J . Chem. Tech. Biofechnol. 1990, 49, 345-35s
Recovery of Metal Ions by Microfungal Filters* David S. Wales & Brian F. Sagar British Textile Technology Group, Shirley Towers, Wilmslow Road, Didsbury, Manchester M20 SRX, U K (Received 29 September 1989; accepted 21 December 1989)
ABSTRACT Many microfungi contain chitinlchitosan as an integral part of the cell wall structure. The binding of toxic and heavy metal ions by chitosan or partly deacetylated chitin is a direct consequence of the base strength of the primary amine group and is most effective for those metals that form complexes with ammonia. Of the microfungi studied, hyphae j - o m Mucor mucedo and Rhizomucor miehei, a f e r treatment with hydroxide to expose the chitin/ chitosan, were found to be most effective in the capture of metal ions. Chemically treated mycelia have so far been shown to bind silver, zinc, lead, copper, nickel, cobalt, cadmium, iron and chromium, with the eficiency of metal-ion binding apparently being inversely proportional to the valency state of the metal ions to be bound. Wet-laid papers produced from mixed slurries of treated mycelia and various conventional paper-making and textile fibres have exceptionally good tensile- and bursting-strength properties, particularly in the wet state. Papers containing 1 g treated mycelia removed up to 90 of various metal ions in solution (50 cm3, 1.5 mmol dm-3) with flow rates of 0.5 cm3 cm-2 min-’. However, the total metal-ion binding capacities of single-thickness microjungal papers are limited under constant flow conditions. The total volume flowing through the system before metal-ion breakthrough occurs increases in direct proportion to the number of sheets of microfungal paper but the concomitant reduction in flow rates becomes a limiting factor. Mycelia-contuining structures that allow eficient metal-ion binding at commercially acceptable flow rates are currently being investigated. Key words: fungi, metal-ion, biosorption, chitin, chitosan. * Paper presented at the meeting ‘Recovery/Removalof Metals by Biosorption-A Commercial Reality or a Scientist’s Dream?’, organised by the Solvent Extraction and lon Exchange Group of the Society of Chemical Industry and held in London on 18 May 1989. 34s J . Chrm. Tech. Biorechnol. 0268-2575/90/$03.500 1990 SCI. Printed in Great Britain
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1 INTRODUCTION The binding of toxic and heavy metal ions by chitosan is well documented.',' This complexing ability is a direct consequence of the base strength of the primary amine group and it is most effective for those metals that form complexes with ammonia, e.g. zinc, copper, mercury and silver. Chitin is present in many fungi as the principal polymer responsible for maintaining the rigidity and shape of the cell wall; a survey of the polysaccharide composition of the cell wall of some 161 different species of fungi3 shows most to be based on glucosamine. Ruiz-Herrera4 assumed the glucosamine to have originated from chitin. However, this is not always the case, for example Mucor mucedo appears to contain both a homopolymer of N acetylglucosamine, i.e. chitin, and a heteropolymer of N-acetylglucosamine interspersed with glucosamine, i.e. ~ h i t o s a n . ~ The ability of microorganisms to accumulate metal ions from aqueous solution has been widely reported6-' and the chelating ability of chitinous material from microfungi has been in~estigated.~ Indeed, a commercial process for treatment of aqueous wastes containing metal ions based on Bacillus subtilis, has already been launched by Advanced Mineral Technologies. ' In this paper the preparation and metal-ion binding properties of filters incorporating various microfungal mycelia selected for their high chitin/chitosan contents are described.
2 MATERIALS AND METHODS
2.1 Growth of microfungi and treatment of mycelia Mucor mucedo, Rhizopus stolonifer, Aspergillus oryzae, Neurospora crassa, Trichoderma viride and Penicillium chrysogenum were grown in shake flasks or stirred fermenters (5 dm3, stirring rate 400 rpm, aeration rate 6 cm-3 s- ') at 30°C in medium containing 17 g dm-3 malt extract (Difco, East Molesey, Surrey) and 3 g dm-3 mycological peptone (Oxoid, Basingstoke, Hampshire) for 48 h. The mycelia were then harvested by filtration through a Saatifil 128 micron filter (Sericol, Broadstairs, Kent) and washed well with water. Rhizomucor miehei was grown in a 20 dm3 bioreactor (MBR, Farnborough, Hampshire) (stirring rate 800 rpm, aeration rate 33 cm-3 s-') at 50°C in the same medium, and again harvested using a Saatifill28micron filter. The mycelia were resuspended in sodium hydroxide (1 g dry weight mycelia per 100 cm3 hydroxide solution, 2 mol dm- 3 , for 2 h at room temperature. After alkali treatment, the mycelia were repeatedly washed through a Saatifil 128 micron filter until a neutral pH was obtained.
