.D. Ulmer, Biochemical effects of mercury, cadmium and lead. Annu. Rev. Biochem., 41 (1972) 91-128. Z. Vaituzis, J.D. Nelson, L.W. Wan and R.R. Colwell, Effects of mercuric chloride on growth and morphology of selected strains of mercury resistant bacteria. Appl. Microbial., 29(2) (1975) 275-286.
of the
Total Environment
I70 (1995) 209-220
AWWA and WPCF, Standard Methods for the 1191 APHA, Examination of Water and Wastewater, 18th Edition, APHA, AWWA, WPCF, 1991. La A.E. Steiner, D.A. McLaren and C.F. Forster, The nature of activated sludge floes. Water Res., 10(l) (1976) 25-30. Dll S.C. Churms and A.M. Stephens, Studies of molecular weight distribution for hydrolysis products from some KIebsiella capsular polysaccharides. Carbohydr. Res., 35(l) (1974) 73-86.
La
[=I
Dl
S.A. Boyd, L.E. Sommers and D.W. Nelson, Infrared spectra of sewage sludge fractions: evidence for an amide metal binding site. Soil Sci. Sot. Am. J., 43(9) (1979) 893-899. I. Ruzic, Theoretical aspects of direct titration of natural waters and its information yield for trace metal speciation. Anal. Chim. Acta, 140(l) (1982) 99-113. M. Irving and R.J.P. Williams, Order of stability of metal complexes. Nature, 162(8) (1948) 746-747.