acute toxicity of heavy metals to aerobic digestion of waste cheese whey

Biological Wastes 33 (1990) 201-210

Acute Toxicity of Heavy Metals to Aerobic Digestion of Waste Cheese Whey

G. Cimino & C. Caristi

Dipartimento di Chimica Organica e Biologica, Universit/t degli Studi, Salita Sperone No. 31, 98166 Vill. S. Agata, Messina, Italy

(Received 29 May 1989; revised version received 30 January 1990: accepted 5 February 1990)

A B S T R A C T

Effects of the acidity and o f metal ions on metabolic activity of activated sludge from waste cheese whey biological treatment have been determined. The 10% and50% inhibitory concentrations ( IClo and lCso) were evaluated

for each ion. In a decreasing order of toxicity two groups were found." Hg z + > Cd z + > CrO]- > C r 3 + > C u z + and P b 4 + > Z n 2 +. The discrepancy in results of other toxicity stufies reveals the impossibility of defining on an absolute scale an order o f toxicity for these ions.

A very strong synergistic effect was noted in the mixed Cu and Zn studies, thus pointing out the difficulty of setting a water-quality standard based on single toxic chemicals.

I N T R O D U C T I O N

Cheese whey constitutes a major waste disposal problem for the dairy industry throughout the world, except for places where local utilization is possible. At present cheese whey is utilized as an animal and human food source. Among emerging technologies another possibility for its utilization is alcohol or single cell protein production (Bernstein, 1979).

A large part of cheese whey (¢. 26% of the about 20 million m 3 produced in the EEC countries) is discharged into soil and watercourses without any t reatment (Moresi, 1983). Because of its high BOD 5 content (30000- 50 000 mg/litre) direct disposal is environmentally unacceptable.

201 Biological Wastes 0269-7483/90/$03'50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain

202 G. Cimino, C. Caristi

To reduce the organic overload of watercourse by wastewaters an aerobic activated sludge process is utilized. This treatment can remove up to 95% of BOD 5, 80% of total nitrogen and phosphorus, and it is simple and economically attractive. But the pH value of the medium and the presence of some metals can modify the performance of the microbial process (Gadd & Griffiths, 1978; Kaspar & Wuhrmann, 1978; A1-Shahwani et al., 1987).

Earlier we investigated activated sludge processes and chemicals regarded as environmental hazards (Cimino & Ziino, 1983; Cimino, 1987). In the present work we examine the influence of acidity and of some elements (Cd, Cr, Cu, Hg, Pb, Zn) on the aerobic biodegradation of waste cheese whey by heterogeneous cultures. The digestion period was limited in order to avoid growth of toxic reaction products, uncontrolled drop in pH value and the possibility of acclimatizing bacteria.

METHODS

Waste cheese whey

Waste cheese whey was sampled from the equalization tank of the biodegradation plant in D. Calogero Dairy, Messina (Italy). In this tank raw whey and industrial washing water are mixed together.

TABLE 1 Composition of Waste Cheese-Whey

Parameter Unit Range

Lactose g/litre 7'5-8.5 Lactic acid g/litre 2"5-3.5 pH - - 4.2-4-5 Settleable solid ml/litre 0-4-0.8 C O D g/litre 1.5-2.5 BOD5 g/litre 1"2-2.0 Dry matter g/litre 3"2-4.9 Ash g/litre 1.0-3.0 Total hardness (as CaCO3) mg/litre 130-140 Total nitrogen (as N) mg/litre 17--44 Total phosphorus (as P205) mg/litre 12-20 Potassium (as K20) mg/litre 60-90 Cd /tg/litre 0" 1~).2 Cr #g/litre 1-4 Cu pg/litre 4-7 Hg pg/litre < 0"01 Pb #g/litre < 1 Zn mg/litre 0.03~).05

Heavy metals and aerobic treatment 203

Its composition range is given in Table 1. It was an acid waste derived from the manufacture of acid casein and cottage cheese. Only traces of heavy metals were present.

Activated sludge

A 10-1itre activated sludge sample was collected from the return sludge of the final settling tank of the biodegradation plant in D. Calogero Dairy.

Its characteristics are given in Table 2. It was aerated fully and stored frozen in 60 ml plastic containers until use.

Whey slurry

The frozen activated sludge was mixed with fresh waste-whey and thawed in a 20°C thermostat. The resultant whey slurry was aerated vigorously before use.

