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Cadmium removal using Cladophora in batch, semi-batch and flow reactors
Bioresource Technology 81 (2002) 249±255
Cadmium removal using Cladophora in batch, semi-batch and ¯ow reactors Steven P.K. Sternberg *, Ryan W. Dorn Department of Chemical Engineering, University of Minnesota at Duluth, Duluth, MN 55812, USA Received 17 August 2000; received in revised form 19 July 2001; accepted 19 July 2001
Abstract This study presents the results of using viable algae to remove cadmium from a synthetic wastewater. In batch and semi-batch tests, a local strain of Cladophora algae removed 80±94% of the cadmium introduced. The ¯ow experiments that followed were conducted using non-local Cladophora parriaudii. Results showed that the alga removed only 12:7 6:4% of the cadmium introduced into the reactor. Limited removal was the result of insucient algal quantities and poor contact between the algae and cadmium solution. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cadmium; Bioremoval; Bioremediation; Cladophora; Macro-algae
1. Introduction The accumulation of heavy metals in the environment has received increased attention because of the threat to public health. The most commonly used procedures for heavy metal removal include chemical precipitation, ion exchange, reverse osmosis, and solvent extraction. Although these processes tend to be ecient, they are generally expensive and require frequent service attention. In addition, the remediation of heavy metals from a surface or ground water by these methods is cost prohibitive. Therefore, the need for economical, eective, and safe methods for metal removal has resulted in the search for alternative materials and methods. Biosorption has emerged as a potential option for heavy metal removal. Biosorption is the accumulation of heavy metals using microorganisms (such as bacteria and fungi) and photosynthetic life (such as algae, aquatic and emergent plants). In shallow bodies of water (1±5 m) having low concentrations of cationic heavy metals (1±20 mg/l), biosorption can be very eective (Roy et al., 1992). Studies have suggested that biosorption occurs in a manner similar to ion exchange for inactivated cells (Aksu et al., 1998; Kuyucak and Volesky, 1989; Nakajima and Sakaguchi, 1986; Tobin et al., *
Corresponding author. E-mail addresses: [email protected] [email protected] (R.W. Dorn).
(S.P.K.
Sternberg),
1987; Tsezos, 1984). In living cells, the ion exchange step may be followed by a metabolism-dependent uptake step in which the metal is transported into the cells. Other important biosorption processes include surface precipitation and surface complexation. The advantage of using living biomass is the rapidly regenerating supply of biomass. The major disadvantage is the toxic effect the metals can have on the organism. For this study, algae were selected because they are ubiquitous and native to almost all parts of the world. Algae grow in fresh or saltwater over a wide range of temperatures and pH. Prior work has shown that Cladophora possess excellent heavy metal bioremoval potential (Ozer et al., 2000; Sobhan and Sternberg, 1999; Oertel, 1991; Vymazal, 1990). This project examined cadmium removal using Cladophora algae in batch and ¯ow reactors. The batch experiments demonstrate the ability of this organism to take up cadmium. The ¯ow experiments demonstrate how this technology may be implemented on a larger scale. Cadmium was selected as the target metal because it is a regulated metal (US EPA, 40CFR 261). Cadmium poses serious health hazards to humans and animals even at concentrations of one milligram per liter. It has been shown to be a non-essential metal ion for growth of plants and animals (Torres et al., 1998). Cadmium can be found in wastewater streams from automobile industries, metal ®nishing, electroplating, and organic chemical industries. It can be leached from storage piles
0960-8524/02/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 3 1 - 6
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and settling basins, and can become mobilized through contact with an acid, such as from acid precipitation. 2. Methods 2.1. Organism Cladophora, a green ®lamentous macro-alga, was used for this work (Dodds and Gudder, 1992). A local strain was used in the batch tests, and a non-local strain was used in the ¯ow reactor tests. The local strain was obtained from a freshwater stream, the English Coulee, running through the campus of the University of North Dakota, Grand Forks, ND. The alga was collected and washed with ¯owing Coulee water to remove dirt and other debris. The samples were rewashed with distilled, deionized water in the lab and then split into smaller portions to initiate individual cultures. To improve the reproducibility and reliability of the test methods, a nonlocal but better identi®ed alga, Cladophora parriaudii, was obtained from the Culture Collection of Algae at the University of Texas, Austin, TX; phone (512) 4714019. 2.2. Water All water used for growing and testing the alga for both batch and ¯ow reactors, was obtained from the English Coulee, in the same location that the alga was collected. The Coulee water was ®ltered using Whatman No. 4 ®lter paper to remove dirt and debris. Distilled and deionized laboratory water was used to wash the freshly harvested alga, and to prepare standards and other solutions. 2.3. Growth and test conditions Cultures were started by adding individual portions of the alga, weighing approximately one gram wet weight, to jars ®lled with 700 ml of Coulee water. 2 ml of fertilizer solution were introduced into each jar once a week during culturing. Samples were allowed to acclimate to the laboratory conditions for two weeks before contamination. Only samples that experienced optimal growth were used in the experiments. Samples that died or failed to grow were discarded. Reagent grade urea (8.4 mg) was added to each jar twice per week during the experiments. Minerals in the fertilizer solution were found to interfere with the cadmium measurements, so urea was used, instead of the plant fertilizer, during the experiments. The pH of each culture was monitored daily, for both the acclimatization period and the actual experiments. The algae modi®ed the water's pH from a background value of 7.8±8.1 to 9.5 in 24 h. The pH change was most
