CONTINUOUS FLOW FERMENTATION TO PURIFY WASTE WATER

BY THE REMOVAL OF CADMIUM

R. C A M P B E L L * and M. H. M A R T I N

Department of Botany, University of Bristol, Bristol BS8 1 UG, U.K.

(Received November 27, 1989; revised February 1, 1990)

Abstract. Fungi have been isolated that can tolerate and absorb high levels of heavy metals, especially Cd, when grown in a cont inuous flow air-lift fermenter with up to 10 throughputs per day. This allows the recovery of metals or the purification of industrial effluent streams. An average of over 97% of the Cd was removed from the simulated effluent during passage through the fermenter, reducing the Cd concentration from over 6 mg L -1 to less than 0.2 mg L-t .The biomass recovered contained up to 2.9% Cd (on a dry weight basis) and can either be disposed of or returned for smelting to recover

the metal.

1. Introduction

Microorganisms have been used in leaching of metal ores and in the removal of metals from waste water (Brierley et al., 1989). The effects of metals on microor- ganisms have been widely studied and reviewed and this has shown the physico- chemical nature of the environment to be very important in determining interactions with and the toxicity of the metal (Collins and Stotzky, 1989).

During studies on the heavy metal pollution close to a smelter (Gingell et al.,

1976; Bewley and Campbell, 1980; Martin et al., 1980) it became clear that many leaf surface and soil microorganisms had considerable tolerance to heavy metals, and that some adsorbed or actively accumulated metals until high levels were present in the microbial biomass. It seemed likely that these organsms could be used to remove metals from aqueous solutions, thus purifying waste water and reducing environmental pollution.

Limits on the amounts to heavy metals permitted in water, especially that to be used subsequently for drinking, have been proposed by various bodies (EEC, 1975); for example in the Federal Republic of Germany the guideline for industrial waste waster is 0.5 mg Cd L -1 for indirect discharge (Rump and Krist, 1989).

Microorganisms have been suggested as a means of removing metals from water by several authors (Curtin, 1983; Hall and Melcer, 1983; Lester et al., 1984; Hutchins et al., 1986; Brierley et al., 1989). Processes for the cleaning of waste waters, and the subsequent recovery of metals, have depended on dead or immobilized biomass as an exchange site, using either mixed cultures (Remacle and Houba, 1983), algae (Darnall et al., 1986), or fungi (Drobot, 1981; Drobot and Lechavalier, 1981). The use of live bacteria has been suggested (Hancock, 1986), but this also depends largely on adsorption rather than the active uptake of the metal. With processes

* Author for all correspondence

Water, Air, and Soil Pollution 50: 397-408, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

398 R. CAMPBELL AND M. H. MARTIN

dependent on adsorption, especially by dead biomass, the system becomes metal saturated relatively quickly, though in some cases the metal can be eluted and the biomass re-used. These are essentially batch systems.

If growing organisms with active uptake, or effective adsorption, could be used the system should retain larger quantities of metal and be better adapted to a continuous flow process. After some preliminary studies, bacteria were considered unsuitable because of the difficulty of retention in the fermenter at high flow rates and the problems of separation of the biomass from the large volumes of liquid. Floc-forming fungi appeared to be suitable organisms in the fermenter and are easily recovered from the effluent. The formation of stable, easily separated flocs by a fungus is dependent on many factors, including the nature of the organism and the conditions within the fermenter (Atkinson and Daoud, 1976).

Cadmium was used as the main test metal in these experiments because it is an important environmental pollutant (Nriagu, 1980) which is known to be adsorbed

and absorbed by microorganisms (Macaskie and Dean, 1982). Microorganisms tolerant to Cd exhibit tolerance to other heavy metals (Silver and Misra, 1988), so the experiments on Cd could have a wider application.

The aim of this project was, therefore, to develop a system using a continuous flow fermenter with a fungus growing in such a form that it would be retained in the fermenter at high flow rates and be easily recovered from the effluent after treatment. In order to be of potential industrial application the fungus should remove Cd from the input (at 5 to 10 mg L -1) and give low levels of metal (<0.5 mg L -1) in the effluent.