Aerobic digesters

Six l'3-1itre dark glass digesters were used, each was equipped with a small magnetic stirring bar and an oxygen electrode (Tecnocontrol SE222). These sensors, connected to a recording analyser, allowed the reading of the oxygen concentration of the air into each digester directly in mg/litre. Electrode standardization was carried out in water-saturated air at 20°C and taking into account the barometric pressure. The digesters, equipped with a

TABLE 2 Composition of Whey Activated Sludge

Parameter Unit Range

pH at 20°C - - 475 Settleable solid ml/litre 855 Dry matter g/litre 20'5 Ash g/litre 1"32 Volatile suspended solid g/litre 18"2 Fixed suspended solid g/litre 0.96 Sludge volume index ml/g 44.3 Sludge density index g/100 ml 2'26 Cd mg/kg dry wt 3'07 Cr mg/kg dry wt 3'69 Cu mg/kg dry wt 10"0 Hg mg/kg dry wt < 0.2 Pb mg/kg dry wt 16.8 Zn mg/kg dry wt 48.4


mercury seal to equalize the inside and outside pressures, were placed in an incubator at 20°C. Their working volumes were calculated by subtracting the volume of the tested whey-slurry sample (nominally 100 ml) from the net volume of each digester, this last determined exactly by distilled water (nominally 1"18 litres).

Under equilibrium conditions, the partial pressure or activity of the oxygen in air-saturated water is equal to that of the oxygen in the humidified air above the water. Therefore, the difference between the oxygen amount into the working volume of each digester, at the beginning and at the end of measurements, allowed calculation of the oxygen demand to carry out the biological metabolism of the test-sample volume. This value, here reported as 'cumulative oxygen consumption' (COC), is expressed in g of oxygen per litre of whey-slurry sample.

All plastic and glassware was rinsed in 6M nitric acid and distilled water prior to use in order to remove adsorbed metals.

Analytical methods

All analytical parameters concerning waste cheese whey and activated sludge were determined as described in Standard Methods (American Public Health Association, 1975). Metals were measured on a Varian AA-1475 atomic absorption spectrophotometer equipped with a Varian GTA-95 graphite furnace, after acid digestion of the samples.

Heavy metal chloride and nitrate salts were used after washing several times with ethyl alcohol and drying under vacuum.

Experimental procedure

For the organic load test, 100 ml whey-slurry samples with various ratios of sludge:waste-whey (1:1, 1:2, 1:4, 1:8, 1:10, 1:15) were added to the six digesters. The oxygen concentration was monitored every 30 min for 48 h.

For the pH dependence test the following routine was carried out. A 600- ml sample of whey-slurry (1:4), was equally distributed into the six digesters. Each whey-slurry was acidified to different pH values by careful slow addition of 0"ly NaOH. Then COC measurements were carried on as previously described.

For the heavy-metal test the operating routine was as follows. A 600 ml sample of whey slurry (1:4), carefully adjusted to pH4.8 by 0.1y NaOH addition, was added to the six digesters. Then 5 ml of solutions, each with a different metal concentration, was added to each digester except the control one, to which only 5 ml distilled water was added. The metals were tested at five concentration levels, 0"01-0-1-1-0-10-100mg/litre in the final volume. Resultant slurries were left to stand in aerobic conditions for 1 h at 20°C to


allow diffusion and chemical reactions between salt and sludge. Then the digesters were sealed and COC was measured. After 36h the trials were stopped.

To show synergistic effects the whey-slurry of each digester contained 1 mg/litre of Zn except for the control where no metals were added. A 5 ml solution with a different copper concentration was then added to each digester, except for the control. The same routine was used as for the zinc test except that whey slurry containing 0" 1 mg/litre copper and 5 ml of solutions with different zinc concentrations were added.

RESULTS AND DISCUSSION

The metabolic activity is shown in Fig. 1 as the amount of oxygen required for unitary biomass in 24 h and 36 h from the start of the test. It appeared to be independent of the sludge organic load. This constant trend at tested times verified the zero-order kinetics for organic load removal, as formerly observed for removal of single compounds in urban wastewater (Wuhrmann & Benst, 1958).

A 1:4 volumetric ratio between the sludge and the waste-whey was considered optimum for subsequent studies. The oxygen into digester did not fall below 60% of saturation in 48h incubation. Thus aerobic conditions were maintained throughout the test. Other main features were: a satisfactory sensitivity of the method (2-4 mg/litre of mean oxygen demand per hour), an organic loading ratio of 0-5kg of COD/kg SSV and a suspended solid content of 4 g/litre MLSS, both values usually regarded as optimum for plants operating at full scale.

K g O 2 ~ K g V S S

0 . 3 . 3 6 h

, - -D-- - - O - . . . . . -~- . . . . . . . . . . . . a . . . . .D- . . . . . . . . . . . . . . e -

0 . 2 " . _ A . . . . . . . . A . . . . . . . . . . . ._& . . . . . . 4~- . . . . . . . . . . . . . . &- -

2 4 h

0 . 1

I i t -- 0 . 5 1 . 0 1 . 5

K g COD/ / K g V S S

Fig. 1. Relation between oxygen consumption and organic load for unit biomass.


coc

100 ~ ~ 4 8 h

50 24h '~x \ \ \

\

2 4 6 8 10 pH Fig. 2. Effect of different pH on the bacterial metabol ic activity.

