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J. K. Afidenyo, W.Afidenyo, W.-T. Yen, R. K. Amankwah and J.A. Ramsay

Contact author:
Mr. James K. Afidenyo
Mining Engineering Department, Queens University,
Kingston, Ontario, Canada K7L 3N6
Tel: (613)767 7659
Fax: (613) 533 6590

Dr. W.-T. Yen

Mining Engineering Department, Queens University,
Kingston, Ontario, Canada K7L 3N6
Tel: (613) 533 2206
Fax: (613) 533 6590

Dr. Richard K. Amankwah

University of Mines and Technology
P. O .Box 237, Tarkwa, Ghana
Tel: +233 243 826262

Dr. Juliana A. Ramsay

Chemical Engineering Department, Queens University,
Kingston, Ontario, Canada K7L 3N6
Tel: (613) 533 2770


J. K. Afidenyo, W.-T. Yen, R. K. Amankwah and J.A. Ramsay


Microbial pretreatment of a double refractory gold ore was investigated using a

white rot fungus, Coriolus versicolor (ATTC 20869) to improve gold extraction. A
series of preliminary tests were conducted to determine the amenability of the
ore to fungus degradation. Pulp density, temperature, pH and retention times
were the process variables considered. Both a single stage and two-step
processes were studied to establish process conditions. During the single stage
process, the ore was contacted with the fungal culture (biotic) for 3 weeks at
30oC and pH 10.0. For the two-step process, the first step involves ore contact
with the culture medium (abiotic) at 45oC and pH 10.0 followed by fungal contact
(biotic) at 30oC. Results indicated that carbonaceous matter was not degraded
but passivated by the fungus; as preg-robbing activities dropped significantly
from 18.1% to below 1.0%. The single stage process resulted in higher gold
recovery than the two-step process. Gold extraction by cyanidation of the
pretreated ore was 78.9% for the single stage compared with 15.0% for the
untreated ore. In the two-step process, only 73.3% gold extraction was achieved.
Application of the two step process conditions to a high gradehigh-grade flotation
concentrate, resulted in 93.3% gold extraction as against untreated concentrate
of 30.5%. The results showed that the microbial pretreatment process degraded
sulfide sulfur, passivated carbonaceous matter and enhanced gold extraction
from double refractory gold ore and concentrate.

Key words: Gold ores; Fungus; Biooxidation; Cyanidation; Preg-robbing

Gold ores and concentrates are classified as refractory when a significant
portiona sizable portion of the gold content cannot be extracted effectively by
conventional cyanidation, even after fine grinding. The refractory behavior of ores
is generally attributed to two main factors: the presence of carbonaceous matter
and the occurrence of sulfides, tellurides and cyanicides [Boyle, 1979; Guay,
1981]. When the refractoriness is due to the presence of sulfides and
carbonaceous matter, the ore or concentrate can be referred to as double
refractory [Nyavor and Egiebor, 1992].
The most important classes of carbonaceous matter are organic carbon
(hydrocarbons, humic acids and other organic substances) and graphitic or
amorphous elemental carbon [Radtke and Scheiner, 1970; Osseo-Asare et al.,
1984; Hausen and Bucknam, 1985; Stenebraten et al. 1999, 2000; Rees and Van
Deventer, 2000]. However, graphitic carbon is the main carbonaceous matter in
double refractory gold ores and poses serious recovery concerns during
leaching. The carbonaceous matter tend to adsorb gold from leach solutions, a
phenomenon known as preg-robbing and the sulfide minerals which may
occlude gold also limits the access of leaching reagents and thereby reducing

ThusThus, pretreatment methods which seek to eliminate or passivate the preg-

robber during gold dissolution [Henley, 1975; Osseo-Asare et al., 1984;
Demopoulos and Pangangelakis, 1987; Afenya, 1991; Linge, 1991] and to
decompose the mineral matrix to liberate gold will have to be employed. The
most common pretreatment processes used for double refractory sulfide ores
include roasting, chlorination, pressure oxidation and bacterial oxidation
[Arriagada and Osseo-Asare, 1984; Berezowsky and Weir, 1989; Marsden and
House, 1992]. However, these afore-mentioned pretreatment steps do not
oxidize or passivate carbonaceous matter significantly and thus continue to act
as preg-robbers in the subsequent gold recovery process. Oxidation or
passivation of the preg-robber is the focus of most research in Biomining in
recent times.