2. Materials and Methods

2.1. INITIAL ISOLATION AND SCREENING

Samples of plant material and soils were collected from close to the Pb-Zn-Cd smelter at Avonmouth, UK and from spoil heaps and soil from old metal mining operations at Shipham, Avon, UK. These samples were used to isolate fungi on

malt agar (2 g malt powder, Oxoid L39, 20 g agar L -1) and potato dextrose agar (Oxoid CM139) each containing 10 mg L -1 Cd as nitrate. Tolerant isolates which grew on these media were subcultured and subsequently tested for growth in liquid culture (1% w/v molasses, plus 10 mg L -l Cd) at 20 °C for 5 days. The fungus was harvested by vacuum filtration, weighed and then dried (80 °C in a forced draught oven) and the Cd content of the mycelium assessed after acid digestion (conc. HNO3) by flame atomic absorption spectroscopy. The fastest growing isolates which retained the most Cd were further tested in the fermenter (see below).

2.2. TESTS FOR TOLERANCE TO OTHER METALS

The fungal isolates selected for Cd tolerance were screened for tolerance to other metals, since tolerance to different heavy metals is often linked (Trevors el al.,

FERMENTATION TO PURIFY WASTE WATER BY THE REMOVAL OF CADMIUM 399

1986; Silver and Misra, 1988). Tryptic soy broth (Difco 0370-17) was prepared

at one tenth the normal strength (3 g powder L -1) and 50 mg L -I of metals were added as soluble salts after autoclaving as follows: Co, Cu, Ni, and Zn as sulphates; Pb and Ag as nitrates; Cr as chromium potassium sulphate; Au as gold III chloride; and U as acetate. Growth was assessed after 2 weeks in shaken culture at 20 °C.

2.3. TI-IE FERMENTER

The laboratory scale air-lift fermenter (modified from Greenshields and Smith, 1971; Smith and Greenshields, 1974) had a capacity of 11 L and was based on a glass column approximately 2 m high (Figure 1). Air, filtered to remove oil aerosols and dust, but not sterile, was supplied at the base of the column at 6 to 10 L rain -1. The column temperature was controlled at 26 °C by a thermostat operating a heater wrapped around the outside of the column. The pH of the liquid was maintained by the addition of sodium hydroxide solution controlled by a pH sensor at the top of the column which activated peristaltic pumps as needed. Early experiments had shown removal of Cd in the biomass to be positively correlated with pH, with optimum conditions at around pH 7.

The supply of mineral salts (potassium dihydrogen phosphate, 0.05% w/v; ammonium sulphate, 0.15% w/v), sucrose (30 g L -1) and Cd nitrate (0.24 g L -l) was controlled by separate pumps so that the total throughflow of liquid and the concentrations of the components could be varied. The concentrations given are nominal values of the stock solutions, these were varied if necessary to achieve the required column concentrations at a given flow rate. None of these solutions was sterile; growth of contaminant organisms was limited in the mineral supply solution by a lack of C. The stock Cd solution was toxic at these high concentrations and the sucrose had too high an osmotic potential for the growth of most organisms. There was occasional yeast contamination in the sucrose, but this was eliminated in later configurations by combining the sucrose and the Cd stock solutions.

There was a sampling port approximately half way up the column for the removal of biomass and liquid during a run. A 500 mL sample was taken daily and was used to determine the concentration of metal in the column, the wet and dry weight of the biomass per unit volume and also to check on the purity and identity of the biomass.

Liquid and biomass (effluent liquid and effluent biomass) continually washed over the top of the column, and were collected and separated on a simple muslin screen. The liquid ran to waste for most of the time, though it was sampled for metal analysis and to determine the efficiency of filtration. The biomass collected from the effluent stream was dewatered by vacuum filtration through Whatman No. 1 filter paper and weighed. Subsequently samples of the biomass were oven dried and analysed for cadmium by atomic absorption spectroscopy as described above.

There were varous valves and connections on the column, not all of which are shown in Figure 1. These allowed draining and cleaning between runs.

400 R. CAMPBELL AND M. H. MARTIN

m mm

minerals

sample port

liquid

trap

me

air in

5 i ~~/ Iter

I IJ '

I

waste when cleaning Co i tt~m

Fig. 1. Diagram of the fermenter system (not to scale). All the pumps, meters etc., except the main mineral feed, are above the liquid level in the column.

FERMENTATION TO PURIFY WASTE WATER BY THE REMOVAL OF CADMIUM 401

The column was inoculated with an axenic shake culture of the fungus in the

same medium as used in the column.

3. Results and Discussion

3.1. I N I T I A L S C R E E N I N G A N D F E R M E N T E R R U N S

The screening procedure produced many fungi with tolerance to Cd, but few of

these grew rapidly in shake culture and most did not accumulate the metal. The most successful fungi in these initial tests were species of Penicillium and Aspergillus, especially A. niger. This latter fungus, in batch shake culture, could remove over 90% of the Cd from the medium and grew well, even at Cd concentrations up to 50 mg L -1.