likely the result of photosynthetic carbon (as HCO3 ) uptake. At higher pH levels, the algae did not grow. A probable reason for the inhibited growth of Cladophora at pH 9.5 is that there was no available HCO3 and it was carbon limited. Availability of carbon is likely more critical than pH. In the scope of the present experiments, carbon supply was not monitored. Very dilute acetic acid (0.01 M) was used to control pH and provide the carbon supply. The target pH was 7.8±8.1. Sylvania Gro-Lux ¯uorescent lamps were used as the light source. Light intensity at the water's surface was maintained at 2150 10% lux (Eaton et al., 1995). Light was provided to the algal jars 24 h a day, seven days a week. 2.4. Batch reactors The batch reactor experiments were carried out in 700 ml Kerr mason jars. The variables included: viable or non-viable alga, and addition of a single large dose or a daily small dose of cadmium. Each experiment was run for 16 days, with daily 3 ml samples taken for metal analysis. Controls consisted of no alga, no metal, and no metal or alga jars. All experiments were run simultaneously in separate jars. Each condition was run in triplicate, with average results reported. The starting amount of alga in each jar was 1350 250 mg wet weight. The same laboratory acclimated cultures were used for the viable and non-viable algae. The non-viable alga was dried in a drying oven for 48 h prior to the experiment. These cultures exhibited no signs of growth during the experiments. They were used to determine if any advantage was gained by using viable biomass. The single large dose added 3.5 mg cadmium, from a 1000 mg/l standard solution, to the jar to create an initial concentration of 5.0 mg/l. The daily dose added 0.7 mg cadmium each day, to instantaneously increase the concentration by 1 mg/l. The cadmium solution was added directly to the alga in the jars. The reactors were stirred with a glass stir rod. The no alga control was used to determine if the alga was the only sink for cadmium. The no metal control was used to determine the eect that cadmium had on growth rate. The no metal or alga control was used to determine if other sources of cadmium were present in the lab environment (cross contamination, leaching from jars, air-borne due to aggressive mixing of other jars) and to verify adequate laboratory technique. Evaporative and sample losses occurred throughout the experiment. Each day the jars were returned to the 700 ml mark through daily additions of ®ltered Coulee water. Careful records accounted for loss of cadmium from each jar caused by sampling. At the end of the experiments, the alga samples were separated from the water by ®ltration. The alga sample and ®lter paper were
S.P.K. Sternberg, R.W. Dorn / Bioresource Technology 81 (2002) 249±255
then digested to determine the amount of cadmium sorbed. 2.5. Plug ¯ow reactor A plug ¯ow reactor (see Fig. 1) was constructed by placing a plastic bae down the middle of a 30-l (50 cm 25 cm 25 cm) glass container. The container was further divided with plastic baes resulting in a seven-cell reactor. Of the seven chambers, ®ve contained algae, one was used to mix and sample the in¯uent, and one was used to mix and sample the euent. The reactor provided an over-and-under ¯ow with triangular cuts in the baes. The experimental design was based on a two-level, three-variable factorial (Keppel, 1982). The center point was also used, and was run in triplicate for use in error estimation. Thus, 11 experiments were conducted. The variables examined include ¯ow rate, inlet concentration, and feed volume. Values are given in Table 1. Flow rate altered the amount of mixing in the system and the contact time between water and algae. The feed volume controlled the total amount of ¯uid and cadmium presented to the system before the algae were harvested and analyzed. During an experimental run, 21 samples (3 ml each) were taken from the inlet and outlet cells at equal time intervals (times varied depending on ¯ow rate and volume fed). A calibrated peristaltic pump was used to introduce the cadmium solution into the reactor. Another peristaltic pump was used to remove the euent from the outlet cell.
Treated Water Out Algae
Algae
Algae
25 cm
Cadmium Solution In Algae
Algae
50 cm
Fig. 1. Top view of the reactor.
Table 1 Experimental design values Variable
High value
Low value
Center value
Flow rate (l/h) Inlet cadmium2 concentration (mg/l) Volume fed (represented by reactor volumes)
7.1 5.0
4.3 1.0
5.8 3.0
5.0
2.0
3.5
251
In addition to the factorial design, two follow-up experiments were performed after completing the factorial design to study the eects of alga quantity and alga±water contact. These experiments were at the center point conditions of the ¯ow reactor factorial design. The ®rst follow-up run used a greater amount of biomass; the second included agitation with increased biomass to each cell during the experimental run. Agitation was performed manually with a glass stir rod. 2.6. Measurements and procedures Wet weights of algae samples were determined by ®rst blotting with commercial-grade paper towels to remove excess water. Samples were weighed using a digital balance (0.1 mg precision). The wet algae contained 91.3% water on average, based on samples dried in a standard drying oven. The wet weighing procedure did not appear to cause any harm to the algae, in terms of growth or bioremoval potential (Dorn, 1998). Growth was measured by simple wet weight measurements recorded before and after the experiment. Measuring intermediate weights would have greatly complicated the analysis, due to water lost during the blotting process. Other indications of growth included a bright green color of the tissue, increases in visible volume of the alga, and the presence of large numbers of bubbles within the alga. Because Cladophora is a macroalga, the wet weight was an easy and direct measurement of growth, and therefore other measures of growth, such as chlorophyll a were perceived as unnecessary to measure growth. The pH was measured using a Cole Parmer (Vernon Hills, Illinois, USA) Model 05668-20 electronic pH meter and pH electrode. Light was measured with a Cole Parmer Model DLM2 light meter. Cadmium concentration was measured throughout the course of the experiments and in the alga biomass at the conclusion of the experiment. All water samples were treated with 2 drops of 1.0 M nitric acid after collection to prevent precipitation before analysis. Metal concentrations in these solutions were measured, in accordance with the procedure described in Standard Methods (Eaton et al., 1995) using a Perkin±Elmer (Shelton, Connecticut, USA) Model 3100 Atomic Absorption Spectrophotometer. The detection limit for cadmium with this equipment was 0.028 mg/l. The alga was collected for metal analysis after completing the batch and continuous ¯ow experiments. Before digestion, samples (with the pre-weighed ®lter paper) were placed in a drying oven at 65±70 °C for two days to determine the dry weight of the biomass. The samples were cooled to room temperature and weighed using a digital balance until a constant weight was obtained. The samples were then placed in a mue furnace at 500 °C for 14 h to remove volatile components and oxidize the adsorbed cadmium.