2.2 Preparation of wet-laid papers The alkali-treated mycelia were mixed with either manilla hemp or ashless clippings in the ratio 60:40 dry weight myce1ia:dry weight conventional fibre, and dispersed in water for 1500 counts (1 count 0.55 s) using a Mark IIIC Standard Pulp Disintegrator (Mavis Engineering, London). Papers (1.7 g dry weight) were then cast using a British Standard sheet paper-making machine (Standard apparatus for pulp evaluation), and dried at 30°C overnight.
Recovery of metal ions by microfingal filters
347
2.3 Preparation of myceliacontaining fleeces A carded web (10 g m-') containing 30% of 4.2 dtex bicomponent polyester binder fibre (Grilon, EMS-Grilon, Switzerland) and 70% of 27 dtex regular polyester fibre (Wellman, UK) was made into a 50 g m-' lightly needle-punched fleece by a typical non-woven cross-laying route. The fleece was then thermally bonded. Circles of the fleece were cut and placed over the grid of the Standard sheet paper-making machine, and an amount of hydroxide-treated mycelia equal to the weight of the fleece impregnated into the fleece by wet-laying.
2.4 Metal-ion determinations The concentrations of metal ions in solution were determined using a Baird Atomic (Braintree, Essex) A3400 atomic absorption spectrophotometer. Total Cu2+-binding capacities were determined by soaking overnight accurately weighed, alkali-treated mycelia filter material (about 1 g dry weight) in saturated copper sulphate solutions. The samples were then washed well in deionised water to remove all traces of unbound metal, allowed to dry and wet-ashed in a mixture of nitric acid (7 cm3)and sulphuric acid (2 cm3). Once all the nitrous oxide fumes had evolved, 4 cm3 of perchloric acid was added, and the wet-ashing was continued to completion. The solution was then diluted to 50 cm3 with deionised water, and the amount of copper present determined by atomic absorption spectrophotometry.
2.5 Measurement of physical properties of wet-laid papers Breaking load was determined using a Testometric strength tester, with a gauge length of 100mm, and cross-head speeds of 7 mm min-' for dry tests and 30 mm min-' for wet tests. The specimen width was 15 mm. Tear strengths were determined using an Elmendorf tear strength tester. 3 RESULTS AND DISCUSSION 3.1 Physical properties of wet-laid papers The wet breaking loads for hybrid wet-laid papers made with untreated mycelia from N. crussu or T. uiride, and manilla hemp (Table 1)wereconsistently better than TABLE 1 Wet and Dry Breaking Loads and Tear Strengths of Wet-laid Papers
N. crassalashless clippings N. crassa/manilla hemp T. uiridelashless clippings T. uiridelmanilla hemp Whatman No. 1 filter paper
Wet breaking load (N)
Dry breaking load (N)
Wet tear strength (9)
Dry tear strength (9)
1.1 1.6 1.6 5.4 1.o
15.2 34.8 13.3 48.7 31.9
14.7 106.7 10.7 76.0 13.3
28.0 244.0 28.0 160.0
85.3
D . S. Wales. B. F . Sayur
348
the wet strength of Whatman No. 1 filter paper. The wet breaking loads of the papers made with mycelia and ashless clippings, however, were similar to Whatman No. 1. Although the dry tensile strengths of the wet-laid papers made with untreated mycelia and ashless clippings were below that of dry Whatman No. 1 filter paper, wet-laid papers from untreated mycelia and manilla hemp were somewhat stronger in the dry state than Whatman No. 1 filter papers. A similar pattern was observed from the results of tear strength measurements. 3.2 Comparison of the metal-ion binding properties of different microorganisms Two methods were used to compare the copper-ion binding properties of wet-laid 60/40 mycelia/manilla hemp papers derived from different microfungi. In a dynamic flow method the concentration of copper remaining in solution after passage of a copper sulphate solution (100 ppm, 50cm3) through three layers of each of the various papers was determined. The experiments were repeated several times at different flow rates. The copper-ion binding efficiencies of the selected microfungal mycelia were compared by plotting copper concentration in the eluate versus flow rate and interpolating the percentage of copper removed at a standard flow rate of 0-5 cm3 cm-' min- I . In a second method the equilibrium binding capacities of the mycelial papers were determined by soaking accurately weighed portions overnight in saturated copper sulphate solution, washing thoroughly in deionised water to remove unbound copper and then wet-ashing the paper to determine the amounts of bound copper. Both methods ranked the microorganisms in the same order with regard to copper binding capability (Table 2), with wet-laid papers containing R . miehei showing the highest copper-binding efficiency,closely followed by those made using M . mucedo or R . stolonifer. The total Cu'+-binding capacity of alkali-treated M . mucedo mycelia (61.2 mg g - ') is close to the value of 79 mg g - claimed for Bacillus subtilis in the Advanced Mineral Technologies patent." However, it should be noted that no attempt was made to optimise fermentation conditions for chitin/ chitosan production by M . mucedo.
