Acta Biotechnol.

16 (1996) 4, 227-235

Akademie Verlag

Heavy Metal Sorption by Microalgae

SANDAU, E., SANDAU. P.,

w, 0.

IGV -Institut f & Getreideverarbeitung GmbH Arthur-Scheunen-Nee 4 0 4 1 14558 Bergholz-Rehbriicke, Germany

Summary
Viable microalgae are known to be able to accumulate heavy metals (bioaccumulation). Against a background of the increasing environmental risks caused by heavy metals. the microalgae Chlorellu vulguris and Spinclina plurensis and their potential for the biological removal of heavy metals from aqueous s o h tions were taken as an example for investigation. Smd-scale cultivation tests (50 1) with Cd-resistant cells of Chlorella vulgaris have shown that approx. 40% of the added 10 mg Cd/l was removed from the solution within seven days. At this heavy metal cqncentration sensitive cells died. Non-viable microalgae are able to eliminate heavy metal ions in a short time by biosorption in uncomplicated systems, withcut any toxicity problems. Compared with original biomasses, the sorption capacity of microalgal by-products changes only insignificantly. Their low price makes them economical.

Introduction

The heavy metal impact on the biosphere is estimated to amount to approx. 5 million tons world-wide every year. The potential toxicity, partly in low concentrations, to humans, animals, plants and microorganisms is the common ecological feature of approx. 40 heavy metals of the periodic table. The future health risks caused by heavy metals, which are already integrated into the food chain, cannot be estimated because heavy metals are released into the atmosphere, the soil and water. In many types of sewage, toxic heavy metals are contained in considerable concentrations. Several conventional methods for the elimination of toxic heavy metals from aqueous solutions are known: precipitation, electrolysis, ion exchange, filtration, evaporation, etc. [l-31. The disadvantages of these methods, especially at low and variable heavy metal concentrations are the high cost and low economic efficiency and, in some cases, the necessary disposal of sludge containing heavy metals. This sludge is a potential danger to ground water. Against this background, we investigated the heavy metal uptake (ppm range) potential of microalgae, which are known under natural conditions to be able to accumulate very low concentrations of heavy metal ions (ppb range) from water without dying [4-71.

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Acta Biotechnol. 1 6 (1996) 4

The following natural resistance mechanisms of viable algal cells with toxic heavy metal ions are known: Extracellular Sorptive Mechanisms [8-121 - Electrostatic interactions of positively charged heavy metal ions with negatively charged groups/ligands of the cell (adsorption, ion exchange) - Micro-precipitation - Surface complexing - Covalent bonds between heavy metal cations and proteins - Bonds to other polymers Intracellular Accumulative Mechanisms [13-1 91 - Complexing of the accumulated heavy metal ions and reduction of the toxic heavy metal concentrations by several species-specific processes (e.g. phytochelatin reaction, hypertrophying and multiplication of the polyphosphate bodies, phlorotannin reaction) - Heavy metal incorporation into vacuoles (demobilization and concentration) - Further bonds to proteins, lipids, DNA, etc. -. Efflux of heavy-metal complexed substances, especially by resistant cells - Membrane reinforcement of cell walls, mitochondriae, chloroplasts and nuclei as heavy metal barriers in adapted cells The tolerance of algae towards toxic heavy metal ions results from a combination of structural collecting, adhesive and barrier mechanisms to protect the cells or cell organelles, and of physiological responses to demobilize and reduce / deactivate the toxic concentration of penetrated heavy metal ions.
Materials and Methods