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The samples were again cooled and weighed. The ashes were dissolved in Aqua Regia acid and placed on a hot plate until completely dry. The procedure was repeated until a clear solution was obtained and no signi®cant mass change was observed. The samples were then resuspended in 1 M HCl, diluted to 1000 ml with laboratory water, and analyzed for cadmium. The standard cadmium solution of 1000 mg Cadmium2 /l was prepared by dissolving 1.6308 g of reagent grade Cadmium Chloride CdCl2 , Fisher Scienti®c (Pittsburgh, PA, USA), in 1000 ml distilled and deionized water. Calibrated micropipettes were used to measure volumes of the standard Cadmium2 solution. The plant fertilizer solution was prepared by dissolving two grams of Stern's Miracle Groâ Products (Port Washington, NY, USA) in 1000 ml of water. Composition of the fertilizer, by weight, is approximately 31% nitrogen, 31% phosphoric acid P2 O5 , 36% potash K2 O, and trace amounts of mineral salts containing Mg, Cu, Fe, Mn, and Fe. 2.7. Calculations All calculations and statistics (average, standard deviation, regression, student t test for signi®cance) were done using the Microsoft Excel spreadsheet program. All batch reactor experiments were triplicated (N 3). The ¯ow reactor error was estimated by running a triplicate of the center point, all other points were run alone. Concentrations were measured directly by AAS using sample standards and calibration curves done internally on the AAS unit. The bioremoval potential was calculated as (Sorbed Cadmium Mass)/(Alga Mass Total Time). The bioremoval was calculated as (Sorbed Cadmium Mass)/(Initial Cadmium Mass) 100%. The mass balance was determined as the sum of cadmium mass in solution, alga, ®lter, and samples.
3. Results and discussion
Viable algae were more eective in removing both the single and daily doses of cadmium when compared to the non-viable algae. This is a result of the regenerating supply of anionic functional groups on the biomass surface due to alga growth or maintenance. The cadmium concentration for the non-viable algae was equivalent to that of the viable algae for the ®rst sample taken (after 12 h). After this time the amounts removed diverged. The ion exchange mechanism proposed for inactivated cells (Kuyucak and Volesky, 1989; Nakajima and Sakaguchi, 1986; Tobin et al., 1987; Tsezos, 1984) gives a possible explanation of this phenomenon. The mechanism states that there are a de®nite and constant number of adsorption sites on the non-viable biomass. After the sites become saturated with metal ions, adsorption equilibrium is established, resulting in no net concentration change. In the scope of the present study, no additional analysis was performed to con®rm this hypothesis. Alga growth was inhibited by the presence of cadmium in the water. The larger the initial cadmium dose, the greater the inhibition. Alga weight tripled over 14 days in the laboratory environment when not exposed to cadmium. The alga exposed to the daily doses (1 mg/l/ day) doubled in weight in 14 days, while the single dose (5 mg/l Cadmium2 ) alga did not increase in biomass during the 14 days. The cadmium concentration in the batch reactors is shown in Fig. 2. Great variability can be seen for the living alga samples, although the overall trend is a decrease over time. The residual cadmium concentration for the no alga controls decreased at the beginning of the experiment. This decrease was the product of cadmium precipitation due to elevated pH of the Coulee water. At higher pH values (greater than 9.0), precipitation was observed in the Coulee water, which was determined to be the cause of the initial decrease. Sampling also tended to reduce the overall concentration in the jars, resulting in an initial 5.0 mg/l concentration to decrease to 4.7 mg/ l in the no alga control jars after 14 days. The cadmium concentration in the semi batch reactors is shown in Fig. 3. The samples were collected just prior to addition of the daily dose of cadmium solution.
3.1. Batch tests Cd Concentration (mg/L
5.00
Results of the batch tests indicated Cladophora effectively removed cadmium from a synthetic wastewater. The viable alga removed cadmium by 80% in the batch and 94% in the semi-batch experiments. The nonviable alga removed cadmium by 65% in the batch and 25% in the semi-batch experiments. Cadmium made up 0.9±2.8% of the algae's ®nal dried cell mass. The mass balance, based on the sum of measured cadmium in the reactor solution, alga, ®lter paper, and the daily sample aliquots, accounted for all the known additions to within 20% (Dorn, 1998).
4.00
3.00
2.00
1.00
0.00 0
50
100
150
200
250
Time (Hours) Viable
Non-viable
No Algae
Fig. 2. Response of algae to single cadmium dose.
300
S.P.K. Sternberg, R.W. Dorn / Bioresource Technology 81 (2002) 249±255 14.00
Cd Concentration (mg/L)
12.00 10.00 8.00 6.00 4.00 2.00 0.00 0
50
100
150
200
250
300
350
400
Time (Hours) Viable
Non-Viable
No Algae
Fig. 3. Response of algae to daily cadmium doses.
The viable and non-viable algae removed cadmium at an equivalent rate for the ®rst day, but quickly diverged after that. The viable alga systems removed approximately the amount added each day, whereas the nonviable alga ceased detectable removal after day 1. The solution pH was shown to have an eect on cadmium removal. In Fig. 3, the residual cadmium concentration increased for the ®rst 150 h. During this time, pH of the solution was adjusted twice daily, resulting in a pH of approximately 8.0±8.3. After 150 h, pH adjustments were made daily and the removal increased, resulting in a lower residual cadmium concentration. During this time, the pH was approximately 8.7±9.0. This supports ®ndings that higher removal rates occur at higher pH values (Gardea-Torresdey et al., 1998; Kratachvil and Volesky, 1998; Ribeyre and Boubou, 1990). Biosorption capacity, de®ned as the mass of cadmium sorbed by the alga per mass of alga per hour, was calculated for both viable and non-viable algae. This is not the same as cadmium removal. It is used to represent the ¯ux of cadmium removal rather than the quantity removed. The biosorption capacity was calculated using the ®nal experimental conditions, which included time, alga mass and mass of cadmium in the alga. The batch reactor biosorption capacity of viable alga was 0:056 0:011 mg cadmium/g alga/hour and for the non-viable alga was 0:045 0:015 mg Cadmium/g alga/ hour. The averages were statistically indistinguishable at the 95% con®dence level (student t test), suggesting there are a limited number of binding sites on a given mass of the algae, regardless if the alga is viable or non-viable. Viable alga however will create more binding sites as it grows, and so can ultimately remove more metal than the non-viable alga, as Figs. 2 and 3 show. 3.2. Continuous ¯ow tests For the continuous ¯ow experiments, the algae removed only a small percentage of cadmium introduced into the system. Only four of the 11 experiments performed in the experimental design showed the C. par-
