Enzyme and Microbial Technology 40 (2006) 107–113

Removal of hexavalent chromium by Trichoderma viride

in an airlift bioreactor
Liliana Morales-Barrera, Eliseo Cristiani-Urbina ∗
Departamento de Ingenierı́a Bioquı́mica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala,
“Centro Operativo Naranjo”, Apdo. Postal C.O.N.—256, Jaime Torres Bodet No. 142, Col. Santa Marı́a la Rivera,
México D.F. C.P. 06401, Mexico
Received 18 March 2005; received in revised form 14 October 2005; accepted 17 October 2005

Abstract
Trichoderma viride was batch-cultivated in a stirred tank bioreactor and in a concentric tube airlift bioreactor in order to determine its capacity
to remove hexavalent chromium [Cr(VI)] from aqueous solutions. The agitation rate (120 rpm) used during the stirred tank bioreactor cultivation
caused mycelial fragmentation, and had a negative effect on the cell growth and Cr(VI) removal. The physical damage inflicted by the agitation rate
was, presumably, sufficient to cause fragmentation of mycelia and as a result, diminish Cr(VI) removal. Compared with stirred tanks, pneumatic
bioreactors are considered more suitable systems for the cultivation of shear sensitive cells, and hence further studies were carried out in a concentric
tube airlift bioreactor. In this type of reactor, the strain exhibited an extraordinary capability to remove high concentrations of the highly toxic
and soluble hexavalent chromium. Therefore, the airlift bioreactor might represent an attractive technological alternative for the treatment of
wastewaters containing high Cr(VI) concentrations, using the T. viride strain. To our knowledge, this is the first report on Cr(VI) removal by a
microbial culture in a pneumatically agitated bioreactor.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Hexavalent chromium; Trichoderma viride; Airlift bioreactor

1. Introduction cessing, etc. [7]. Improper handling and storage of chromium-

Chromium exists in nine valence states ranging from −2 to
+6. However, only hexavalent chromium [Cr(VI)] and trivalent
chromium [Cr(III)] are ecologically important because they are
the most stable oxidation forms in the natural environment [1,2].
Cr(VI) is known to be highly toxic, mutagenic and carcinogenic
to living organisms including mammals. Cr(III), on the other
hand, is more stable and is approximately 100 times less toxic
and 1000 times less mutagenic than Cr(VI) [3,4]. Furthermore,
Cr(III) is an essential trace element necessary for glucose, lipid
and aminoacid metabolism [4], as well as a popular dietary sup-
plement [5].
Hexavalent chromium is the major chromium species used
in industrial processes [6], including chrome plating, petroleum
refining, manufacture of pigments, leather tanning, wood pro-
∗ Corresponding author. Tel.: +52 55 57 29 63 00x62454;
fax: +52 55 53 96 35 03.
E-mail address: ecristia@encb.ipn.mx (E. Cristiani-Urbina).
laden effluents or wastes has led to Cr(VI) contamination of
surface water, groundwater, soil and sediment [8,9]. Focusing
on its toxicity and exposure potential, the United States Environ-
mental Protection Agency (USEPA) lists hexavalent chromium
as a priority pollutant [10,11].
Conventional methods for the treatment of Cr(VI)-
contaminated wastewaters include chemical reduction followed
by precipitation, ion exchange and adsorption on coal, acti-
vated carbon, alum, kaolinite and flyash [12]. Nevertheless, these
methods have several disadvantages, such as high operating
costs, the requirement of preliminary treatment steps, the diffi-
culty of treating the solid waste subsequently generated, and the
requirement of large quantities of chemical adsorbents [12,13].
In recent years, there has been an increasing researcher inter-
est in microorganisms that are able to transform the highly toxic
and water-soluble hexavalent chromium to the less toxic and
insoluble trivalent chromium. A wide variety of bacterial pure
and mixed cultures have been reported to be capable of reducing
Cr(VI), under aerobic and/or anaerobic conditions [1–3,12–22].
However, little information about the capability of filamentous