'
TABLE 2 Comparison of Copper-ion Binding Properties of Different Microfungi Flow methodCu2+ removed from I 0 0 ppm solution at 0.5 cm3 cm-= min-' (%)
Wet laid papers (60/40) R. miehilmanilla M . mucedo/manilla R. stoloni$r/manilla A . oryzae/manilla P. chrysoymum/manilla 100% alkali-treated M. mucedo
96 86 82 58 18 -
Overnight immersionTotal Cu2+ binding capacity
(ms g - ' )
16.8 15.4 10.9 7.3 61.2
Recovery of meral ions by microfingal jlters
349
3.3 Effect of pH on copper-ion binding Copper sulphate solutions (100 ppm Cu2+)were prepared at pH 2.0, 3.0, 3.5, 4.0 and 5.0 by adjustment with sulphuric acid. Samples (50cm3) of each solution were passed through wads of three 60/40 M. mucedolmanilla hemp papers at four different flow rates. The copper-ion binding profiles of the wet-laid papers were similar at the two highest pH values (Fig. 1). At pH 3.5 the percentages of Cu” removed from solution were similar to those obtained at the higher pH values when low flow rates (less than 0.83 cm3 cm-’ min-’) were employed, but a much lower value was obtained at the higher flow rate. The copper-ion binding efficiency of the papers was significantly reduced at all flow rates at pH 3-0 and was negligible at pH 2.0. It would appear, therefore, that hydroxide-treated M. mucedo mycelia is capable of efficiently binding Cu2+ions, providing the pH of the solution is not much less than 40. 3.4 Efficiency of binding different metal ions Wet-laid papers made from M. mucedolmanilla hemp (60/40) were used to compare the behaviour of different metal ions. Portions (50 cm3) of solutions of the various metal ions (1.5 mmol dm-3) were passed through three layers of the microfungal paper at five different flow rates, and the percentage removal of metal ion normalised at two flow rates (0.5 and 1.0 cm3 cm-’ min- ’) (Table 3).
’T 90
I
1
2 3 4 rate (crn3 cm-2min-1)
5
I
1 FIOW
I
Fig. 1. Effect of pH on removal ofcopper ions by 60/40 M. mucedo/manilla hemp wet-laid papers. (0) pH 5.0. (0) pH 4.0, (A)pH 3.5, ( 0 )pH 3.0 and (A) pH 2.0.
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TABLE 3
Percentage Removal of Metal Ions from Aqueous Solutions (1.5 mmol dm- 3, by Wet-laid Papers from 60/40 M. mucedo/Manilla Hemp at Two Flow Rates Flow rate
Metal ion
0.5 cm3 cm-' min-' Ag Zn2'
PbZ cu2
Ni2'
coz Cd2 Fe2+ Fe3' Cr3 +
91 82 74 74 69 71 69 69 67 61
1.0 cm3 cm-
min-
'
87 I0 12 63 66 62 63 63 62 51
Silver ions were the most eficiently bound of all the metal ions examined. Zinc was more efficiently removed from solution at the lower flow rate than any of the remaining metals but there was little difference in the binding efficiency of the papers with regard to the other divalent metal ions (lead, cupric, nickel, cobalt, cadmium and ferrous). Of the two trivalent metal ions examined the binding emciency of ferric ions by these wet-laid papers was only marginally less than that for the divalent metal ions. However, the binding efficiency of chromium ions was reduced at the higher flow rate, when compared to the divalent metal ions. Thus, the efficiency of metal-ion binding by these hybrid mats appears to be inversely proportional to their valency state.
3.5 Further comparison of the metal-ion binding properties of wet-laid papers containing R. miehei or M . mucedo Experiments were again carried out using three layers of 60/40 mycelia/manilla hemp wet-laid papers to filter 50cm3 aliquots of aqueous solutions (1.5 mmol dm-3) of salts of copper, chromium, nickel and zinc at five different flow rates. The percentages of metal ion removed at a flow rate of 0.5 cm' cm-2 min-' were interpolated from plots similar to Fig. 1. The papers containing R . miehei were clearly much more effective at removing all four metal ions from solution compared to the M. mucedo paper (Table 4), presumably associated with the higher chitin/ chitosan content of R . miehei, as determined by Kjeldahl nitrogen (nitrogen values M. mucedo, 4.42% and R . miehei, 5.16%).