253
riaudii adsorbing more than 10% of the cadmium fed to the reactor. The average adsorption for the 11 experiments was 12:7 6:4%. One factor contributing to the limited removal was the small biomass to reactor volume ratio that existed during the experimental runs. The mass of alga in a cell, and the cell volume were approximately equivalent to the values in the batch experiments. However, the total volume of contaminated water in the reactor was approximately 9.2 l or 13 times the amount in any single jar. Therefore, it is probable that much of the cadmium had limited exposure to the alga, resulting in low removals. The experimental data were analyzed using Excel's data analysis regression option. The response (Y) was calculated from the experimental values and was designed to analyze data objectively and to be independent of any variables not controlled or tested. For this study, Y was the percentage of cadmium adsorbed by the alga per mass cadmium fed divided by the total ®nal mass (dry weight) of alga in the reactor (in grams). This response was chosen to nullify the eect that mass of alga might have on the results, since alga growth was not a controlled variable. Of the variables tested (¯ow rate, inlet concentration, and volume of feed), none had a statistically signi®cant eect on the response. A plot of total mass of algae in the reactor versus response (Y ) was constructed and no detectable relationship was found (results not shown). Another important indication of reactor eectiveness is comparing the measured breakthrough residence time (de®ned as the time when cadmium concentration ¯owing out is one-half of that ¯owing in) with the calculated residence time (calculated by dividing the volume of the reactor by the volumetric ¯ow rate). If the measured breakthrough residence time is less than the calculated, it indicates dead zones exist within the reactor. If the breakthrough residence time is greater than the calculated, it indicates the ¯ow of contaminant is retarded or slowed because of the reactor. For the experimental design, the breakthrough residence times and the theoretical residence times were similar. This result would be expected if the reactor contained only water, i.e. no algae present to slow the transport of cadmium. However, since this data was obtained when the reactor was full of algae, it suggests the algae did not slow cadmium contaminant transport. As a result, a followup experiment was performed where a larger quantity of algae (approximately three times more on a weight basis)were used. The center point experiment was redone. Results showed there was an increase in the amount of cadmium adsorbed versus center point experiments done in the experimental design. However, the breakthrough residence time was earlier than seen in the previous experiments (60 min versus approximately 95 min). This suggested the algae on the surface created dead volume that was not available for bulk ¯ow. It is
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believed that removal occurred only when diusion caused the cadmium to contact the biomass. A second follow-up experiment was then performed in which the algae were again grown in the reactor for a week. The center point experiment was run with cell agitation every 15 min to increase the contact (mass transfer) between cadmium and algae. Results showed that the breakthrough residence time was later than in any of the previous experiments (approximately 187 min), suggesting interaction between the cadmium and the algae. The outlet cadmium concentrations for the three dierent types of experiments performed at the center point conditions (normal, full non-agitated, and full agitated) are compared in Fig. 4. The dark, solid vertical line indicates the theoretical residence time of 96 min. The dashed vertical lines show breakthrough residence times for the two follow-up experiments (non-agitated RT 60 min; agitated RT 187 min). As with the batch tests, biosorption capacities were calculated for the continuous ¯ow experiments. Again, ®nal conditions (masses and times) were used for all calculations. The average biosorption capacity was 1:20 0:47 mg Cadmium/g alga/h for the experimental design (11 runs). This value is 20 times higher than the values calculated for the batch tests. This suggests the sorption process occurs rapidly. Therefore, with sucient algae, eective removal could be obtained within hours, not days as indicated by the batch tests. For the non-agitated and agitated tests, the biosorption capacities were 0.56 and 1.34, respectively. The relatively low value for the non-agitated test suggested that the algae did not reach its sorptive capacity because of poor contact. The agitated test showed the highest biosorption capacity, indicating better contact, and therefore better mass transfer. 4. Summary and conclusions
Cladophora, a green, ®lamentous algae. The batch experiments introduced a one-time charge of 3.5 mg cadmium to a 700 ml system. The semi-batch experiments introduced 0.7 mg of cadmium daily to a 700 ml system. The ¯ow experiments examined a 9.2 l system where the alga was exposed to a continuous supply of cadmium. In the batch and semi-batch experiments, the living alga removed 80±94% of the cadmium introduced. Also in the living alga, cadmium made up 1:73 0:33% of the dried cellmass. The biosorption capacity, de®ned as the mass of cadmium in the cellmass divided by the mass of alga per unit time, was 0:056 0:011 mg Cadmium/g alga/h. Based on the positive results obtained from the semibatch experiments, continuous ¯ow experiments were performed using C. parriaudii. Results showed that the alga adsorbed little cadmium. In the experimental design, the alga removed only 12:7 6:4% of the cadmium introduced. The biosorption capacity was 1:20 0:47 mg Cadmium/g alga/h, which was 20 times larger than in batch tests, indicating that contact time need not be long for eective removal. The major problem with the reactor was determined to be lack of mass transfer (contact) between the soluble cadmium and alga. This was due to the relatively small alga mass and that the alga was not as permeable to the bulk ¯ow. As a result of the experimental design, follow-up experiments were performed to address some reactor inadequacies encountered in the original design. More algae were used and agitation was provided to allow for better contact between the cadmium and biomass. Results of the non-agitated system showed the percent of cadmium sorbed increased to 17.1% over the original experiments, but the biosorption capacity dropped to 0.56 mg Cadmium/g alga/h, indicating lack of contact between the algae and cadmium. For the agitated experiment, the percent sorption increased further to 26.6% and the biosorption capacity rose to 1.34 mg Cadmium/g alga/h.
Batch, semi-batch and continuous ¯ow experiments were used to examine the adsorption of cadmium by References
Fig. 4. Comparison of center point experiments (¯ow rate 5.76 l/h, inlet concentration 3 ppm, reactor volumes 3.5).