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.10.044
108 L. Morales-Barrera, E. Cristiani-Urbina / Enzyme and Microbial Technology 40 (2006) 107–113

fungi [23] and yeasts [7,8] to reduce Cr(VI) to Cr(III) is found

in the available literature. Recently, a newly Cr(VI)-reducing
fungal strain, identified as Trichoderma viride, was isolated and
studied in our laboratory for its capability to remove Cr(VI). The
fungus showed a remarkable ability to remove high concentra-
tions of Cr(VI) when grown in flasks containing glucose as the
sole carbon and energy source, under aerobic conditions.
The main purpose of this work was to evaluate the Cr(VI)
removal potential of T. viride in sparged stirred tank and airlift
bioreactors.

2. Material and methods

2.1. Microorganism

A fungal strain that had been isolated from tannery waste and identified as T.
viride was used throughout this work. This strain was obtained from the Culture
Collection of the Biochemical Engineering Department of the National School
of Biological Sciences, National Polytechnic Institute (Mexico City, Mexico).
The fungal strain was maintained on malt extract agar and routinely subcultured
every 4 weeks. Inoculated plates were incubated at 30 ◦ C for 4–6 days and then
stored at 4 ◦ C.

2.2. Culture media

Growth liquid medium contained, per liter of distilled water, 10 g of glucose,

3 g of (NH4 )2 SO4 , 1 g of KH2 PO4 , 0.3 g of MgSO4 ·7H2 O, 0.1 g of KCl, 0.1 g
of yeast extract, 0.05 g of CaCl2 , and 1 mg of FeCl3 . After sterilization, a small
volume of 20 g/l K2 CrO4 solution was added in order to obtain a particular initial
Cr(VI) concentration. Stock Cr(VI) solution was prepared by dissolving 20 g of
99.99% K2 CrO4 in about 250 ml of distilled water and diluting to 1 l. The initial
pH of the culture media was 6.0 ± 0.1.

2.3. Inoculum preparation

Mycelial biomass was produced in 1000 ml Erlenmeyer flasks containing

200 ml of the aforementioned culture medium with an initial Cr(VI) concentra-
tion of 1 mM. The flasks were shaken continuously at 54 rpm, at 30 ◦ C. Mycelia
of T. viride were harvested in the late exponential phase of growth by filtration
through nylon cloth and successively washed with the culture medium without
Cr(VI). The biomass was re-suspended in a small volume of the culture medium
without Cr(VI) and the resulting suspension was homogenized. A sample of the
biomass suspension was used to inoculate the bioreactors used for the Cr(VI)
removal experiments.