The effects of the pH value on bacterial activity are shown in Fig. 2 after 24 h and 48 h. Both profiles showed a significant increase in the bacterial metabolic activity at pH values between 3.5 and 5.5, with an optimum at pH near 4-8. As a general rule the growth of bacteria has been reported to be optimal under neutral to slightly acid conditions (Beaubien & Jolicoeur, 1984). The reason for this discrepancy must be related to difference in acid tolerance of the sludge micro-organisms selected for acid enzyme-systems.

T A B L E 3 Coefficient o f Relative Biological Activi ty at Different Con-

cent ra t ions of Ions

Chemical Concentration (mg/litre)

0"01 0"1 1"0 10 100

C d 2 + 0"98 0"88 0"45 0"26 0"20 Cr 3 + 0"96 0'95 0"90 0"75 0"33 C r O 2- 0"90 0"80 0'65 0'19 0"05 Cu 2 + 0"98 0-96 0"90 0'73 0-08 Hg 2 - 0"68 0"55 0"30 0"09 0"04 Pb 4+ 0-99 0-97 0-96 0"90 0-17 Zn 2 + 0"97 0-96 0-91 0-80 0"60


Cr04 -I ,.2 C u ~ i

A

Hg I pb4 ~

Z n I . . , Cu I

i i I I i ~ r -2 -1 0 1 2 -2 -1 0 1 2 Log C

Fig. 3. Variations in the biological activity with increasing concentration of ions (C = rng/litre).

Studying the effects of Cd 2 +, Cr 3 +, CrO~- , Cu 2 +, Hg 2÷, Pb 4 ÷, Zn 2 ÷ on the performance of microbial activity a clear decrease in the bacterial metabol ism was observed. This was due to direct metal toxicity not substrate limitation.

To quantify the inhibitory effects of metal applicat ion the coefficients of relative activity for each metal were calculated by plott ing the COC values of each metal trial vs those of the control, and these are given in Table 3. In all cases the analysis of variance of the linearized equat ion showed a high index of regression (R > 0.9990). The slopes of the straight lines obtained by linear regression showed no influence (n = 1) or inhibitory effect (n < 1) of some metals on the ability of bacteria to carry out further metabolism. Plott ing 'n ~ value vs the logar i thm of the formal concentra t ion of the ion (mg/litre) approximately sigmoid curves were obtained (Fig. 3). These show some initial ability of the biological system to acclimate to initial toxic levels. This


T A B L E 4 The 10% and 50% Inhibitory Concentrat ions (mg/litre)

o f Various Ions

Chemical IC~ o 1C5 o

Cd 2 + 0"05 0"5

Cr 3 + 0"6 2 0

CrO, 2 - 0.01 4

Cu 2 + 0-4 25

Hg 2 + 0"001 O" 1

P b * + 2"5 4 0

Zn 2 + 2 5 150

tendency may reflect a variety of processes such as enzyme induction, development of tolerance, changes in metabolism, etc. (Gadd & Griffiths, 1978). Above some value of element concentration the digesters were severely inhibited, and showed a significant drop in biological activity. When heavy metal concentration was increased suddenly, the digesters were quickly and irreversibly inhibited.

Table 4 shows the IC~o and ICso values obtained graphically for each metal. The 0.9 and 0"5 points of the 'n' axis gave, from the sigmoid curves of Fig. 3, the IC~o and ICso concentrations respectively. Table 4 clearly shows two groups of metal ions according to the magnitude of the toxic effect. In the first group we find in decreasing order of toxicity Hg 2+ > Cd 2 + > CrO]- > Cr 3+ > Cu2+; in the second group we have Pb 4+ > Zn 2+.

It is not surprising to find significant discrepancy among published results of toxicity studies. In fact it is obvious that each of the microbial toxicity procedures depends on both the bacterial species and the chemical composition of the media: this latter by influencing the availability of metal ions to the biota also affects their toxicity. Also, each micro-organism has its own pattern of sensitivity to toxic elements which cannot be readily

T A B L E 5 Coefficients o f Relat ive Biological Act iv i ty at Different Con-

centrations o f Copper and Zinc Ions

Chemical Concentration (mg/litre)

0"01 O" 1 1.0 10 100

Cu 2 + (at presence

o f 1 ppm Zn z +) 0.80 0.70 0.54 0.30 0.24 Zn z + (at presence

o f 0.1 ppm Cu e +) 0"85 0.80 0.71 0.09 0-07


TABLE 6 Toxicity of Cu and Zn Ions in the Presence of Other Metals

Chemical 1C1 o lCs o (mg/litre) (mg/litre)