Aksu, Z., Ozer, D., Ozer, A., Kutsal, T., Caglar, A., 1998. Investigation of the column performance of cadmium (II) biosorption by Cladophora crispate ¯ocs in a packed bed. Separat. Sci. Technol. 33 (5), 667±682. Dodds, W.K., Gudder, D.A., 1992. The Ecology of Cladophora. J. Psycol. 28, 415±427. Dorn, R.W., 1998. Cadmium removal using Cladophora in a ¯ow reactor. M.S. Thesis, University of North Dakota, Grand Forks, ND, pp. 53±55. Eaton, A.D., Clesceri, L.S., Greenberg, A.E. (Eds.), 1995. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, pp. 3-11±8-36. Gardea-Torresdey, J.L., Gonzalez, J.H., Tiemann, K.J., Rodriguez, O., 1998. Biosorption of cadmium, chromium, lead, and zinc by biomass of Medicago sativa (Alfalfa). J. Hazardous Mater. 57, 29±39.
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Cadmium removal using Cladophora in batch, semi-batch and ¯ow reactors Steven P.K. Sternberg *, Ryan W. Dorn Department of Chemical Engineering, University of Minnesota at Duluth, Duluth, MN 55812, USA Received 17 August 2000; received in revised form 19 July 2001; accepted 19 July 2001
Abstract This study presents the results of using viable algae to remove cadmium from a synthetic wastewater. In batch and semi-batch tests, a local strain of Cladophora algae removed 80±94% of the cadmium introduced. The ¯ow experiments that followed were conducted using non-local Cladophora parriaudii. Results showed that the alga removed only 12:7 6:4% of the cadmium introduced into the reactor. Limited removal was the result of insucient algal quantities and poor contact between the algae and cadmium solution. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cadmium; Bioremoval; Bioremediation; Cladophora; Macro-algae
1. Introduction The accumulation of heavy metals in the environment has received increased attention because of the threat to public health. The most commonly used procedures for heavy metal removal include chemical precipitation, ion exchange, reverse osmosis, and solvent extraction. Although these processes tend to be ecient, they are generally expensive and require frequent service attention. In addition, the remediation of heavy metals from a surface or ground water by these methods is cost prohibitive. Therefore, the need for economical, eective, and safe methods for metal removal has resulted in the search for alternative materials and methods. Biosorption has emerged as a potential option for heavy metal removal. Biosorption is the accumulation of heavy metals using microorganisms (such as bacteria and fungi) and photosynthetic life (such as algae, aquatic and emergent plants). In shallow bodies of water (1±5 m) having low concentrations of cationic heavy metals (1±20 mg/l), biosorption can be very eective (Roy et al., 1992). Studies have suggested that biosorption occurs in a manner similar to ion exchange for inactivated cells (Aksu et al., 1998; Kuyucak and Volesky, 1989; Nakajima and Sakaguchi, 1986; Tobin et al., *
Corresponding author. E-mail addresses: [email protected] [email protected] (R.W. Dorn).
(S.P.K.
Sternberg),
1987; Tsezos, 1984). In living cells, the ion exchange step may be followed by a metabolism-dependent uptake step in which the metal is transported into the cells. Other important biosorption processes include surface precipitation and surface complexation. The advantage of using living biomass is the rapidly regenerating supply of biomass. The major disadvantage is the toxic effect the metals can have on the organism. For this study, algae were selected because they are ubiquitous and native to almost all parts of the world. Algae grow in fresh or saltwater over a wide range of temperatures and pH. Prior work has shown that Cladophora possess excellent heavy metal bioremoval potential (Ozer et al., 2000; Sobhan and Sternberg, 1999; Oertel, 1991; Vymazal, 1990). This project examined cadmium removal using Cladophora algae in batch and ¯ow reactors. The batch experiments demonstrate the ability of this organism to take up cadmium. The ¯ow experiments demonstrate how this technology may be implemented on a larger scale. Cadmium was selected as the target metal because it is a regulated metal (US EPA, 40CFR 261). Cadmium poses serious health hazards to humans and animals even at concentrations of one milligram per liter. It has been shown to be a non-essential metal ion for growth of plants and animals (Torres et al., 1998). Cadmium can be found in wastewater streams from automobile industries, metal ®nishing, electroplating, and organic chemical industries. It can be leached from storage piles
0960-8524/02/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 3 1 - 6
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and settling basins, and can become mobilized through contact with an acid, such as from acid precipitation. 2. Methods 2.1. Organism Cladophora, a green ®lamentous macro-alga, was used for this work (Dodds and Gudder, 1992). A local strain was used in the batch tests, and a non-local strain was used in the ¯ow reactor tests. The local strain was obtained from a freshwater stream, the English Coulee, running through the campus of the University of North Dakota, Grand Forks, ND. The alga was collected and washed with ¯owing Coulee water to remove dirt and other debris. The samples were rewashed with distilled, deionized water in the lab and then split into smaller portions to initiate individual cultures. To improve the reproducibility and reliability of the test methods, a nonlocal but better identi®ed alga, Cladophora parriaudii, was obtained from the Culture Collection of Algae at the University of Texas, Austin, TX; phone (512) 4714019. 2.2. Water All water used for growing and testing the alga for both batch and ¯ow reactors, was obtained from the English Coulee, in the same location that the alga was collected. The Coulee water was ®ltered using Whatman No. 4 ®lter paper to remove dirt and debris. Distilled and deionized laboratory water was used to wash the freshly harvested alga, and to prepare standards and other solutions. 2.3. Growth and test conditions Cultures were started by adding individual portions of the alga, weighing approximately one gram wet weight, to jars ®lled with 700 ml of Coulee water. 2 ml of fertilizer solution were introduced into each jar once a week during culturing. Samples were allowed to acclimate to the laboratory conditions for two weeks before contamination. Only samples that experienced optimal growth were used in the experiments. Samples that died or failed to grow were discarded. Reagent grade urea (8.4 mg) was added to each jar twice per week during the experiments. Minerals in the fertilizer solution were found to interfere with the cadmium measurements, so urea was used, instead of the plant fertilizer, during the experiments. The pH of each culture was monitored daily, for both the acclimatization period and the actual experiments. The algae modi®ed the water's pH from a background value of 7.8±8.1 to 9.5 in 24 h. The pH change was most