3.6 Copper binding by wet-laid papers under constant flow conditions A wad of six M. mucedolmanilla hemp wet-laid papers was used to filter a copper solution (100ppm) at a constant flow rate (6.3 cm3 cm-2 min-'). Fractions (10 cm3)were collected and the copper remaining in the filtrate determined (Fig. 2).
Recowry o / nietul ions by microfungal ,filters
351
TABLE 4 Percentage Removal of Metal Ions from Aqueous Solutions (1.5 mmol dm-3) by Wetlaid Papers made from 40/60 Manilla Hemp and R . miehei or M . mucedo (flow rate 0.5 cm3 cm-2 min-') Metal ion
R. miehei
M. mucedo
cu2+
96 90 82 74
74 82 69 67
Zn2 NiZ Cr3
+
+
+
120r
-- -
100-
-
0.
r
.
.
80. 80-
L
P
0
40
80
I
I
120
160
I
I
I
I
I
200 2 40 280 320 Fraction v o l u m e km3)
360
I
I I
400 440
Fig. 2. Concentration ofcopper after passage of 400 cm3 of a 100 ppm copper solution through six layers of 60/40 M . rnucedo/manilla hemp wet-laid papers (flow rate 6.3 cmz cm-' min-I).
The proportion of Cu2+passing through the papers was less than 10% for the first 30 cm3 but thereafter it rose rapidly; 75 7; of the Cu2+ passed through the mycelial paper in the 130cm3 fraction. By simply increasing the number of layers of these wet-laid papers to delay breakthrough of the Cu2+the flow rates were reduced to unacceptable levels and alternative, less dense, structures containing mycelia were therefore sought. 3.7 Copper removal by polyester fleece containing treated mycelia
Discs of polyester fleece impregnated with mycelia were packed in a perspex column (19 cm long, i.d. 5.0cm), and a solution (approximately 2 dm3) containing Cu2+ (100 ppm) was pumped upwards through the column. Fractions (50cm3) were collected, and the copper remaining in solution after passage through the column
D.S. Wales, B . F . Sagar
352
was determined. At the end of each run the copper bound to the mycelia was removed by flushing the column with sulphuric acid (0.5%) and the pH of the column adjusted with sodium bicarbonate (3.0%). Plots of copper concentration in the column effluent versus volume of 100 ppm CuZcsolution passed through the column for the M. mucedo impregnated polyester fleece are presented in Fig. 3 for ten successive cycles (data for cycle 7 are missing).
Fraction volume (crn3)
Fig. 3. Concentration of copper in the effluent after repeated passage of copper solution (l00pprn) throughacolurnn of 50/50M.mrccrdo/polyester fleece. Runs I-5,flow rate 5.1 crn3ern-' rnin- ';runs 610, flow rate 8.9 cm3 ern-' min-'.
Recouery o / metal ions by microfungal ,filters
353
During the first five filtration/regeneration cycles, carried out at a flow rate of 5.1 cm3 cm-2 min-', the concentration of copper in the column emuent did not exceed 10 ppm until between 800 and I250 cm3 of solution had passed through the column (Table 5). After five filtration/generation cycles, the flow rate was increased to 8.9cm3cm-' min-', and the concentration of copper now exceeded 10ppm only after between 550 and 900cm3 of Cu2+ solution had passed through the column. The copper removal profiles became somewhat more irregular as the number of filtration/regeneration cycles increased, although the efficiency of copper binding did not seem to be affected. The efficiency of regeneration of the column was measured by calculating the amount of copper absorbed by the column, determining the amount of copper removed from the column by sulphuric acid treatment, and expressing the results as a percentage recovery (Table 5 ) . These data confirm that all the copper is removed from the column by the regeneration process. The experiment was repeated using discs of polyester fleece impregnated with treated R . miehei mycelia, with a single flow rate of 8-9cm3cm-2 min-'. Six filtration/regeneration cycles were carried out (Fig. 4). Similar copper-ion removal TABLE 5 Repeated Removal of Cuz+ (100 ppm) by M . mucedo Trapped in a Polyester Fleece Cycle
1
2 3
4 5 6 8 9 10
Flow rate (cm3 c m - min- )
Volume passed before Cu2+ in effluent exceeded 1 0 ppm (cm3)
Cuzt recovered ,tiom column b y acid regerierarion ( 0;)
5.1 5.1 5.1 5.1 5.1 8.9 8.9 8.9 8.9
800 1250
86 91 106 104 96 122 104 I32 117
'
1100
lo00 1 100 700 900 550 900
TABLE 6 Repeated Removal of Cuz+ (100 ppm) by R . miehei in a Polyester Fleece Cycle
Flaw rate (cm3 cm-' min-')
Volume passed before Cuz+ in efluent exceeded I0 ppm (cm3)
Cu2+ recovered jrom column b y acid regeneration ( :
Recovery of Metal Ions by Microfungal Filters* David S. Wales & Brian F. Sagar British Textile Technology Group, Shirley Towers, Wilmslow Road, Didsbury, Manchester M20 SRX, U K (Received 29 September 1989; accepted 21 December 1989)