None of the isolates could tolerate Ag or Au, and only a few grew in Cu or Co ammended media. However, Aspergillus spp., Penicillium spp. and Cladosporium spp. showed tolerance to Cd, Cr, Zn, Pb, Ni, and U in shaken batch culture, though growth in combinations of metals was often slow.

Organisms which seemed tolerant to metals and which grew well under these

conditions were tested in the air-lift fermenter, using Cd as the test metal.

Cladosporium did not grow fast enough and was washed out of the column at all but the slowest flow rates, and so did not remove much Cd from the inftuent.

Aspergillus grew well and removed up to 80% of the Cd, but it did not persist in the column. After a few days the dominant organism in the column changed to any one of a number of contaminants in different runs. Penicillium, Mucor and

a wide range of bacteria, were isolated from the column biomass, but most did not remove Cd from the flow. On one occasion the column was colonized by a

protozoan, Colpoda sp., which was feeding on contaminating bacteria. The Aspergillus which we used clearly could not compete with incoming organisms in this non-

sterile system, even though it started with a substantial inoculum. The Penicillium used competed much better and survived for up to 10 days, though the level of biomass fluctuated considerably. Cadmium recovery was good as long as there

was sufficient biomass in the column, but in general the erratic growth resulted

in overall recovery rates of only 33.6%. It was also difficult to recover the biomass because the flocs were not discrete and filtration took a very long time.

In several different runs with Penicillium and the Aspergillus it was noted that

the major contaminating organism in the biomass was a fungus, identified as Fusarium sp. This apparently originated as a contaminant in the mineral feed tank, but the

same organism, as judged by morphology of mycelium and spores, took over the fermenter column on many different occasions. Isolates of this fungus were screened

and they appeared to accumulate Cd and other metals. This fungus was therefore used as initial inocolum since it seemed to grow well in the column and fulfil

all the requirements for Cd removal. Modifications of the nutrients gave a good floc format ion and recovery of the biomass was easily achieved.

402 R. CAMPBELL AND M. H. MARTIN

FUSARIUM AS THE INITIAL INOCULUM

Several runs of the fermenter for continuous periods of 4 to 5 weeks were performed

and one of these will be used to illustrate the functioning of the column. The biomass production (Figure 2), measured by that collected as effluent biomass,

was essentially zero after the inocolum was added, except for a little wash-over

of inoculum, but rapidly rose to about 30 g dry weight per day. The flow rate

for this period was 40 L per day (Figure 2), but on day 8 the flow rate of the standard mineral solution was doubled and the concentrations of sucrose and Cd

doubled without altering the flow rates. The total volume throughput was therefore doubled without changing any of the concentrations of nutrients or Cd within the column (Figure 2). This resulted in wash-out of the biomass, reducing that in the column, but increasing the effluent biomass a little at first (Figure 2). The effluent biomass was then reduced as there was insufficient biomass production in the column to supply biomass to the effluent. On day 12 therefore the flow rates and

concentrations were reduced to the standard values (Figure 2) and the column biomass then increased. Over the rest of the run the flow rate was then slowly

increased, and the stock concentrations changed to keep column concentrations of nutrients and Cd constant until by day 38 the throughput was 110 L day -1

(Figure 2) at which point some washout was occurring, as shown by the drop in the column biomass (Figure 2). Maximum biomass production (effluent biomass,

% Cd removed 250 120

200

150

100

50

. . . . . . . . . . . . . . . . . . L...!._J__.L....L..J_L_.J_..I ............. !.._.[ _L.J_.L.._L_...L_L.. !...! .!.._ J...! . . . . . . .

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Days

100

80

60

40

20

0

I % Cd removal x effluent dry wt g/d

J J F low rate L / d a y ~ co lumn b iomasa gxlO0

Fig. 2. The percent removal of Cd (supply concentration - effluent liquid concentration) during passage through the column, the flow rate, the effluent biomass dry weight production per day, and the column

biomass (g x 100 per 500 mL sample) for a fermenter run of 38 days.

F E R M E N T A T I O N TO PURIFY WASTE WATER BY T H E REMOVAL OF C A D M I U M 403

Figure 2) was 60 g dry weight per day. The decrease in column biomass f rom

days 15 to 19 coincided with an increase in bacteria in the column, but the Fusarium

recovered spontaneously. I t seems that the biomass can increase in response to

increasing flow rates, and therefore to increasing total amounts of nutrients and

Cd (though the concentrations remain the same), provided that the changes were made gradually. Sudden changes were not tolerated and also lead to increases in contaminating organisms within the column, presumably because there were spare nutrients in the column as the Fusarium failed to respond quickly enough to the altered conditions. With steady flow rates or slow changes Fusarium remained the

dominant organism in the column, even though the conditions were not axenic.