likely the result of photosynthetic carbon (as HCO3 ) uptake. At higher pH levels, the algae did not grow. A probable reason for the inhibited growth of Cladophora at pH 9.5 is that there was no available HCO3 and it was carbon limited. Availability of carbon is likely more critical than pH. In the scope of the present experiments, carbon supply was not monitored. Very dilute acetic acid (0.01 M) was used to control pH and provide the carbon supply. The target pH was 7.8±8.1. Sylvania Gro-Lux ¯uorescent lamps were used as the light source. Light intensity at the water's surface was maintained at 2150 10% lux (Eaton et al., 1995). Light was provided to the algal jars 24 h a day, seven days a week. 2.4. Batch reactors The batch reactor experiments were carried out in 700 ml Kerr mason jars. The variables included: viable or non-viable alga, and addition of a single large dose or a daily small dose of cadmium. Each experiment was run for 16 days, with daily 3 ml samples taken for metal analysis. Controls consisted of no alga, no metal, and no metal or alga jars. All experiments were run simultaneously in separate jars. Each condition was run in triplicate, with average results reported. The starting amount of alga in each jar was 1350 250 mg wet weight. The same laboratory acclimated cultures were used for the viable and non-viable algae. The non-viable alga was dried in a drying oven for 48 h prior to the experiment. These cultures exhibited no signs of growth during the experiments. They were used to determine if any advantage was gained by using viable biomass. The single large dose added 3.5 mg cadmium, from a 1000 mg/l standard solution, to the jar to create an initial concentration of 5.0 mg/l. The daily dose added 0.7 mg cadmium each day, to instantaneously increase the concentration by 1 mg/l. The cadmium solution was added directly to the alga in the jars. The reactors were stirred with a glass stir rod. The no alga control was used to determine if the alga was the only sink for cadmium. The no metal control was used to determine the eect that cadmium had on growth rate. The no metal or alga control was used to determine if other sources of cadmium were present in the lab environment (cross contamination, leaching from jars, air-borne due to aggressive mixing of other jars) and to verify adequate laboratory technique. Evaporative and sample losses occurred throughout the experiment. Each day the jars were returned to the 700 ml mark through daily additions of ®ltered Coulee water. Careful records accounted for loss of cadmium from each jar caused by sampling. At the end of the experiments, the alga samples were separated from the water by ®ltration. The alga sample and ®lter paper were
S.P.K. Sternberg, R.W. Dorn / Bioresource Technology 81 (2002) 249±255
then digested to determine the amount of cadmium sorbed. 2.5. Plug ¯ow reactor A plug ¯ow reactor (see Fig. 1) was constructed by placing a plastic bae down the middle of a 30-l (50 cm 25 cm 25 cm) glass container. The container was further divided with plastic baes resulting in a seven-cell reactor. Of the seven chambers, ®ve contained algae, one was used to mix and sample the in¯uent, and one was used to mix and sample the euent. The reactor provided an over-and-under ¯ow with triangular cuts in the baes. The experimental design was based on a two-level, three-variable factorial (Keppel, 1982). The center point was also used, and was run in triplicate for use in error estimation. Thus, 11 experiments were conducted. The variables examined include ¯ow rate, inlet concentration, and feed volume. Values are given in Table 1. Flow rate altered the amount of mixing in the system and the contact time between water and algae. The feed volume controlled the total amount of ¯uid and cadmium presented to the system before the algae were harvested and analyzed. During an experimental run, 21 samples (3 ml each) were taken from the inlet and outlet cells at equal time intervals (times varied depending on ¯ow rate and volume fed). A calibrated peristaltic pump was used to introduce the cadmium solution into the reactor. Another peristaltic pump was used to remove the euent from the outlet cell.
Treated Water Out Algae
Algae
Algae
25 cm
Cadmium Solution In Algae
Algae
50 cm
Fig. 1. Top view of the reactor.
Table 1 Experimental design values Variable
High value
Low value
Center value
Flow rate (l/h) Inlet cadmium2 concentration (mg/l) Volume fed (represented by reactor volumes)
7.1 5.0
4.3 1.0
5.8 3.0
5.0
2.0
3.5
251
In addition to the factorial design, two follow-up experiments were performed after completing the factorial design to study the eects of alga quantity and alga±water contact. These experiments were at the center point conditions of the ¯ow reactor factorial design. The ®rst follow-up run used a greater amount of biomass; the second included agitation with increased biomass to each cell during the experimental run. Agitation was performed manually with a glass stir rod. 2.6. Measurements and procedures Wet weights of algae samples were determined by ®rst blotting with commercial-grade paper towels to remove excess water. Samples were weighed using a digital balance (0.1 mg precision). The wet algae contained 91.3% water on average, based on samples dried in a standard drying oven. The wet weighing procedure did not appear to cause any harm to the algae, in terms of growth or bioremoval potential (Dorn, 1998). Growth was measured by simple wet weight measurements recorded before and after the experiment. Measuring intermediate weights would have greatly complicated the analysis, due to water lost during the blotting process. Other indications of growth included a bright green color of the tissue, increases in visible volume of the alga, and the presence of large numbers of bubbles within the alga. Because Cladophora is a macroalga, the wet weight was an easy and direct measurement of growth, and therefore other measures of growth, such as chlorophyll a were perceived as unnecessary to measure growth. The pH was measured using a Cole Parmer (Vernon Hills, Illinois, USA) Model 05668-20 electronic pH meter and pH electrode. Light was measured with a Cole Parmer Model DLM2 light meter. Cadmium concentration was measured throughout the course of the experiments and in the alga biomass at the conclusion of the experiment. All water samples were treated with 2 drops of 1.0 M nitric acid after collection to prevent precipitation before analysis. Metal concentrations in these solutions were measured, in accordance with the procedure described in Standard Methods (Eaton et al., 1995) using a Perkin±Elmer (Shelton, Connecticut, USA) Model 3100 Atomic Absorption Spectrophotometer. The detection limit for cadmium with this equipment was 0.028 mg/l. The alga was collected for metal analysis after completing the batch and continuous ¯ow experiments. Before digestion, samples (with the pre-weighed ®lter paper) were placed in a drying oven at 65±70 °C for two days to determine the dry weight of the biomass. The samples were cooled to room temperature and weighed using a digital balance until a constant weight was obtained. The samples were then placed in a mue furnace at 500 °C for 14 h to remove volatile components and oxidize the adsorbed cadmium.