ABSTRACT Many microfungi contain chitinlchitosan as an integral part of the cell wall structure. The binding of toxic and heavy metal ions by chitosan or partly deacetylated chitin is a direct consequence of the base strength of the primary amine group and is most effective for those metals that form complexes with ammonia. Of the microfungi studied, hyphae j - o m Mucor mucedo and Rhizomucor miehei, a f e r treatment with hydroxide to expose the chitin/ chitosan, were found to be most effective in the capture of metal ions. Chemically treated mycelia have so far been shown to bind silver, zinc, lead, copper, nickel, cobalt, cadmium, iron and chromium, with the eficiency of metal-ion binding apparently being inversely proportional to the valency state of the metal ions to be bound. Wet-laid papers produced from mixed slurries of treated mycelia and various conventional paper-making and textile fibres have exceptionally good tensile- and bursting-strength properties, particularly in the wet state. Papers containing 1 g treated mycelia removed up to 90 of various metal ions in solution (50 cm3, 1.5 mmol dm-3) with flow rates of 0.5 cm3 cm-2 min-’. However, the total metal-ion binding capacities of single-thickness microjungal papers are limited under constant flow conditions. The total volume flowing through the system before metal-ion breakthrough occurs increases in direct proportion to the number of sheets of microfungal paper but the concomitant reduction in flow rates becomes a limiting factor. Mycelia-contuining structures that allow eficient metal-ion binding at commercially acceptable flow rates are currently being investigated. Key words: fungi, metal-ion, biosorption, chitin, chitosan. * Paper presented at the meeting ‘Recovery/Removalof Metals by Biosorption-A Commercial Reality or a Scientist’s Dream?’, organised by the Solvent Extraction and lon Exchange Group of the Society of Chemical Industry and held in London on 18 May 1989. 34s J . Chrm. Tech. Biorechnol. 0268-2575/90/$03.500 1990 SCI. Printed in Great Britain
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1 INTRODUCTION The binding of toxic and heavy metal ions by chitosan is well documented.',' This complexing ability is a direct consequence of the base strength of the primary amine group and it is most effective for those metals that form complexes with ammonia, e.g. zinc, copper, mercury and silver. Chitin is present in many fungi as the principal polymer responsible for maintaining the rigidity and shape of the cell wall; a survey of the polysaccharide composition of the cell wall of some 161 different species of fungi3 shows most to be based on glucosamine. Ruiz-Herrera4 assumed the glucosamine to have originated from chitin. However, this is not always the case, for example Mucor mucedo appears to contain both a homopolymer of N acetylglucosamine, i.e. chitin, and a heteropolymer of N-acetylglucosamine interspersed with glucosamine, i.e. ~ h i t o s a n . ~ The ability of microorganisms to accumulate metal ions from aqueous solution has been widely reported6-' and the chelating ability of chitinous material from microfungi has been in~estigated.~ Indeed, a commercial process for treatment of aqueous wastes containing metal ions based on Bacillus subtilis, has already been launched by Advanced Mineral Technologies. ' In this paper the preparation and metal-ion binding properties of filters incorporating various microfungal mycelia selected for their high chitin/chitosan contents are described.
2 MATERIALS AND METHODS
2.1 Growth of microfungi and treatment of mycelia Mucor mucedo, Rhizopus stolonifer, Aspergillus oryzae, Neurospora crassa, Trichoderma viride and Penicillium chrysogenum were grown in shake flasks or stirred fermenters (5 dm3, stirring rate 400 rpm, aeration rate 6 cm-3 s- ') at 30°C in medium containing 17 g dm-3 malt extract (Difco, East Molesey, Surrey) and 3 g dm-3 mycological peptone (Oxoid, Basingstoke, Hampshire) for 48 h. The mycelia were then harvested by filtration through a Saatifil 128 micron filter (Sericol, Broadstairs, Kent) and washed well with water. Rhizomucor miehei was grown in a 20 dm3 bioreactor (MBR, Farnborough, Hampshire) (stirring rate 800 rpm, aeration rate 33 cm-3 s-') at 50°C in the same medium, and again harvested using a Saatifill28micron filter. The mycelia were resuspended in sodium hydroxide (1 g dry weight mycelia per 100 cm3 hydroxide solution, 2 mol dm- 3 , for 2 h at room temperature. After alkali treatment, the mycelia were repeatedly washed through a Saatifil 128 micron filter until a neutral pH was obtained.