Culrivution
Chloreh vulgaris (AM/ 1979, M m ) fmm the strain c o k t i o n of the htitute of cereal Processing. Ltd., was cultivated at 25 O C in the following nutritive solution: 0.3 g urea, 0.4 g N W 0 3 . 0.34 g KHzPOc 0 . 5 g MgS04 x 7 H20.0.014 g FeSO4 x 7 H20.0.74 mg ZnS04 x 7 H20,0.057 mg H3B03. 0.238 mg CoS04 x 7 H20, 0.236 mg CuSO4 x 5 H20,4 . 1 mg MnS04 x 4 H20, 0.092 mg (NH,)&lo-& x 4 HzO,0.025 mg W V q , 1 1 distilled water. Cultivation was performed in stin-ed cuvettes with a volume of 2 1 or in an algal laboratory fermenter (ALF) with a volume of 50 1in coiled pipe configuration. The photosynthetically active part of the ALF had a length of 51 m [20,21].The cultures (viable microalgae) were illuminated by a light cassette a t an average intensity of 120 pE/m2 x s . After the addition of heavy metals, the cultures were kept over night in darkness in order to reduce stress. The pH value was adjusted to 6 . 5 6 . 8 by automatic C Q addition. For the prevention of heavy metal precipitations during the addition of the heavy metals, the pH value was reduced to 6.0 for two hours. The growth of the cultures was determined by measuring the extinction in the SPEKOL (wavelength 750 nm) or measuring the optical density (OD) in the ALF. The resistant ecotypes were produced by four additions of 0.1 m a . followed by one addition of 0 . 2 mg/l of t h e appropriate heavy metal to the algal suspension over a period of five weeks. The algal suspension was diluted continuously by the nutritive solution, when high biomass (BM) concentrations (> 1 g BMA) we= achieved After the 5th addition, the resulting biomass was centrifuged, washed and used as a resistant inoculum.

SANDAU, E . , SANDAU, P. et a l . , Heavy Metal Sorption by Microalgae

229

Non-Viable Biomusjes
B i o m a s s of the microalgae Chlorella vulgaris and Spimlinaphtensis were centrifuged (at 6,000 rpm) from the suspension, washed in deionized water and dried [22]. CER (Chlorella extnction &dues) and SER (Spirulinaextraction residues) are by-products of a high pressure extraction using supercritical gases or further extraction processes to obtain valuable biomaterials (colouring agents. polyunsaturated fatty acids. plysaccharides and antioxidants) from microalgae.

Heavy Metal Sorption Experiments

The heavy metals were added in their nitrate forms [Cd(NQ>, x 4 HzO, Zn(NO& x 6 H20, Ph(NQ)2 and Cu(NQk X 3 HzO]. Within the heavy metal uptake by microorganisms, variahle terms are applicable. Bioaccumulation is an active, metabolism-dependent process of viable cells and biosorption a more passive adsorption of nonviable biomass [I 1,221. The heavy metal bioaccumulation or hiosorption was determined in several experiments: - During cultivation, addition of heavy metals into the algal suspension (viahle algae) and determination of the heavy metal removal during cultivation - Separation of the viable algae from the suspension, re-suspension in a solution of distilled water and heavy metals and determination of the heavy metal removal - Experiments with non-viahle microalgal cells and by-products in a solution of distilled water and heavy metals, analogously to the second experiment.

Heavy Metal Analytics

The heavy metals were determined by the aid of an atomic ahsorption spectrophotometer (AAS)with a background compensator and an air-acetylene flame. Low concentntions were determined by graphite tube technique and deuterium hackground compensation, according to the standard additive method [23]. For the determination of the total accumulation of heavy metals, the suspension was centrifuged. The heavy metal content was determined either in the supernatant or in the separated biomass aftex treatment with aqua regia. l l wall or penetnted into the cells, the In order to determine how many heavy metals adhered to the e biomass was washed with EDTA.The heavy metals separated were identifed in the solution, and t h e heavy metals in the cells were identified in the biomass.

Results

Effect of Different Heavy Metul Cations on the Growth of Sensitive and Resistant Cells

Cultivation tests with living Chlorellu vulgaris microalgae have shown that small additions of 1.5 mg Cd/l were found to restrict the physiological activity (cell division) of the aIgae, but a metai-specific resistance was aIso observed (Fig. 1). The 2 1 volume cuvette tests have clearly shown that at comparable heavy metal additions (Cd 10 mg/l, Zn 15 mg/l) the cells which adapt to heavy metal ions (Cd, Zn, Cd/Zn resistant types) have higher division rates than the sensitive cells. The sensitive, Cd treated cultures died after a short time. Zn proved to be less toxic than Cd.