2.4. Culture conditions

Cultivations of T. viride were performed in a magnetically stirred fermenter

and in a concentric tube airlift bioreactor. The stirred tank reactor was made
of PyrexTM glass and had a working volume of 1.4 l; its internal diameter and
total height was 11.5 and 22 cm, respectively. This reactor was aerated through
a porous glass diffuser with a pore size of 30–40 ␮m. The airflow rate was set
at 5.0–5.3 l/min and the rotation speed of the stirrer at 120 rpm.
The airlift bioreactor used in this work was of the draft-tube internal-loop
type, with a working volume of 1.4 l. A scheme of the reactor is shown in Fig. 1
and their dimensions are given in Table 1. The lid, base, external tube and inner
tube of the bioreactor were made of PyrexTM glass. The lid had ports for the
addition of the inoculant, acid and alkali, for the supply of nutrients and to
introduce oxygen and pH electrodes. The external tube also had ports through
which samples could be taken. The airlift bioreactor was sparged in the draft
tube by means of a porous glass diffuser with a 7.2 cm diameter and a pore size
of 30–40 ␮m.
The external tube was joined to the lid and base by stainless steel screws that
Fig. 1. Schematic view of the concentric tube airlift bioreactor.
connected segmented Nylamid flanges, so that the glass lips of the external tube
 L. Morales-Barrera, E. Cristiani-Urbina / Enzyme and Microbial Technology 40 (2006) 107–113
Table 1
Geometrical characteristics of the concentric tube airlift bioreactor
Dimension (symbol) Value (cm)
External tube height (HC ) 27.0
External tube diameter (DC ) 9.5
Inner tube height (HR ) 12.0
Inner tube diameter (DR ) 7.8
Bottom clearance (d1 ) 1.5
Top clearance (d2 ) 6.25
were fastened between the lid or the base and the flange. The internal surfaces
of the flanges were grooved to accommodate sterilizable silicon rubber O-rings.
The input air was controlled at 4 kg/cm2 through a pressure-regulating
valve and the gas flow rate was measured with a rotameter. An airflow rate
of 5.0–5.3 l/min was used. The superficial gas velocity referred to the riser
cross-sectional area was of about 0.017–0.018 m/s. Under this condition, the
volumetric oxygen transfer coefficient (KLa ) of the reactor was about 310 h−1 .
The cross-sectional areas of the riser and downcomer were 47.78 and
23.1 cm2 , respectively. Thus, the riser-to-downcomer cross-sectional area ratio
was 2.07. Before using it, the bioreactor was sterilized by autoclaving at 121 ◦ C
for 20 min.
Cr(VI) removal experiments were carried out in the stirred tank and airlift
bioreactors at room temperature (20 ± 2 ◦ C); the pH was not controlled dur-
ing the experiments. The initial biomass concentration of all the batch cultures
carried out in both bioreactors was of approximately 1 mg/ml.
Experiments without biomass and with heat-killed biomass were conducted
under the same conditions in order to estimate Cr(VI) loss in the absence of living
biomass. The heat-killed controls were autoclaved twice at 121 ◦ C for 20 min.
The biomass-free controls were used to detect possible abiotic Cr(VI) reduction
brought about by medium components. In the autoclaved controls, any loss in
the Cr(VI) content would be attributable to Cr(VI) reduction and/or adsorption
on fungal biomass. Measurements of Cr(VI) and total chromium concentrations
at the outset and at regular intervals throughout the test period were used to
establish any loss of Cr(VI) due to transformation of Cr(VI) to Cr(III) and/or
adsorption on microbial biomass.
2.5. Analytical techniques
2.5.1. Cell concentration
The cell concentration was estimated by dry cell measurements. The cul-
ture samples were filtered through preweighed 1.2 ␮m filters (Whatman GF/A),
washed twice with sterile distilled water and dried at 95 ◦ C to reach a constant
weight. The filtrates obtained were used to determine the Cr(VI), total chromium
and glucose concentrations.
2.5.2. Glucose concentration
The glucose concentration of the culture samples was estimated enzymati-
cally using a glucose assay kit from Sigma.
2.5.3. Cr(VI) and total chromium concentrations
Cr(VI) and total chromium concentrations were determined by colorimetric
methods using a Bausch and Lomb spectrophotometer, following the proce-
dures described in the Hach Water Analysis Handbook 2002 [24]. Cr(VI) in
the filtrates was determined by the 1,5-diphenylcarbohydrazide method using a
single dry powder formulation called ChromaVer3TM Chromium Reagent. This
reagent contains an acidic buffer combined with 1,5-diphenylcarbohydrazide
which reacts to give a purple color when hexavalent chromium is present. Color
development was directly proportional to the amount of hexavalent chromium
present.