Microbial pretreatment of refractory gold using chemolithotrophic bacteria to

break down the sulfide matrix to liberate gold particles is becoming popular since
the advent of the Biox Technology in 1986. Several types of bacteria are used
in bio-mining but the prominent ones that are known to be involved in the
oxidation of sulfide ores include Acidithiobacillus ferrooxidans, Acidithiobacillus
thiooxidans and Leptospirillum ferrooxidans [Bierley and Brans, 1994; Bierley,
1995; Hackl, 1997; Rawlings, 1998]. However, carbonaceous materials are not
oxidized significantly by this pretreatment step and continue to serve as preg-
robbers in the subsequent gold leaching process.

Some investigations into the microbial degradation of carbonaceous matter have

been reported. A recent novel two stage approach to the degradation of sulfide
sulfur and carbonaceous matter in double refractory ore was investigated
(Amankwah and Yen, 2003). In this study, Streptomyces setonii, a coal degrading
bacteria, was used to degrade the carbonaceous matter after sulfide oxidation in
the first stage by employing the three well known chemolithotrophic biomining
bacteria. Gold recovery during cyanidation increased from 81.1% to 94.7% after
carbonaceous matter degradation.

A mixed regime of heterotrophic bacteria, many of which are from the

Pseudomonas family and naturally associated with gold ores, could deactivate
the active sites on carbonaceous components leading to increase in gold
extraction during cyanidation [Brierley and Kulpa, 1992; Kulpa and
Brierley ,Brierley, 1993]. Portier (1991) used other heterotrophic bacteria and
some fungi to degrade carbonaceous matter and reported increases in gold
recovery due to microbial action.

The use of fungi in the field of mineral processing has not been studied much
until recent times. Some recent research into the use of fungi include leaching
and precipitation of nickel and iron from laterites (Alibhai et al 1993),
solubilisation of manganese from ores (Baglin et al, 1992), leaching of lateritic
nickel ores (Bosecker ,Bosecker, 1985; Sukla et al, 1993), and mineral leaching
of non sulfide nickelferrous ores (Agatzini and Tzeferis, 1994). The fungal
strains used in all above investigation were Aspergillus sp and Penicilliun sp.
Results of the studies on nickel oxide ores (Bosecker, 1985; Tzeferis et al 1991)
have shown the amenability of this mineral to leaching by fungal strains. The
efficiency of leaching depended on the type of ore and the kind and
concentration of organic metabolite produced by the fungi (Bosecker, 1985;
Alibhai, et al 1993)

Most of the investigations have been in processing of non-auriferous ores.

ThusThus, this present investigation into the biodegradation of double refractory
gold ores by a versatile white-rot fungus presents a new challenge in the
application of fungi in mineral processing.

In this study, sulfide sulfur and carbon degrading abilities of a white rot fungus
was investigated for double refractory gold ore and concentrate with the aim of
improving gold extraction.

Both a single stage and two-step microbial pretreatment processes were used to
oxidize the sulfides and to passivate/oxidize carbonaceous matter in a double
refractory gold ore. During the single stage process, the ore was contacted with
the fungus culture (biotic) for 3 weeks at 30 oC and pH range, 9.5 -10.5 during
which carbonaceous matter passivation and sulfide oxidation occur
simultaneously. For the two steptwo-step process, the first step is abiotic and it
involves the use of the culture medium adjusted to an alkaline pH range of 9.5
10.5 for oxidation of the sulfides minerals; followed by fungal contact (biotic) at
30oC to further oxidize the sulfides and passivate the carbonaceous matter. The
single stage process conditions resulted in higher recovery. Results indicate that
sulfides were oxidized and carbonaceous matter was partially degraded and
passivated at the same time; as preg-robbing activities dropped significantly gold
extraction in the subsequent cyanidation step increased.