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Cd Sorption of Resistant Chlorellu vulgaris in Smnll-Scale Tests

Acta Biotechnol. 16 (19%) 4

In a small-scale test, the Cd-resistant ecotype of Chlorella vulguris was used as an inoculum with the aim of determining division rate and Cd-accumulation performance. A 50 1 volume algal laboratory fermenter (ALF) was used as the cultivation system. During the adaptation of the resistant microalgae in the photobioreactor, 0.6 mg/l Cd was added in three stages during a period of ten days. Before the beginning of the exponential growth of the cells, on the 12th day, lQmg Cd/l was added at a pH of 6.0. Despite the normally toxic Cd concentration of the addition, within seven days, the algal biomass almost trebled from 0.52 g / l to 1.43 g/l. During the same period, the algae filtered out 4 mg Cd/l from the cultivation suspension. The test has shown that over a longer period, resistant microalgae are able to eliminate low concentrations of toxic heavy metal cations in relevant quantities from water. For practical approaches, extended experiments to investigate more economical cultivation systems and the disposal of the heavy metal contaminated biomass have to be performed. The determination of the Cd distribution on and in the cells has shown that an average of 53% Cd is accumulated on the outer cell wall and an average of 47% penetrates into the cytosol or is bound to solid components within the cell. These findings are either confirmed by [24] or differ from other published results [25].

1 . 2

-Is] without a d d i i
1 .o

* [S] cdlznaddidM
8 [s]

E
Y

m addition

+ [s]cd addition

0.8
0.6
0.4

0 .-

E c
5
C

X[s] cd addition,strain B cdlzna d d i i 8 [r] Zn addition

4 [r]

0 . 2
_.

/
--.J

.. chlorosis

0.0

5
Cultivation time [d]

Fig. 1. Growth of resistant (r) and sensitive (s) Clilorefla vulgiuis under comparable heavy metal stress

SANDAU,E . , SANDAU, P. eta[., Heavy Metal Sorption by Microalgae

Non- Viable Microa/gue

23 1

Sorption tests with biomasses of Chlorella vulgaris and Spirulina platensis and their resulting residues (CER, SER) after high-pressure extraction have resulted in remarkable loads [Tab. 11. In practice, the algal extraction residues are cost-effective by-products.
Tab. 1. Biosorption of non-viable microalgae and microalgal by-products Initial concentration [mdll Heavy metal Sorption capacity

[mdg BMI Spirulina phtensis SER 0.74 0.76 0.81 0.77 3.73 2.86 4.08 4.26 6.28 4.11 5.72 8.26 10.48 6.30 8.38 16.54
C,

Chlorelh vulgaris 0.79 0.88 0.77 0.76 0.02 3.11 3.73 4.54 7.53 5.02 7.59 8.74 12.45 6.42 10.90 17.13

CER 0.79 0.96 0.88 0.78 3.61 2.88 4.42 4.91 6.63 4.8 1 7.01 9.13 10.76 6.92 9.46 17.84

1.o

Cd

cu
Pb 5.0

zn

0.69 0.65 0.67 0.72 3.59 3.16 3.40 3.82 6.89 4.90 6.21 9.33 12.08 7.36 10.33 16.97

zn
Ph

Cd

cu
10.0 Cd

cu
Pb 20.0 Cd

zn

cu
Ph t-60min pH-6.0

zn

BM - 1.06 sfl

These results reveal that the capacity of the four samples of biomass to adsorb the metals Pb, Cd, Cu and Zn increased significantly with rising initial ion concentration. In addition, they show that there were only low sorption differences between the original microalgae and their extraction residues. Time-dependent sorption tests (Tab. 2) reveal an increased accumulation in viable cells after 24 h in contrast to non-viable cells, in which no significant sorption was observed after 1 h. The increase is almost complete due to the intracellular bioaccumulation of living cells. Within 1 h, non-viable cells adsorb higher metal quantities than viable ones at high initial ion concentrations (Fig. 2). The flattened initial isotherms of viable microalgae vs. the linear isotherms of nonviable microalgae confirm that not only adsorption processes take place in living cells.