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The samples were again cooled and weighed. The ashes were dissolved in Aqua Regia acid and placed on a hot plate until completely dry. The procedure was repeated until a clear solution was obtained and no signi®cant mass change was observed. The samples were then resuspended in 1 M HCl, diluted to 1000 ml with laboratory water, and analyzed for cadmium. The standard cadmium solution of 1000 mg Cadmium2 /l was prepared by dissolving 1.6308 g of reagent grade Cadmium Chloride CdCl2 , Fisher Scienti®c (Pittsburgh, PA, USA), in 1000 ml distilled and deionized water. Calibrated micropipettes were used to measure volumes of the standard Cadmium2 solution. The plant fertilizer solution was prepared by dissolving two grams of Stern's Miracle Groâ Products (Port Washington, NY, USA) in 1000 ml of water. Composition of the fertilizer, by weight, is approximately 31% nitrogen, 31% phosphoric acid P2 O5 , 36% potash K2 O, and trace amounts of mineral salts containing Mg, Cu, Fe, Mn, and Fe. 2.7. Calculations All calculations and statistics (average, standard deviation, regression, student t test for signi®cance) were done using the Microsoft Excel spreadsheet program. All batch reactor experiments were triplicated (N 3). The ¯ow reactor error was estimated by running a triplicate of the center point, all other points were run alone. Concentrations were measured directly by AAS using sample standards and calibration curves done internally on the AAS unit. The bioremoval potential was calculated as (Sorbed Cadmium Mass)/(Alga Mass Total Time). The bioremoval was calculated as (Sorbed Cadmium Mass)/(Initial Cadmium Mass) 100%. The mass balance was determined as the sum of cadmium mass in solution, alga, ®lter, and samples.
3. Results and discussion
Viable algae were more eective in removing both the single and daily doses of cadmium when compared to the non-viable algae. This is a result of the regenerating supply of anionic functional groups on the biomass surface due to alga growth or maintenance. The cadmium concentration for the non-viable algae was equivalent to that of the viable algae for the ®rst sample taken (after 12 h). After this time the amounts removed diverged. The ion exchange mechanism proposed for inactivated cells (Kuyucak and Volesky, 1989; Nakajima and Sakaguchi, 1986; Tobin et al., 1987; Tsezos, 1984) gives a possible explanation of this phenomenon. The mechanism states that there are a de®nite and constant number of adsorption sites on the non-viable biomass. After the sites become saturated with metal ions, adsorption equilibrium is established, resulting in no net concentration change. In the scope of the present study, no additional analysis was performed to con®rm this hypothesis. Alga growth was inhibited by the presence of cadmium in the water. The larger the initial cadmium dose, the greater the inhibition. Alga weight tripled over 14 days in the laboratory environment when not exposed to cadmium. The alga exposed to the daily doses (1 mg/l/ day) doubled in weight in 14 days, while the single dose (5 mg/l Cadmium2 ) alga did not increase in biomass during the 14 days. The cadmium concentration in the batch reactors is shown in Fig. 2. Great variability can be seen for the living alga samples, although the overall trend is a decrease over time. The residual cadmium concentration for the no alga controls decreased at the beginning of the experiment. This decrease was the product of cadmium precipitation due to elevated pH of the Coulee water. At higher pH values (greater than 9.0), precipitation was observed in the Coulee water, which was determined to be the cause of the initial decrease. Sampling also tended to reduce the overall concentration in the jars, resulting in an initial 5.0 mg/l concentration to decrease to 4.7 mg/ l in the no alga control jars after 14 days. The cadmium concentration in the semi batch reactors is shown in Fig. 3. The samples were collected just prior to addition of the daily dose of cadmium solution.
3.1. Batch tests Cd Concentration (mg/L
5.00
Results of the batch tests indicated Cladophora effectively removed cadmium from a synthetic wastewater. The viable alga removed cadmium by 80% in the batch and 94% in the semi-batch experiments. The nonviable alga removed cadmium by 65% in the batch and 25% in the semi-batch experiments. Cadmium made up 0.9±2.8% of the algae's ®nal dried cell mass. The mass balance, based on the sum of measured cadmium in the reactor solution, alga, ®lter paper, and the daily sample aliquots, accounted for all the known additions to within 20% (Dorn, 1998).
4.00
3.00
2.00
1.00
0.00 0
50
100
150
200
250
Time (Hours) Viable
Non-viable
No Algae
Fig. 2. Response of algae to single cadmium dose.
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S.P.K. Sternberg, R.W. Dorn / Bioresource Technology 81 (2002) 249±255 14.00
Cd Concentration (mg/L)
12.00 10.00 8.00 6.00 4.00 2.00 0.00 0
50
100
150
200
250
300
350
400
Time (Hours) Viable
Non-Viable
No Algae
Fig. 3. Response of algae to daily cadmium doses.