Ore Characterization

Ore samples were sourced from plants which process double refractory gold ore.
The ore was milled to 95% with particle size less than 75 um. Sulfide sulfur and
carbonaceous matter in the ore sample were determined using the combustion
and volumetric method. A Leco titrator, SC-444DR was employed for the
analysis. The conventional fire assay method was used to determine the grade of
gold. The main sulfide minerals present is pyrite. The grade of the main
components in the ores is indicated in TABLE 1:

TABLE1: Partial chemical analysis of the ore samples used

Component Grade
Whole OreWhole Ore Flotation Concentrate
Total Sulfide sulfur (%) 3.80 8.95
Total Carbon (%) 0.63 6.84
Carbonate (%) 0.038 1.56
Elemental carbon (%) 0.592 5.28
Gold (g/t) 2.33 65.75

Microorganisms and inoculum preparation

The fungal strain used for the studies was Coriolus versicolor (ATTC 20869)
which was maintained at 4 oC on plates containing Kirk's medium, 1.5% agar and
3% malt extract. Several agaragar plugs of the actively growing fungus were
used to inoculate 500 ml of Kirk's medium in 1000 ml Erlenmeyer flasks. The
medium contained 10 g/l glucose, 1.2 g/l ammonium tartrate, 0.05 g/l MnSO 4 .
7H2O, 0.01 g/l CaCl2 .2H2O, 1 mg/l thiamin, 1 ml/l of trace minerals (Kirk et al.
1978). The medium was also supplemented 2.92 g/l 2, 2-dimethylsuccinate. The
trace mineral solution contained 30 g/l MnSO 4. 7H2O, 10 g/l NaCl, 5g/l
MnSO4.H2O, 1 g/l CoSO4, 1 g/l FeSO4 .7H2O, 1 g/l ZnSO4 .7H2O, 0.82 g/l CaCl2,
0.1g/l CuSO4 .5H2O, 0.1 g/l NaMoO4. 2H2O, 0.1 g/l H3BO3 and 1 g/l EDTA.
Shake-flask cultures were grown at 30 oC with continuous agitation (180 rpm) for
10 14 days.

Microbial- Medium Ore interactions

A series of single stage preliminary tests (biotic) during which the whole ore was
contacted with the fungal culture were conducted to investigate the amenability to
fungal sulfide sulfur and carbon degradation. The first batch of test was
conducted at the natural pH (unadjusted) of the fungal-medium ore system at
pulp density of 10% for 2 weeks. Further investigations at various adjusted pH
values of 3.5, 4.5, 7.0 and 10.0 were conducted. Other process variables
investigated were pulp density, retention time and temperature at the established
optimum pH of 10.0.

Control tests (abiotic) to investigate the effect of the culture medium on the ore at
the established conditions were also studied. The fungus Coriolus versicolor is
reported to thrive optimally at temperatures of 28 to 30 oC.

For the single stage process, the sulfide ore was contacted with the fungal
medium in 2000 liters Erlenmeyer flasks for 2 weeks at 20% pulp density and
temperatures of 30oC at alkaline conditions. The flask was agitated continuously
on an orbital shaker at 180 rpm. The initial pH of 4.5 to 5.0 was adjusted to
between 9.5 and 10.5 and maintained daily with 10 M sodium hydroxide solution.
The oxidized product of the biotic leaching was filtered and solids washed
several times with water to get rid of the alkali, dried and subjected to gold
extraction by cyanidation. Duplicate experiments were run and the difference in
values was within 1-2%.

For the two steptwo-step process, the ore was first contacted with the culture
medium (abiotic) in 2000 liters Erlenmeyer flasks for 2 weeks at a pulp density of
20% and temperatures of 45oC at alkaline conditions. The product of the abiotic
leaching was filtered and repulped to 20% solids with the fungal culture in
Erlenmeyer flasks and agitated continuously on an orbital shaker at 180 rpm for a
further 7 days. Daily monitored pH for the second step was between 5 and 6. At
the end of the biotic contact time, the ore samples were filtered, washed and

Samples of bioleach filtrate for both pretreatment processes were taken for gold
analysis by atomic absorption spectrometer

Post - fungal treatment investigations

Determination of changes in carbonaceous matter content

Percentage reduction in carbonaceous matter and sulfide were determined

quantitatively using the Leco volumetric combustion technique. To eliminate the
interference of the fungal biomass (organic carbon) from the pretreated ore
analysis, a sample of the product was contacted with 5-6% hypochlorite for 1
hour as described by Berger et al (1989) and Ramsay et al (1990) and
thoroughly rinsed with water.
Evaluation of gold extraction after microbial degradation

Preg-robbing and cyanidation tests were used to evaluate the gold extraction
properties of products obtained from the microbial pretreatment processes. All
pretreated samples were washed with water and dried before evaluation.