2.2 Preparation of wet-laid papers The alkali-treated mycelia were mixed with either manilla hemp or ashless clippings in the ratio 60:40 dry weight myce1ia:dry weight conventional fibre, and dispersed in water for 1500 counts (1 count 0.55 s) using a Mark IIIC Standard Pulp Disintegrator (Mavis Engineering, London). Papers (1.7 g dry weight) were then cast using a British Standard sheet paper-making machine (Standard apparatus for pulp evaluation), and dried at 30°C overnight.
Recovery of metal ions by microfingal filters
347
2.3 Preparation of myceliacontaining fleeces A carded web (10 g m-') containing 30% of 4.2 dtex bicomponent polyester binder fibre (Grilon, EMS-Grilon, Switzerland) and 70% of 27 dtex regular polyester fibre (Wellman, UK) was made into a 50 g m-' lightly needle-punched fleece by a typical non-woven cross-laying route. The fleece was then thermally bonded. Circles of the fleece were cut and placed over the grid of the Standard sheet paper-making machine, and an amount of hydroxide-treated mycelia equal to the weight of the fleece impregnated into the fleece by wet-laying.
2.4 Metal-ion determinations The concentrations of metal ions in solution were determined using a Baird Atomic (Braintree, Essex) A3400 atomic absorption spectrophotometer. Total Cu2+-binding capacities were determined by soaking overnight accurately weighed, alkali-treated mycelia filter material (about 1 g dry weight) in saturated copper sulphate solutions. The samples were then washed well in deionised water to remove all traces of unbound metal, allowed to dry and wet-ashed in a mixture of nitric acid (7 cm3)and sulphuric acid (2 cm3). Once all the nitrous oxide fumes had evolved, 4 cm3 of perchloric acid was added, and the wet-ashing was continued to completion. The solution was then diluted to 50 cm3 with deionised water, and the amount of copper present determined by atomic absorption spectrophotometry.
2.5 Measurement of physical properties of wet-laid papers Breaking load was determined using a Testometric strength tester, with a gauge length of 100mm, and cross-head speeds of 7 mm min-' for dry tests and 30 mm min-' for wet tests. The specimen width was 15 mm. Tear strengths were determined using an Elmendorf tear strength tester. 3 RESULTS AND DISCUSSION 3.1 Physical properties of wet-laid papers The wet breaking loads for hybrid wet-laid papers made with untreated mycelia from N. crussu or T. uiride, and manilla hemp (Table 1)wereconsistently better than TABLE 1 Wet and Dry Breaking Loads and Tear Strengths of Wet-laid Papers
N. crassalashless clippings N. crassa/manilla hemp T. uiridelashless clippings T. uiridelmanilla hemp Whatman No. 1 filter paper
Wet breaking load (N)
Dry breaking load (N)
Wet tear strength (9)
Dry tear strength (9)
1.1 1.6 1.6 5.4 1.o
15.2 34.8 13.3 48.7 31.9
14.7 106.7 10.7 76.0 13.3
28.0 244.0 28.0 160.0
85.3
D . S. Wales. B. F . Sayur
348
the wet strength of Whatman No. 1 filter paper. The wet breaking loads of the papers made with mycelia and ashless clippings, however, were similar to Whatman No. 1. Although the dry tensile strengths of the wet-laid papers made with untreated mycelia and ashless clippings were below that of dry Whatman No. 1 filter paper, wet-laid papers from untreated mycelia and manilla hemp were somewhat stronger in the dry state than Whatman No. 1 filter papers. A similar pattern was observed from the results of tear strength measurements. 3.2 Comparison of the metal-ion binding properties of different microorganisms Two methods were used to compare the copper-ion binding properties of wet-laid 60/40 mycelia/manilla hemp papers derived from different microfungi. In a dynamic flow method the concentration of copper remaining in solution after passage of a copper sulphate solution (100 ppm, 50cm3) through three layers of each of the various papers was determined. The experiments were repeated several times at different flow rates. The copper-ion binding efficiencies of the selected microfungal mycelia were compared by plotting copper concentration in the eluate versus flow rate and interpolating the percentage of copper removed at a standard flow rate of 0-5 cm3 cm-' min- I . In a second method the equilibrium binding capacities of the mycelial papers were determined by soaking accurately weighed portions overnight in saturated copper sulphate solution, washing thoroughly in deionised water to remove unbound copper and then wet-ashing the paper to determine the amounts of bound copper. Both methods ranked the microorganisms in the same order with regard to copper binding capability (Table 2), with wet-laid papers containing R . miehei showing the highest copper-binding efficiency,closely followed by those made using M . mucedo or R . stolonifer. The total Cu'+-binding capacity of alkali-treated M . mucedo mycelia (61.2 mg g - ') is close to the value of 79 mg g - claimed for Bacillus subtilis in the Advanced Mineral Technologies patent." However, it should be noted that no attempt was made to optimise fermentation conditions for chitin/ chitosan production by M . mucedo.