In the analysis for total chromium, the samples were heated to the boiling
point under strong alkaline conditions in the presence of hypobromite. Under
these conditions, trivalent chromium in the samples was oxidized to the hex-
avalent form. After the oxidation was complete, total chromium content was
determined by the 1,5-diphenylcarbohydrazide method. Test results were mea-
sured at 540 nm.
109
Cr(VI) and total chromium concentrations were proportional to their
absorbance, and were quantified by use of external standards with a 10-point
calibration curve. Sterile distilled water was used as the diluent.
3. Results and discussion
3.1. Cr(VI) removal by T. viride in flasks and in a stirred
tank bioreactor
When T. viride was cultivated in unbaffled flasks contain-
ing culture medium with initial Cr(VI) concentrations of 1, 1.5
and 2.0 mM, it was capable of growing and removing almost
completely the Cr(VI) present in the medium. However, at
higher initial Cr(VI) concentrations, the fungus neither grew nor
removed measurable amounts of Cr(VI). The cell mass increase
and the Cr(VI) removal efficiency achieved in flasks at initial
Cr(VI) concentrations ranging from 1 to 2 mM were of about
2.8–3.2 g/l and 97–100%, respectively. In contrast, lower values
of cell mass increase (1.4 g/l) and of Cr(VI) removal efficiency
(60%) were attained in the stirred tank bioreactor, when an initial
Cr(VI) concentration of 1.5 mM and an agitation rate of 120 rpm
were used.
During the experiments in the stirred tank bioreactor, it was
observed that the fungus mycelia length was shorter than that
commonly found in flasks. These observations suggest that
the agitation rate used during the bioreactor cultivation caused
mycelial fragmentation, and this may have been due to the shear
stress created by the magnetic stirrer. It would be reasonable to
postulate that the physical damage inflicted by the shear forces
was sufficient to cause fragmentation of mycelia, and as a con-
sequence, to diminish the Cr(VI) removal capacity of the fungal
strain. It is well known that shear fields cause physical damage
to mycelial organisms and to filamentous bacteria [25], and have
a strong influence on microbial morphology, which controls the
performance of a microbial culture [25,26].
The results obtained in the stirred tank bioreactor suggest that
the fungal strain used in this study is very sensitive to shear stress
and therefore, it would be important to optimize the agitation rate
in order to minimize physical damage and increase the efficiency
of Cr(VI) removal.
Concentric tube airlift bioreactors are frequently used in aer-
obic fermentation processes and have some advantages over the
mechanically agitated reactors. The main advantages are the
ease of construction and maintenance, no mechanical moving
parts, low power input, and besides they provide a hydrodynamic
environment suitable for fragile biocatalysts that are susceptible
to physical damage by fluid turbulence or mechanical agita-
tion [25,26]. Due to the above-mentioned advantages over the
mechanically agitated reactors, the concentric tube airlift biore-
actors represent a more attractive technological alternative for
the biological treatment of wastewaters than the conventional
activated sludge systems and sparged stirred reactors [25], and
there are currently numerous successful industrial applications
of these bioreactors [26]. Taking into account the above facts,
further studies on Cr(VI) removal by T. viride were performed
in a concentric tube airlift bioreactor.
110 L. Morales-Barrera, E. Cristiani-Urbina / Enzyme and Microbial Technology 40 (2006) 107–113
3.2. Cr(VI) removal by T. viride in an airlift bioreactor
Batch cultures of T. viride were carried out in an airlift
bioreactor, using initial Cr(VI) concentrations of 1.3, 1.6, and
1.94 mM. The fungal strain was able to grow at all the initial
Cr(VI) concentrations tested and formed big and heavy masses
of aggregated mycelium, which led to an heterogeneous biomass
concentration in the airlift bioreactor. For this reason, it was not
possible to measure appropriately the fungus growth. The above
results suggest that the hydrodynamic conditions in the airlift
bioreactor affected the growth pattern of T. viride, resulting in
the formation of big mycelial aggregates. It has been reported
that cultivation conditions affect the morphological and physico-
chemical characteristics of fungal hyphal elements and thereby
their tendency to aggregate [27–29].
There was no significant inhibition of glucose consumption
by growing cells of T. viride in the presence of 1.3–1.94 mM
Cr(VI), and as a result, the overall efficiency of glucose con-
sumption was 100%. These results are in agreement with those
found in unbaffled flasks (data not shown).