Preg-robbing tests were conducted on both pretreated and untreated ore

samples. 3 g samples were placed in 27 ml solution of potassium gold cyanide
containing 5.0 mg Au/l in 50-ml flasks. The pH was kept at 10.5 and free cyanide
concentration of 0.5 g/l. Gold dissolution was not observed in the course of the
preg-robbing tests. The samples were agitated at 75 rpm for 24 hours. The ore
was then separated from the solution by filtration and the final solution gold
determined using an atomic absorption spectrophotometer. The difference in gold
concentration before and after the solution-ore contact is the gold preg-robbing

Cyanide leaching was used to determine the effect of fungal pretreatment on

gold extraction. Cyanidation was conducted on 75 to 100 g each of ore after both
carbonaceous matter degradation/passivation and sulphur sulphide oxidation
Samples were leached at a pulp density of 33% by weight for 24 hours at pH
10.5 11.0. The pH was adjusted using industrial grade calcium oxide (lime) and
cyanide strength was 1.0 g/l. The dissolved gold concentration was determined
using atomic absorption and the solid residual gold was determined by
conventional fire assay method followed by atomic absorption analysis.


Biooxidation of sulfides

Biooxidation of refractory gold ore sulfides using the fungal pretreatment is novel.
The motivation for this research into the microbial pretreatment of a double
refractory gold ore stems from the abilities of a versatile fungus Trametes
versicolor whose strains, Polyporius and Coriolus versicolor are known to
degrade coal by the generation of a regime of mixed oxidative enzymes including
manganese peroxidase and lignin peroxidase (Kirk and Farrel,
1987).Managanese peroxidase and lignin peroxidase have been known to
produce strong oxidants for the oxidation of coal and other carbon bearing matter
in wood. Peroxidases require H2O2 to complete their catalytic cycle degrading
activities. Extracellular H2O2-generating enzymes are secreted simultaneously
with lignin modifying enzymes. Hydrogen peroxide has also been reported to
have the ability to deactivate active sites on carbonaceous matter [Nyavor and
Egiebor, 1992].

In this study, the two-step process achieved a sulfide oxidation 76.3% for the
2 week abiotic step compared with 54.1% for 2 weeks biotic treatment at 30 oC.
Sulfide oxidation may be enhanced by the presence of hydroxyl ions in solution
and the increase in sulfide oxidation during the 45 oC abiotic step may be due to
improved kinetics. Generally, sulfide oxidation for the abiotic test was higher than
the biotic step as indicated in the FIG 1. This may be due to build up of
metabolites on the sulfide surfaces during the biotic process. Analysis of the
filtrate after both biotic and abiotic steps at alkaline conditions revealed that some
gold had been leached into solution. The gold dissolution can be attributed to
formation of thiosulfate due to the alkaline conditions as indicated by equation 1
for arsenopyrite oxidation.

6FeAsS +1302 +22 NaOH = 6Fe (OH) 3 +2Na3AsO3S +4Na3AsO4 +2Na2S2O3 +2H2O (1)

Thiosulfate has been confirmed to be the dominant product of pyrite oxidation

and from the results, the gold present in the samples dissolved simultaneously
during alkaline oxidation of the sulfides. The mechanism of gold solubilization
appears to be associated with the in situin-situ formation of thiosulfate as a

FIG 1: Sulfide sulfur oxidation for abiotic and biotic process at alkaline conditions
and pulp density 10% solids at 30oC

Fungal action on carbonaceous matter

The components of carbonaceous matter associated with gold ores include

hydrocarbons, humic acids and elemental carbon. Maturity of the elemental
carbon fraction ranges from high rank lignite to anthracite and the distribution of
these components vary from one deposit to another (Radtke and Scheiner, 1970;
Osseo-Asare et al., 1984; Hausen and Bucknam, 1984; Stenebraten et al., 1999,
2000). Analysis of samples from a deposit similar to those used in this study
revealed the presence of hydrocarbons, humic acid and elemental carbon
(Afenya, 1976; Osseo-Asare et al., 1984).