The viable and non-viable algae removed cadmium at an equivalent rate for the ®rst day, but quickly diverged after that. The viable alga systems removed approximately the amount added each day, whereas the nonviable alga ceased detectable removal after day 1. The solution pH was shown to have an eect on cadmium removal. In Fig. 3, the residual cadmium concentration increased for the ®rst 150 h. During this time, pH of the solution was adjusted twice daily, resulting in a pH of approximately 8.0±8.3. After 150 h, pH adjustments were made daily and the removal increased, resulting in a lower residual cadmium concentration. During this time, the pH was approximately 8.7±9.0. This supports ®ndings that higher removal rates occur at higher pH values (Gardea-Torresdey et al., 1998; Kratachvil and Volesky, 1998; Ribeyre and Boubou, 1990). Biosorption capacity, de®ned as the mass of cadmium sorbed by the alga per mass of alga per hour, was calculated for both viable and non-viable algae. This is not the same as cadmium removal. It is used to represent the ¯ux of cadmium removal rather than the quantity removed. The biosorption capacity was calculated using the ®nal experimental conditions, which included time, alga mass and mass of cadmium in the alga. The batch reactor biosorption capacity of viable alga was 0:056 0:011 mg cadmium/g alga/hour and for the non-viable alga was 0:045 0:015 mg Cadmium/g alga/ hour. The averages were statistically indistinguishable at the 95% con®dence level (student t test), suggesting there are a limited number of binding sites on a given mass of the algae, regardless if the alga is viable or non-viable. Viable alga however will create more binding sites as it grows, and so can ultimately remove more metal than the non-viable alga, as Figs. 2 and 3 show. 3.2. Continuous ¯ow tests For the continuous ¯ow experiments, the algae removed only a small percentage of cadmium introduced into the system. Only four of the 11 experiments performed in the experimental design showed the C. par-
253
riaudii adsorbing more than 10% of the cadmium fed to the reactor. The average adsorption for the 11 experiments was 12:7 6:4%. One factor contributing to the limited removal was the small biomass to reactor volume ratio that existed during the experimental runs. The mass of alga in a cell, and the cell volume were approximately equivalent to the values in the batch experiments. However, the total volume of contaminated water in the reactor was approximately 9.2 l or 13 times the amount in any single jar. Therefore, it is probable that much of the cadmium had limited exposure to the alga, resulting in low removals. The experimental data were analyzed using Excel's data analysis regression option. The response (Y) was calculated from the experimental values and was designed to analyze data objectively and to be independent of any variables not controlled or tested. For this study, Y was the percentage of cadmium adsorbed by the alga per mass cadmium fed divided by the total ®nal mass (dry weight) of alga in the reactor (in grams). This response was chosen to nullify the eect that mass of alga might have on the results, since alga growth was not a controlled variable. Of the variables tested (¯ow rate, inlet concentration, and volume of feed), none had a statistically signi®cant eect on the response. A plot of total mass of algae in the reactor versus response (Y ) was constructed and no detectable relationship was found (results not shown). Another important indication of reactor eectiveness is comparing the measured breakthrough residence time (de®ned as the time when cadmium concentration ¯owing out is one-half of that ¯owing in) with the calculated residence time (calculated by dividing the volume of the reactor by the volumetric ¯ow rate). If the measured breakthrough residence time is less than the calculated, it indicates dead zones exist within the reactor. If the breakthrough residence time is greater than the calculated, it indicates the ¯ow of contaminant is retarded or slowed because of the reactor. For the experimental design, the breakthrough residence times and the theoretical residence times were similar. This result would be expected if the reactor contained only water, i.e. no algae present to slow the transport of cadmium. However, since this data was obtained when the reactor was full of algae, it suggests the algae did not slow cadmium contaminant transport. As a result, a followup experiment was performed where a larger quantity of algae (approximately three times more on a weight basis)were used. The center point experiment was redone. Results showed there was an increase in the amount of cadmium adsorbed versus center point experiments done in the experimental design. However, the breakthrough residence time was earlier than seen in the previous experiments (60 min versus approximately 95 min). This suggested the algae on the surface created dead volume that was not available for bulk ¯ow. It is
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S.P.K. Sternberg, R.W. Dorn / Bioresource Technology 81 (2002) 249±255
believed that removal occurred only when diusion caused the cadmium to contact the biomass. A second follow-up experiment was then performed in which the algae were again grown in the reactor for a week. The center point experiment was run with cell agitation every 15 min to increase the contact (mass transfer) between cadmium and algae. Results showed that the breakthrough residence time was later than in any of the previous experiments (approximately 187 min), suggesting interaction between the cadmium and the algae. The outlet cadmium concentrations for the three dierent types of experiments performed at the center point conditions (normal, full non-agitated, and full agitated) are compared in Fig. 4. The dark, solid vertical line indicates the theoretical residence time of 96 min. The dashed vertical lines show breakthrough residence times for the two follow-up experiments (non-agitated RT 60 min; agitated RT 187 min). As with the batch tests, biosorption capacities were calculated for the continuous ¯ow experiments. Again, ®nal conditions (masses and times) were used for all calculations. The average biosorption capacity was 1:20 0:47 mg Cadmium/g alga/h for the experimental design (11 runs). This value is 20 times higher than the values calculated for the batch tests. This suggests the sorption process occurs rapidly. Therefore, with sucient algae, eective removal could be obtained within hours, not days as indicated by the batch tests. For the non-agitated and agitated tests, the biosorption capacities were 0.56 and 1.34, respectively. The relatively low value for the non-agitated test suggested that the algae did not reach its sorptive capacity because of poor contact. The agitated test showed the highest biosorption capacity, indicating better contact, and therefore better mass transfer. 4. Summary and conclusions
Cladophora, a green, ®lamentous algae. The batch experiments introduced a one-time charge of 3.5 mg cadmium to a 700 ml system. The semi-batch experiments introduced 0.7 mg of cadmium daily to a 700 ml system. The ¯ow experiments examined a 9.2 l system where the alga was exposed to a continuous supply of cadmium. In the batch and semi-batch experiments, the living alga removed 80±94% of the cadmium introduced. Also in the living alga, cadmium made up 1:73 0:33% of the dried cellmass. The biosorption capacity, de®ned as the mass of cadmium in the cellmass divided by the mass of alga per unit time, was 0:056 0:011 mg Cadmium/g alga/h. Based on the positive results obtained from the semibatch experiments, continuous ¯ow experiments were performed using C. parriaudii. Results showed that the alga adsorbed little cadmium. In the experimental design, the alga removed only 12:7 6:4% of the cadmium introduced. The biosorption capacity was 1:20 0:47 mg Cadmium/g alga/h, which was 20 times larger than in batch tests, indicating that contact time need not be long for eective removal. The major problem with the reactor was determined to be lack of mass transfer (contact) between the soluble cadmium and alga. This was due to the relatively small alga mass and that the alga was not as permeable to the bulk ¯ow. As a result of the experimental design, follow-up experiments were performed to address some reactor inadequacies encountered in the original design. More algae were used and agitation was provided to allow for better contact between the cadmium and biomass. Results of the non-agitated system showed the percent of cadmium sorbed increased to 17.1% over the original experiments, but the biosorption capacity dropped to 0.56 mg Cadmium/g alga/h, indicating lack of contact between the algae and cadmium. For the agitated experiment, the percent sorption increased further to 26.6% and the biosorption capacity rose to 1.34 mg Cadmium/g alga/h.
Batch, semi-batch and continuous ¯ow experiments were used to examine the adsorption of cadmium by References
Fig. 4. Comparison of center point experiments (¯ow rate 5.76 l/h, inlet concentration 3 ppm, reactor volumes 3.5).
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