During the Cr(VI) removal experiments carried out in the air-
lift bioreactor, it was observed that although residual Cr(VI) con-
centration decreased as incubation progressed, total chromium
in solution remained almost constant. These results suggest that
the fungus was capable of transforming the highly toxic and
soluble hexavalent chromium to the much less toxic and less
mobile trivalent form, and that the cells did not adsorb measur-
able amounts of Cr(VI).
Cr(VI) removal in our experiments was due to viable T. viride
cells because no Cr(VI) was removed by heat-killed cells or
in the absence of cells, nor was a measurable change in total
chromium concentration under those conditions. Moreover, no
Cr(VI) removal occurred in the experiments with viable cells
in the absence of an electron donor (culture medium with-
out glucose and yeast extract). The above observations suggest
that neither adsorptive removal of Cr(VI) by intact cells nor
Cr(VI) reduction by medium components were significant in
these experiments.
Fig. 2 shows the Cr(VI) concentration profiles for the dif-
ferent initial Cr(VI) concentrations tested. A complete removal
Fig. 2. Residual Cr(VI) concentration–time curves for the batch cultures of T.
viride.
of 1.3 and 1.6 mM Cr(VI) by T. viride was observed within
30 and 80 h of incubation, respectively. In contrast, a complete
Cr(VI) removal was not observed after more than 160 h of incu-
bation when an initial Cr(VI) concentration of 1.94 mM was
used; however, a very high overall efficiency of Cr(VI) removal
was achieved (94.3%).
The concept of finite Cr(VI) reduction capacity has been
used to quantitatively characterize the toxicity effect of Cr(VI)
on Cr(VI) reduction by microorganisms in batch cultures. The
finite capacity indicates the maximum amount of Cr(VI) that a
batch culture can reduce, and the loss of Cr(VI) reduction capac-
ity in microbial cells may be attributed to the mutagenic and
toxic effects of Cr(VI) [30]. Based on Cr(VI) reduction experi-
ments performed at a range of Cr(VI) concentrations, Wang and
Shen [30] observed that the batch cultures of Bacillus sp., Pseu-
domonas fluorescens LB300, and Escherichia coli ATCC 33456
reduced Cr(VI) at rates which decreased progressively with the
amount of Cr(VI) reduced and that the Cr(VI) reduction ceased
whenever the maximum capacity (0.4–1.25 mM) was reached. A
similar behavior was observed in the present work, which indi-
cates that the Cr(VI) reduction capacity of T. viride was reached
at the highest Cr(VI) concentration tested.
As shown in Fig. 2, the time required for complete reduction
of Cr(VI) increased as the initial Cr(VI) concentration increased.
Similar observations were previously reported for cultures of
Enterobacter cloacae [21], E. coli [31], P. fluorescens [32],
Bacillus sp. [32], and activated sludge [33].
For comparison purposes, Table 2 shows a summary of
reported data on Cr(VI) reduction by microorganisms in batch
experiments. It can be seen that the highest concentration
of Cr(VI) that was removed by T. viride in our experiments
was greater than those commonly found with pure cultures of
microorganisms, as well as for a number of microbial consortia.
However, it is important to take into account that the microbial
chromate resistance and chromate-removal parameters are cor-
related with the medium composition and the cell density [4,7].
As it was mentioned before, the biomass concentration could
not be measured appropriately, and hence, it was not possible
to report the specific Cr(VI) removal rate. However, from the
point of view of comparing the rate of Cr(VI) removal in an
airlift bioreactor to its performance, volumetric rates are more
meaningful than specific rates.
The volumetric rate of Cr(VI) removal was dependent on
the initial concentration of Cr(VI) (Fig. 3). The Cr(VI) removal
rate decreased with increasing the initial Cr(VI) concentration,
which might have been due to toxicity effects. A similar result
has been previously reported for Phanerochaete chrysosporium
[23]. Several authors have also informed that after an optimum
Cr(VI) concentration, the average Cr(VI) reduction rate began
to decrease and eventually microorganisms lost the capability
of reducing Cr(VI) [3,47]. It is believed that Cr(VI) is toxic to
microorganisms due to its ability to induce several cellular stress
responses as well as to damage different cellular components,
such as membranes, proteins, and nucleic acids [48,49].
The results obtained in this work show convincingly that T.
viride has a remarkable capacity to remove Cr(VI) when cul-
tivated in a concentric tube airlift bioreactor. This finding is
 L. Morales-Barrera, E. Cristiani-Urbina / Enzyme and Microbial Technology 40 (2006) 107–113 111