In this investigation, no clear trend was observed for carbon degradation for both
the abiotic and biotic steps as data was rather erratic. However, preg-robbing
trends were clearly defined at the pHs investigated. There was no appreciable
change in carbonaceous matter content after the fungal-medium contact with ore
but preg-robbing activity reduced drastically showing that there was passivation
of carbonaceous matter. Preg-robbing activity dropped considerably by 82.5%
after the abiotic process and 98-99% reduction was achieved after fungal
contact. The culture medium itself had some carbon passivating abilities which is
worth investigating. Passivation in the presence of fungus may be attributed to
enzymatic secretions by the fungus.

Effects of process variables on sulfide oxidation and carbon passivation

Effect of Pulp Density

Sulfide sulfur oxidation was studied at various pulp densities between 5% and
50% solids for the biotic process at alkaline condition and 30 oC for 2 weeks. The
changes in sulfide oxidation with increasing pulp density were not very significant
as all were in the range 48% to 44% as density increased from 5% to 50%. This
indicates that pulp density is not a major a variable in the biotreatment of sulfides
using Coriolus versicolor.

Carbon degradation trends were not well defined and was characterised by
fluctuations. Sorption of gold was remarkably low at 0.7% at 5% solids. It was
equally low for all pulp densities used and even after pretreatment at 50% solids
pregrobbing was 6.5%. Increasing preg-robbing with percent solids might be due
to high attrition rate and thereby hindering the passivating effect of the fungal
metabolites. It was observed that preg-robbing values did not correlate with
undegraded carbonaceous matter.

Effect of pH

The effect of pH was investigated for unadjusted (natural), 3.5, 4.5 and 10.5 for
both abiotic and biotic process for 2 weeks at a temperature of 30 oC. Sulfide
oxidation for both processes followed the same trend but values for the abiotic
were slightly higher than the biotic. The alkaline pH gave the overall best sulfide
degradation followed by the unadjusted (averaged 4.3), 4.5 and 3.5 in that order.
The lower sulfur sulfide oxidation by the culture alone may be due to coating of
sulfide mineral surfaces by the metabolites produced by the fungus. The higher
oxidation at the alkaline conditions may be attributed to the presence of hydroxyl
ions in solution.
Carbon degradation was erratic but generally values were higher than the
original ore due to introduction organic carbon by the fungi biomass and culture
medium. However, preg-robbing trends were clear. GenerallyGenerally, preg-
robbing data for the biotic were better than the abiotic as shown in FIG 2. This
indicates that there is microbial involvement in the carbon passivation process.

FIG 2: Sorption of gold by ore sample after microbial pre-treatment at different

pH values

Effect of Contact Time

Sulfide oxidation increased with retention time. About 45% sulfide oxidation was
achieved after 2 weeks and 73% after 9 weeks during the preliminary studies.
The rate however, slowed down as the retention time increased. Since a batch
culture was used, the decrease in oxidation rate can be attributed to reduction in
microbial activity due to exhaustion of growth media.

Passivation of carbonaceous matter was higher at shorter contact times.

ThusThus, as the contact time increased, passivation decreased and the ability
of the biotreated material to preg-rob increased. Since pasivation is a surface
phenomenon, it is possible that the continuous stirring and subsequent increase
in attrition led to a reduction in passivating effect of the fungal metabolites over
the period of time.

Effect of Temperature

Temperature effect on sulfide oxidation was studied at alkaline pH for both biotic
and abiotic process at room temperature (26 oC), 30 and 45oC. Sulfide oxidation
tends to increase with temperature. 76.7% sulfide oxidation was achieved for
abiotic process at 45oC as against 44.74% and 50.0% at 26 oC and 30oC
respectively. Biotic experiments achieved 42.7% and 45.8% sulfide oxidation at
room temperature and 30oC respectively. The increasing trend can be attributed
to increased kinetics. These results were pivotal in investigating the two steptwo-
step process of abiotic at 45oC followed by biotic at 30oC.

Effect of fungal action on gold extraction

Preg-robbing activity dropped from 18.1% for the untreated ore to below 1.0% for
both the 3 weeksweeks single stage and the two steps pre-treatment process of
2 weeks abiotic at 45oC and 7days biotic at 30oC. When the single stage
pretreated whole ore was subjected to cyanidation, gold extraction after 24 hours
was 78.9% compared with 73.3% for the two-step process. The single stage pre-
treatment process thus gave better results than the two steps process. Gold
extraction for the untreated ore was however, 15% (FIG 3).