The form of the biomass is important for efficient filtering, and this fungus under

the standard nutrient conditions and in the presence of Cd, produced discrete flocs of irregular shape (Figure 3a) and of variable size because they were of different

ages when removed from the column. Fungal material collected on the muslin filter

Fig. 3(A) The floc form of the fungus in the column. Note the dense biomass in flocs of various sizes. (B) The biomass as collected on the muslin filter. (C and D) The biomass after vacuum filtration to

produce a fibrous, odourless mass of mycelium which is easily handled for disposal or metal recovery.

404 R. C A M P B E L L A N D M. H . M A R T I N

as a wet but coherent mass (Figure 3b) which was dewatered to a solid, fibrous mass (Figure 3c and d). The biomass was easy to handle, had no unpleasant smell and did not produce spores or other airborne particles. The level of biomass in

the column was 2 to 4 g dry weight L -1, sufficient to give a dense growth while still allowing the passage of air up the column and the mixing of the column contents.

The efficiency of biomass production is quite low, on average ( + / - SE) 0.2 (0.02) g

biomass g-] of sucrose, but it fluctuated considerably and the maximum was 0.4 g g-1.

The mean concentration Of Cd in the biomass, both in the column and the effluent, was 12 (0.7 and 1.0, respectively) mg g-1 (1.2% by dry weight) with a maximum

of 29 mg g-1. A total of 84% of the Cd supplied during the run was accounted for in the biomass. The mean concentration of Cd in the supply was 6.4 (0.07)

mg L -~ and this was reduced to 0.16 (0.05) mg L -1 in the effluent liquid (Figure 4). The average removal was 98 (0.8) % (w/w) and the variations in this value (Figure 4) were not due to any obvious factor except washout at day 11 and at the end of

the run (Figure 2). Washout causes reductions in column biomass leaving insufficient fungus for the removal of Cd as the liquid passes through the column, so the

level of Cd in the effluent increases (Figure 4). Removal efficiency is not dependent on flow rate through the column except at extreme values or during sudden changes

which affect the biomass (Figure 2).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 - ~ i i ; [ z ~ ~ I I I [ i z I t z [ i I z I i I J T I I ] i i ~ T I ' l ! "J

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Days

"" Cd mg/L In effluent Cd supplied mg/L ~ Column biomass g I

Fig. 4. The concentration of Cd supplied in the input and washed over in the effluent liquid from the column. The column biomass (g dry weight per 500 mL sample) is also shown to illustrate the

correlation with Cd removal.

FERMENTATION TO PURIFY WASTE WATER BY T H E REMOVAL OF CADMIUM 405

4. General Discussion

This fermentation process has possibilities for the treatment of industrial effluent for the removal of heavy metals, particularly Cd. It produces effluent, on an experimental scale, within the standards for Cd in discharges of industrial waste waters (Rump and Krist, 1989). This fermenter has been scaled-up to industrial proportions for the reduction in the BOD of distillery and fruit processing waste (Satec Ltd., Crewe, England, personal communication) and there seems no a priori

reason why it should not be suitable for metal removal at this size. Other air- lift fermenters are used on an industrial scale for the production of single cell protein (Reed, 1982) and for the growth of mammalian cells for the production of antibodies (Carvell and Chapman, 1989). The scaled-up fermenter will need an industrially available, cheap C and N source. Sucrose used in this study is possible, and similar materials such as molasses or various sugar syrups are available commercially. It is possible that waste products from the food industry with a high BOD may also be used. These possibilities depend on, and dictate, the economics of the process. At present, the requirements for cleaning effluents are often dependent on dilution to a required concentration which is a relatively cheap alternative. Until absolute pollution levels are required to be reduced, the demand or economic pressure for such a fermenter system seems limited. These experiments used Cd as the polluting metal in the form of the nitrate, which was chosen for its high solubility to allow a wide range of concentrations to be used. Any Cd in the biomass was therefore absorbed or adsorbed rather than being passively entrapped particulate material. There was insufficient nitrate in the Cd supply to significantly influence the overall supply of N, and Cd frequently occurs in this form in industrial effluents, though Cd sulphate may also be present. The pH (see below) and other components of the waste, affect Cd toxicity. The toxicity is greatest at pH ) 7 because of the presence of monovalent CdOH + (Collins and Stozky, 1989). Competitive adsorption of different metal ions (Collins and Stotzky, 1989) may occur in industrial waste streams where Pb and Zn, and Ni and Cu are often present. In the experiments described above only Cd, the most toxic of these metals, was used and so would be more damaging than a mixture; the system thus tested the fungus under the most difficult conditions it would meet with respect to metal ions. The Cd concentrations used were realistic for many industrial waste streams, and the fungus grew well at twice the nominal concentration of 6 mg L -1. It tolerated much higher levels, though growth slowed with prolonged exposure. In industrial conditions the fungus would not therefore be adversely affected by temporary fluctuations in metal concentration.