'
TABLE 2 Comparison of Copper-ion Binding Properties of Different Microfungi Flow methodCu2+ removed from I 0 0 ppm solution at 0.5 cm3 cm-= min-' (%)
Wet laid papers (60/40) R. miehilmanilla M . mucedo/manilla R. stoloni$r/manilla A . oryzae/manilla P. chrysoymum/manilla 100% alkali-treated M. mucedo
96 86 82 58 18 -
Overnight immersionTotal Cu2+ binding capacity
(ms g - ' )
16.8 15.4 10.9 7.3 61.2
Recovery of meral ions by microfingal jlters
349
3.3 Effect of pH on copper-ion binding Copper sulphate solutions (100 ppm Cu2+)were prepared at pH 2.0, 3.0, 3.5, 4.0 and 5.0 by adjustment with sulphuric acid. Samples (50cm3) of each solution were passed through wads of three 60/40 M. mucedolmanilla hemp papers at four different flow rates. The copper-ion binding profiles of the wet-laid papers were similar at the two highest pH values (Fig. 1). At pH 3.5 the percentages of Cu” removed from solution were similar to those obtained at the higher pH values when low flow rates (less than 0.83 cm3 cm-’ min-’) were employed, but a much lower value was obtained at the higher flow rate. The copper-ion binding efficiency of the papers was significantly reduced at all flow rates at pH 3-0 and was negligible at pH 2.0. It would appear, therefore, that hydroxide-treated M. mucedo mycelia is capable of efficiently binding Cu2+ions, providing the pH of the solution is not much less than 40. 3.4 Efficiency of binding different metal ions Wet-laid papers made from M. mucedolmanilla hemp (60/40) were used to compare the behaviour of different metal ions. Portions (50 cm3) of solutions of the various metal ions (1.5 mmol dm-3) were passed through three layers of the microfungal paper at five different flow rates, and the percentage removal of metal ion normalised at two flow rates (0.5 and 1.0 cm3 cm-’ min- ’) (Table 3).
’T 90
I
1
2 3 4 rate (crn3 cm-2min-1)
5
I
1 FIOW
I
Fig. 1. Effect of pH on removal ofcopper ions by 60/40 M. mucedo/manilla hemp wet-laid papers. (0) pH 5.0. (0) pH 4.0, (A)pH 3.5, ( 0 )pH 3.0 and (A) pH 2.0.
D. S . Wales, B . F . Sugar
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TABLE 3
Percentage Removal of Metal Ions from Aqueous Solutions (1.5 mmol dm- 3, by Wet-laid Papers from 60/40 M. mucedo/Manilla Hemp at Two Flow Rates Flow rate
Metal ion
0.5 cm3 cm-' min-' Ag Zn2'
PbZ cu2
Ni2'
coz Cd2 Fe2+ Fe3' Cr3 +
91 82 74 74 69 71 69 69 67 61
1.0 cm3 cm-
min-
'
87 I0 12 63 66 62 63 63 62 51
Silver ions were the most eficiently bound of all the metal ions examined. Zinc was more efficiently removed from solution at the lower flow rate than any of the remaining metals but there was little difference in the binding efficiency of the papers with regard to the other divalent metal ions (lead, cupric, nickel, cobalt, cadmium and ferrous). Of the two trivalent metal ions examined the binding emciency of ferric ions by these wet-laid papers was only marginally less than that for the divalent metal ions. However, the binding efficiency of chromium ions was reduced at the higher flow rate, when compared to the divalent metal ions. Thus, the efficiency of metal-ion binding by these hybrid mats appears to be inversely proportional to their valency state.
3.5 Further comparison of the metal-ion binding properties of wet-laid papers containing R. miehei or M . mucedo Experiments were again carried out using three layers of 60/40 mycelia/manilla hemp wet-laid papers to filter 50cm3 aliquots of aqueous solutions (1.5 mmol dm-3) of salts of copper, chromium, nickel and zinc at five different flow rates. The percentages of metal ion removed at a flow rate of 0.5 cm' cm-2 min-' were interpolated from plots similar to Fig. 1. The papers containing R . miehei were clearly much more effective at removing all four metal ions from solution compared to the M. mucedo paper (Table 4), presumably associated with the higher chitin/ chitosan content of R . miehei, as determined by Kjeldahl nitrogen (nitrogen values M. mucedo, 4.42% and R . miehei, 5.16%).