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Acta Biotechnol. 16 (1996) 4

O - . " " 0

10
Initial metal concentration [mg/l]

15

20

1: 60 rnin

c,BM: 2.19 g l

pH: 6.0

Ftg.2. Heavy m e t a l socption by viahle and non-viable Chlorella vulgaris cells

Tab. 2. Time dependent heavy metal sorption of Chlorella vulgaris

~ ~~~~~ ~

Initial concentration

Heavy metal

Sorption capacity

[m@I

[mdg BMI
Viable biomass Ih 24h
Non-viable biomass

lh

24h

~~

~~

1.0

CU

zn

0.44 0.47 0.38 0.41

0.36 0.38 0.45 0.48 1.97 2.09 3.86 3.81 7.48 6.62 2.01 2.11 3.91 3.85 7.51 6.68

5.0
10.0

CU

zn
zn

2.09 1.69 3.76 2.78 4.85 3.52

2.27 2.03 4.31 3.06 6.68 4.07

CU
CU

20.0 pH - 6 . 0

zn

C,

BM - 2.19 f l

Several authors have reported that heavy metal biosorption takes place on or in the cell wall of non-viable cells [ l l , 26, 271. Our ultrastructural examinations (see Micrograph 1) revealed that the heavy metal cations can even diffuse into the interior of the cell and adsorb to the negatively charged ligands (electron-dense deposits onto and in the ChZoreZZa vulgaris cell).

SANDAU, E., SANDAU, P. et al., Heavy Metal Sorption by Microalgae

233

Micrograph 1. Pb biosorption of non-viable Chlorellrr vulgaris (x 18,000)

Despite the favourable sorption results of non-viable microalgae, their price (20 to 40 DM/kg) and availability remain a problem. Microalgal by-products, however, are well suited and cost-effective. Compared with viable microalgal cells, the non-viable biomasses have the following practical advantages: - complicated and expensive cultivation conditions are not necessary - no toxicity limit - better technological handling - competitive ions of the algal suspension cannot affect sorption - use of cost-effective algal degradation products - shorter reaction times - high sorption capacity, dependent on several external factors (e.g. pH value, heavy metal and biomass concentration, competitive ions) An economical solution to the disposal problem of viable and non-viable biomasses contaminated by heavy metals is necessary and is determined by the metal value. Both materials can be disposed of in a special combustion process. Non-viable sorbents can be regenerated by reducing the pH value as well as by eluting the sorbed metals from the cells [28,29].
Conclusions

The small scale cultivation tests have shown that over a long period heavy-metal adapted cells of Chlorellu vulgaris are able to bioaccumulate heavy metal cations in low concentrations and to remove them from the cultivation suspension, utilizing the

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Acta Biotechnol. 16 (1996) 4

extra- and intracellulq potentials of biofixation and detoxification. However in practice this approach, via closed photobioreactors and resistant microalgae, is not advisable. The application of inclined open systems and sessile algae could be more effective and should be tested. Non-viable microalgae adsorb heavy metal cations much more quickly, more effectively, and without any problems of toxicity. Compared with the original products, the biosorption capacity of microalgal by-products changed only insignificantly. They are low-cost by-products, as opposed to the original products. For practical applications, further experiments will be necessary, e.g. regarding the more economical disposal of the contaminated biomass.
Acknowledgement

This project was sponsored by the Federal M d t r y of Education, Science, Research and Technology, Germany (No. 0310403 A).
Received 14 February 1996 Received in revised form 16 September 1996 Accepted 1 October 1996

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