Table 2
Summary of reported data on Cr(VI) reduction by microorganisms
Organism Cr(VI) concentration [mM] Cr(VI) reduction efficiency [%] Reference

Acinetobacter lwoffii 1 13.2–36.9 [2]

Acinetobacter sp. 1 24.7 [2]
Activated sludge 0.096 100 [34]
Aureobacterium esteroaromaticum 1 22.3–33.1 [2]
Bacillus subtilis 0.1–0.5 100 [12]
1–2 0
Bacterial consortium 0.048 84 [1]
Candida maltosa 1.15 85 [8]
Cellulomonas sp. 0.2 99.5 [35]
Deinococcus radiodurans R1 0.5 100 [36]
Desulfovibrio desulfuricans 0.5 86–96 [37]
Enrichment consortium 0.6 98.5 [14]
0.04–0.38 100 [3]
0.96 60
Enterobacter cloacae HO-1 0.25 100 [38]
0.5 75
1.5 13
Escherichia coli ATCC 33456 0.38 100 [31]
1.5 75
3.07 38.5
Hydrogenophaga pseudoflava 1 24.8 [2]
Microbacterium liquefaciens MP30 0.1 100 [19]
Microbacterium sp. 0.1 100 [18]
0.5 40
Ochrobactrum anthropi 1 25.6–34.2 [2]
Phanerochaete chrysosporium 0.58 100 [23]
0.96 0
Pseudomonas aeruginosa A2Chr 0.77 60 [39]
0.96 16 [40]
0.19 96
Pseudomonas fluorescens var. pseudo-iodinum P-11 0.58 57 [22]
Pseudomonas fragi B-184 0.58 83 [22]
Pseudomonas mendocina P-13 0.58 50 [22]
Pseudomonas putida B-117 0.58 97 [22]
LB303 0.02 100 [41]
Pseudomonas “rhatonis” P-17 0.58 100 [22]
Pseudomonas stutzeri 0.1 88 [42]
Pyrobaculum islandicum 0.42 100 [43]
Shewanella oneidensis MR-1
(a) Anaerobic conditions 0.05 29.4 [5]
(b) Aerobic conditions 0.4 35 [11]
(c) Fumarate-reducing conditions 0.04 94 [44]
Streptomyces griseus 0.096 100 [45]
1.15 100
Sulfate-reducing bacteria (isolate TKW) 0.36 94.5 [14]
Sulfidogenic and nonsufidogenic microbial consortia 0.85 100 [46]
1.35 100
Thiobacillus ferrooxidans 0.19 65 [20]
Trichoderma viride 1.3 100 This work
1.6 100
1.94 94.3
112 L. Morales-Barrera, E. Cristiani-Urbina / Enzyme and Microbial Technology 40 (2006) 107–113
Fig. 3. Volumetric rates of Cr(VI) reduction achieved in the batch cultures of T.
viride, for the different initial Cr(VI) concentrations tested.
potentially useful since this fungus could be employed in detox-
ification of wastewaters with high Cr(VI) concentrations. To our
knowledge, this is the first report on Cr(VI) removal by a strain
of the genus Trichoderma, and also the first one in using an airlift
bioreactor for this purpose.
4. Conclusion
Cr(VI) removal by T. viride was studied in a stirred tank
bioreactor and in an airlift bioreactor. Experimental results sug-
gest that shear stress created by the magnetic stirrer used during
the bioreactor cultivation caused mycelial fragmentation, which
in turn affected negatively the capability of the fungal strain to
remove Cr(VI). In contrast, almost a complete removal of high
concentrations of Cr(VI) was achieved in the airlift bioreactor,
which suggests that this type of reactor might be a promising
alternative for the biological treatment of Cr(VI)-laden wastew-
aters, especially when shear sensitive microorganisms are used.
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