When the two steptwo-step pre-treatment process was applied to a high

gradehigh-grade flotation concentrate, 93.3% gold dissolution was achieved
during cyanidation as against the untreated concentrate of 30.5%. The increase
in the gold extraction during cyanidation is due to improved sulfide oxidation and
reduction in preg-robbing activity by carbonaceous matter passivation and
degradation. The single stage process for the flotation concentrate is being

FIG 3: Gold extraction from pretreated samples. Samples 1-4 are ores while 5
and 6 are concentrates; (1) Untreated ore, (2) 14 days biotic at 30 oC, (3) 14 days
biotic at 30oC (4) 2 weeks abiotic at 45oC + 1 week biotic at 30oC, (5) untreated
concentrate, (6) 2 weeks abiotic at 45oC + 1 week biotic at 30oC. All experiments
were conducted at pH 10.0


This novel approach to extracting gold from ore or concentrate can be

implemented via the following flow sequences:

Single stage:

- Biotic carbonaceous matter passivation and sulfide sulfur oxidation at 30 oC

- Filtering of oxidized/passivated product
- Recovery of gold from filtrate by adsorption.
- Repulping and thickening of solid residue.
- Cyanidation and gold recovery from solid residue


- Abiotic sulfide sulfur oxidation carbon degradation at 45 oC

- Filtering of oxidized product and recovery of gold from filtrate by adsorption.
- Washing and thickening of solid residues
- Passivaton of carbonaceous matter by using fungi Coriolus versicolor
- Washing of product and safe disposal of effluent
- Cyanidation and gold recovery from solid product

The result of this study is a new technical process for recovering gold from
double refractory gold ores. This novel process is a potential for pretreating
double refractory gold ores. The fungal passivation of carbonaceous matter can
be used to complement the traditional processes like pressure oxidation and
bacterial leaching where the carbonaceous matter is not oxidized significantly.


It could be concluded from the above results and discussion that both the single
and theand the two steptwo-step microbial pre-treatment process for the
treatment of double refractory gold ores and concentrates led to a substantial
increase in gold extraction. The fungus, Coriolus versicolor passivated
carbonaceous matter in the double refractory gold ore while alkaline medium
enhanced sulfide sulfur oxidation significantly. The single stage process will be a
preferred choice due to its simplicity.
Further investigations into this novel pretreatment option is in progress and
results may alter the processing proposal outlined above.

Gratefully acknowledge funding of the research by National Sciences and
Engineering Research Council of Canada (NSERC), Barrick Gold Corporation
and AngloGold Ashanti (Ghana) Limited. We are also grateful to Mr Paul
Philippe-Champagne, a PhD candidate of the Biochemical Engineering Research
group, Queens University for his guidance and advice during the research

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Ore Characterization

Microorganisms and inoculums preparations

Microbial Mineral interactions

Post-bacterial pretreatment investigations

Determination of changes in ore carbonaceous matter content

Evaluation of gold extraction after microbial pretreatment


Biooxidation of sulfides

Fungal action on carbonaceous matter

Carbon analysis before and after hypochlorite digestion of biomass

Effects of Process Variables on sulfide and carbon degradation

Effect of pH
Effect of pulp density
Effects of contact time
Effects of temperature

Effect of fungal action on preg-robbing and gold extraction





Table captions

Partial Chemical analysis of the ore samples used

Component Grade
Ore Concentrate
Total Sulfide sulfur (%) 3.80 8.89
Total Carbon (%) 0.63 6.84
Carbonate (%) 0.138 5.38
Elemental carbon (%) 0.592 1.28
Gold(g/t) 2.33 65.75

Figure Captions

FIG 1: Sulfide sulfur oxidation for abiotic and biotic process at alkaline conditions
and pulp density 10% solids
FIG 2: Sorption of gold by ore sample after microbial pre-treatment at different
pH values

FIG 3: Gold extraction from untreated and pretreated samples.

Samples 1-4 are ores while 5 and 6 are concentrates; (1) Untreated ore, (2) 14
days biotic at 30oC, (3) 14 days biotic at 30 oC (4) 2 weeks abiotic at 45 oC + 1
week biotic at 30oC, (5) untreated concentrate, (6) 2 weeks abiotic at 45 oC + 1
week biotic at 30oC. All experiments were conducted at pH 10.0