3.6 Copper binding by wet-laid papers under constant flow conditions A wad of six M. mucedolmanilla hemp wet-laid papers was used to filter a copper solution (100ppm) at a constant flow rate (6.3 cm3 cm-2 min-'). Fractions (10 cm3)were collected and the copper remaining in the filtrate determined (Fig. 2).
Recowry o / nietul ions by microfungal ,filters
351
TABLE 4 Percentage Removal of Metal Ions from Aqueous Solutions (1.5 mmol dm-3) by Wetlaid Papers made from 40/60 Manilla Hemp and R . miehei or M . mucedo (flow rate 0.5 cm3 cm-2 min-') Metal ion
R. miehei
M. mucedo
cu2+
96 90 82 74
74 82 69 67
Zn2 NiZ Cr3
+
+
+
120r
-- -
100-
-
0.
r
.
.
80. 80-
L
P
0
40
80
I
I
120
160
I
I
I
I
I
200 2 40 280 320 Fraction v o l u m e km3)
360
I
I I
400 440
Fig. 2. Concentration ofcopper after passage of 400 cm3 of a 100 ppm copper solution through six layers of 60/40 M . rnucedo/manilla hemp wet-laid papers (flow rate 6.3 cmz cm-' min-I).
The proportion of Cu2+passing through the papers was less than 10% for the first 30 cm3 but thereafter it rose rapidly; 75 7; of the Cu2+ passed through the mycelial paper in the 130cm3 fraction. By simply increasing the number of layers of these wet-laid papers to delay breakthrough of the Cu2+the flow rates were reduced to unacceptable levels and alternative, less dense, structures containing mycelia were therefore sought. 3.7 Copper removal by polyester fleece containing treated mycelia
Discs of polyester fleece impregnated with mycelia were packed in a perspex column (19 cm long, i.d. 5.0cm), and a solution (approximately 2 dm3) containing Cu2+ (100 ppm) was pumped upwards through the column. Fractions (50cm3) were collected, and the copper remaining in solution after passage through the column
D.S. Wales, B . F . Sagar
352
was determined. At the end of each run the copper bound to the mycelia was removed by flushing the column with sulphuric acid (0.5%) and the pH of the column adjusted with sodium bicarbonate (3.0%). Plots of copper concentration in the column effluent versus volume of 100 ppm CuZcsolution passed through the column for the M. mucedo impregnated polyester fleece are presented in Fig. 3 for ten successive cycles (data for cycle 7 are missing).
Fraction volume (crn3)
Fig. 3. Concentration of copper in the effluent after repeated passage of copper solution (l00pprn) throughacolurnn of 50/50M.mrccrdo/polyester fleece. Runs I-5,flow rate 5.1 crn3ern-' rnin- ';runs 610, flow rate 8.9 cm3 ern-' min-'.
Recouery o / metal ions by microfungal ,filters
353
During the first five filtration/regeneration cycles, carried out at a flow rate of 5.1 cm3 cm-2 min-', the concentration of copper in the column emuent did not exceed 10 ppm until between 800 and I250 cm3 of solution had passed through the column (Table 5). After five filtration/generation cycles, the flow rate was increased to 8.9cm3cm-' min-', and the concentration of copper now exceeded 10ppm only after between 550 and 900cm3 of Cu2+ solution had passed through the column. The copper removal profiles became somewhat more irregular as the number of filtration/regeneration cycles increased, although the efficiency of copper binding did not seem to be affected. The efficiency of regeneration of the column was measured by calculating the amount of copper absorbed by the column, determining the amount of copper removed from the column by sulphuric acid treatment, and expressing the results as a percentage recovery (Table 5 ) . These data confirm that all the copper is removed from the column by the regeneration process. The experiment was repeated using discs of polyester fleece impregnated with treated R . miehei mycelia, with a single flow rate of 8-9cm3cm-2 min-'. Six filtration/regeneration cycles were carried out (Fig. 4). Similar copper-ion removal TABLE 5 Repeated Removal of Cuz+ (100 ppm) by M . mucedo Trapped in a Polyester Fleece Cycle
1
2 3
4 5 6 8 9 10
Flow rate (cm3 c m - min- )
Volume passed before Cu2+ in effluent exceeded 1 0 ppm (cm3)
Cuzt recovered ,tiom column b y acid regerierarion ( 0;)
5.1 5.1 5.1 5.1 5.1 8.9 8.9 8.9 8.9
800 1250
86 91 106 104 96 122 104 I32 117
'
1100
lo00 1 100 700 900 550 900
TABLE 6 Repeated Removal of Cuz+ (100 ppm) by R . miehei in a Polyester Fleece Cycle
Flaw rate (cm3 cm-' min-')
Volume passed before Cuz+ in efluent exceeded I0 ppm (cm3)
Cu2+ recovered jrom column b y acid regeneration ( :