ELl2 +

(in presence of 1 ppm Zn 2 +) 0.001 2-5 Zn 2 +

(in presence of 0.1 ppm Cu 2 ~) 0.01 30

correlated with others. In the literature the following sequences in decreasing order of toxicity are reported: Hg > Zn > Cu > Pb (Dutka & Kwan, 1984), Hg > Cd > Cu > Zn > Cr > Pb (Beaubien & Jolicoeur, 1984), Cu > CrO 2- > Cr 3+ (Lamb & Tollefson, 1973), Cr > Cu > Zn (Barth et al., 1965), Cu > Cr 3+ > Cd > Zn > CrO 2- (Heukelekion & Gellman, 1955). Such variations discourage any at tempt to define an absolute scale of toxicity for heavy metals to micro-organisms in biodegradat ion processes.

To study the synergistic effect of metals on inhibitory activity we tested Zn 2 + and Cu 2 +, which, in small doses and singly, were only mildly toxic. In Table 5 the coefficients of relative activity at different concentrations of metal are reported. In Table 6 we have shown the IClo and ICso values obtained by plots of 'n' vs logarithm of formal concentration of the ion. Comparing the values of Tables 4 and 6, a significant increase in inhibitory effect of Cu and Zn ions is found. The results obtained make these elements similar to cadmium or mercury which are regarded as the most important of the environmental hazards. However, even though these data represent only one of the combinations of chemicals in waters, they show that it is unwise to try to assess the toxicity in effluents of a single metal species.

R E F E R E N C E S

Al-Shahwani, M. F., A1-Ghazali, M. R. & Al-Rawi, E. A. (1987). Effect of different pH, temperatures and Fe concentration on Sphaerotilus natans growth and clarity performance index. Appl. Microbiol. Biotechnol., 25, 379-83.

APHA (American Public Health Association) (1975). Standard Methods for Examination of Water and Wastewater, 14th edn. American Public Health Association, Washington, DC.

Barth, E. F., Ettinger, M. B., Solotto, B. V. & McDermott, G. N. (1965). Summary report on the effects of heavy metals on the biological treatment processes. J. Water Pollut. Control Fed., 37, 86-96.

Beaubien, A. & Jolicoeur, C. (1984). The toxicity of various heavy metal salts, alcohols and surfactants to microorganisms in a biodegradation process: a flow microcalorimetry investigation. In Toxicity Screening Procedures Using

210 G. Cimino, C. Car&ti

Bacterial Systems, ed. D. Lin & B. J. Dutka. Marcel Dekker, New York, pp. 261-81.

Bernstein, S., Tzeng, C. H. & Sisson, D. (1979). The commercial fermentation of cheese whey for the production of protein and/or alcohol. Symp. Biotechnol. Bioeng., 7, 1-9.

Cimino, G. (1987). Sperimentazioni impiantistiche e gestionali. Liquami da mattatoi: la depurazione biologica. AES rivista dell'antinquinameto, IX, 44-9.

Cimino, G. & Ziino, M. (1983). Heavy metal pollution. Part VII: Emissions from Mt Etna Volcano. Geophys. Res. Lett., 1, 31-4.

Dutka, B. J. & Kwan, K. K. (1984). Studies on asynthetic activated sludge toxicity screening procedure with comparison to three microbial toxicity tests. In Toxicity Screening Procedures Using Bacterial Systems, ed. D. Lin & B. J. Dutka. Marcel Dekker, New York, pp. 125-38.

Gadd, G. M. & Griffiths, A. J. (1978). Microorganism and heavy metal toxicity. Microbial Ecol., 4, 303-17.

Heukelekian, H. et al. (1955). Studies of biochemical oxidation by direct methods. IV. The effect of toxic metal ions on oxidation. Sewage and Industrial Wastes, 27, 70-84.

Kaspar, H. F. & Wuhrmann, K. (1978). Product inhibition in sludge digestion. Microbial Ecol., 4, 241-8.

Lamb, A. & Tollefson, E. L. (1973). Toxic effects of cupric, chromate and chromic ions on biological oxidation. Water Res., 7, 599-609.

Moresi, M. (1983). SCP production from whey: scale up of a process. In Production and Feeding of Single Cell Protein, ed. M. P. Ferranti & A. Fiechter. Applied Science Publishers, London, pp. 163-5.

Wuhrmann, K. & Benst, F. V. (1958). Zur theorie des belebtschlammverfahreus. Schweizerische zeitschrift fiir hydrologie, XX, 311-18.

acute toxicity of heavy metals to aerobic digestion of waste cheese whey

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