To evaluate the efficiency and economics of metal removal under industrial conditions, information is needed from pilot scale plants with real industrial effluent rather than the simulation given above. It is not possible to predict performance from laboratory studies with these fermenters (Greenshields and Smith, 1971; Smith and Greenshields, 1974) since the controlling factors in the dynamics of through-

406 R. CAMPBELL AND M. H, MARTIN

flow tower fermenters are less well understood than for batch processes (Atkinson and Daoud, 1976; Cocker, 1980). For example Carvell and Chapman (1989) used a 100 L laboratory fermenter and then 300 L and 600 L trials to reach the 1000 L

industrial model. Using growing organisms in a continuous process (rather then batch) is desirable

for the efficient recovery of the metal, but the organism is subject to toxic substances or other unfavorable parameters in the waste. Very low pH or the presence of fluorine, cyanide or As pose problems and may require pretreatment o f the effluent, though organisms tolerant to at least some of these pollutants are known (Silver and Misra, 1988). We have not found organisms able to tolerate or grow in the presence of some metals, such as gold and silver, but this may well reflect the very brief search and experimentation which we have conducted, rather than the absence of such organisms from the environment. Other authors have found silver tolerant organisms (Hutchins et al., 1986; Silver and Misra, 1988) and there seems no reason why a search for fungi which would tolerate and take up Ag in the

conditions in our fermenter, should not be successful. The stability of the organism in the non-sterile column has been shown to be

good, since the Fusarium was dominant for the 38 days of the run described and for other runs of a similar length, but it is not guaranteed to survive rapid perturbations in the fermenter conditions. The microbiological contamination of the feed stock was kept low during these runs by good hygiene, but the air was not sterilized. If severely contaminated supplies were used it is likely that the desired organism would not be maintained. It would be possible to run the system axenically, but on an industrial scale this would be very expensive, though it is possible for the growth of mammalian cells in air-lift fermenters (Carvell and Chapman, 1989).

The stability of the floc form, on which the simple recovery of the biomass depends, does not present a problem. The flocs of this fungus are very tolerant of a variety of nutrient and fermenter conditions, which is fortunate since the form of the floc is vital for the operation of the fermenter and to maintain a suitable retention time and viscosity characteristics (Cocker, 1980). Similarly there was no problem with the growth of the fungus on the walls of the fermenter or on the various

probes and pipes etc. projecting into the liquid stream. The end products of the process are a liquid with much reduced metal levels

and a fungal biomass with very high metal level (up to 2 to 3% by weight when dried). This biomass could be disposed of as waste, but since it contains a higher concentration of Cd than many ore sources, it could also be used to recover the Cd in smelting, though it would need to be thoroughly dewatered to reduce its thermal capacity. We see the recovery of Cd as a useful bonus to the cleaning of the effluent, but with other metals the recovery may be the prime object of the fermentation. The process described both recovers the metal and cleans the effluent; which is the most important function will depend on economics and on

the particular metal considered.

F E R M E N T A T I O N TO PURIFY WASTE WATER BY T H E REMOVAL OF C A D M I U M 407

Acknowledgments

We are v e r y g r a t e fu l to Sa tec Ltd . , C rewe , E n g l a n d fo r f inanc ia l s u p p o r t fo r this

r e sea r ch a n d fo r the l o a n o f the f e r m e n t e r ; we h a d v a l u a b l e d i scuss ions wi th m a n y

m e m b e r s o f t he c o m p a n y , bu t w o u l d espec ia l ly l ike to t h a n k A. W e b b a n d P. Wall .

We w o u l d a lso l ike to t h a n k M. J. C a h a l a n a n d W. L. L i n t o n o f R T Z Serv ices

Ltd . fo r d i s cus s ion at the ea r ly s tages o f the p ro jec t .

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408 1~. CAMPBELL AND M. H. MARTIN

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