Biochemical Engineering Journal 28 (2006) 295298

Short communication

A novel treatment system of wastewater contaminated

with copper by a moss
Fumihisa Kobayashi a, , Rumiko Kofuji b , Yuya Yamashita a ,
Yoshitoshi Nakamura a
a

Division of Material Engineering, Graduate School of Natural Science & Technology, Kanazawa University,
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
b Division of Biological Science, Graduate School of Natural Science & Technology, Kanazawa University,
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
Received 19 October 2005; received in revised form 18 November 2005; accepted 18 November 2005

Abstract
A novel treatment system of wastewater contaminated with copper was developed by using some mosses that are demonstrably metal tolerant
and accumulate heavy metals into the cells. Scopelophila cataractae could remove copper more efficiently than other mosses, i.e. Physcomitrella
patens and Polytrichum formosum. One hundred milligram per liter of copper ion was removed completely for 9 d using the suspended cultivation
system flowing air coupled with intermittent mechanical disruption of the protonema filaments of S. cataractae by a homogenizer.
2005 Elsevier B.V. All rights reserved.
Keywords: Bioaccumulation; Heavy metals; Moss; Plant cell bioreactors; Submerged culture; Wastewater treatment

1. Introduction
The development of better designed and operated wastewater
treatment system is desired because the permissible legal limit
concentration of toxic material in the wastewater discharged
into rivers and oceans has decreased annually [1,2]. Biological treatment has been thought to be a lower cost treatment for
the removal of toxic heavy metals, i.e. mercury, cadmium, and
copper, from industrial wastewater.
Some mosses are known as demonstrably metal tolerant being
able to withstand high levels of heavy metals that are toxic to
other species [3]. One famous group of species is the copper
mosses [4,5] and Oda and Honjyo [6] have investigated the
characterization of copper, lead, and zinc in metal tolerance
mosses, i.e. Atrichum undulatum, Scopelophila cataractae, and
Pohlia bulbifera. They reported that copper, lead, and zinc were
detected in the cells of these mosses using X-ray fluorescence
method and about 20 mg g1 of heavy metals accumulated in the
cell wall. If the mosses could absorb and accumulate heavy metals, the simple treatment process could be proposed for removing

Corresponding author. Tel.: +81 76 234 4820; fax: +81 76 234 4829.
E-mail address: fumihisa@t.kanazawa-u.ac.jp (F. Kobayashi).

1369-703X/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2005.11.013

heavy metals from wastewater. However, very few papers on the

application of mosses for the treatment of wastewater contaminated with heavy metals have been published.
In this work, the removal method of copper by the moss was
developed. The mosses, Physcomitrella patens (P. patens), Polytrichum formosum (P. formosum), and Scopelophila cataractae
(S. cataractae), were used to test their abilities for removing
copper in the tissue cultivation using the multiple well plates.
Furthermore, the suspended cultivation system flowing air coupled with intermittent mechanical disruption of the protonema
filaments of mosses by a homogenizer was attempted for the
efficient removal of copper.

2. Materials and methods

2.1. Plant materials
Three mosses, P. patens, P. formosum, and S. cataractae, were
used in this study. The wild-type strain of Physcomitrella patens
ssp. patens [7] was used. P. formosum and S. cataractae were
collected in Kanazawa City. The protonema of P. formosum was
obtained from spores using the following method. The capsules
of P. formosum were soaked in 10% (v/v) sodium hypochlorite

296

F. Kobayashi et al. / Biochemical Engineering Journal 28 (2006) 295298

solution for 5 min, and then washed with sterile distilled water.
Subsequently, they were crushed out with sterile tweezers. The
spores were scattered on spore germination medium solidified
with 0.8% agar, based on BCDAT medium (containing 1 mM
MgSO4 , 10 mM KNO3 , 45 M FeSO4 , 1.8 mM KH2 PO4 ,
10 mM CaCl2 , and 5 mM diammonium (+)-tartrate) and cultured under continuous light [8]. After approximately 1 week the
surface of the solidified medium was covered with green mats
of growing protonemata. In the case of S. cataractae, it hardly
forms sporophyte [5], and difficultly obtains the protonema by
spore germination. Therefore, gametophytes were collected and
treated with the following method. S. cataractae plants were
washed with running water for 5 h. The washed plants soaked in
0.1% Plant Preservative Mixture (PPMTM , Nakalai Tesque Co.
Ltd., Kyoto, Japan) for 1 h and then rinsed five times in sterile
distilled water. After being crushed and dispersed using the
homogenizer with sterilized shaft (POLYTRON PT-MR2100,
KINEMATICA Co. Ltd., Luzern, Switzerland), they were
planted to solidified BCDAT medium with the addition of
0.01% Plant Preservative Mixture. After approximately 2 weeks
the plants grew with other microorganisms on the solidified
medium. The portion of protonemata were picked up and
replanted on the fresh solidified medium, avoiding the other
microorganism. After being five times repeated the procedure,
obtained pure protonemata were used as S. cataractae samples.
2.2. Culture media and cultivation
BCDATG medium with several copper ion concentrations as
CuCl2 was used for the moss culture in this study [8]. BCDATG
medium contains 1 mM MgSO4 , 10 mM KNO3 , 45 M FeSO4 ,
1.8 mM KH2 PO4 , 1 mM CaCl2 , 5 mM diammonium (+)-tartrate,
and 5 g l1 glucose. The glucose as an organic component was
added into the medium because the organic component seems to
exist in the real wastewater and enhance the moss growth [2,9].
The pH of the media was adjusted to 6.5 with KOH and the
temperature of culture was maintained at 25 C in compliance
with the standard method of moss tissue culture [10].
For testing the removal ability of copper by using three
mosses, the tissue cultivation was carried out using the multiple
well plates (six well plates: well volume 16 ml, Asahi Technograss Co. Ltd., Tokyo, Japan). Fifty milligram protonema of
three species mosses, P. patens, P. formosum, and S. cataractae were added into each well containing 10 ml sterilized liquid
medium. These mosses were incubated under continuous illumination (4590 lx). After 3 weeks, the sample of the medium
was used for the analysis of copper ion concentration.
The treatment operation of copper by moss was carried out
using the suspended cultivation system flowing air as shown in
Fig. 1. 0.5 g protonema as dry weight was transferred to 100 ml
of liquid BCDATG medium containing 100 mg l1 copper ion
as CuCl2 in 150 ml-test tube for plant culture. The cap had two
narrow glass tubes for an aspiration and exhaust. The aspirating air was sterilized by membrane filter and the flow rate was
1.0 l min1 . The test tubes with the caps were placed in rotary
shaker (BR-30L, TAITEC Co. Ltd., Koshiya, Japan) and incubated while shaking at 50 rpm under continuous illumination

Fig. 1. Schematic diagram of a suspended cultivation system flowing air for the
treatment of copper by moss.

(4590 lx) supplied by five fluorescent lights. Two milliliter samples withdrawn from the culture at suitable intervals were used
for measurement of the dry weight of mosses, the copper ion
concentration, and the glucose concentration. The time course
of glucose concentration was analyzed as the removal index of
an organic component in wastewater.
2.3. Analysis
The dry weight of mosses was measured after drying at
80 C for 24 h. The copper ion concentration was determined
by HPLC [11,12]. The stainless steel column used was TSKGEL ODS-80TS (TOSO Co. Ltd., Tokyo, Japan). The eluent was
mixture of methanolwaterdichloromethaneacetylacetone
(58/35/6/1). The flow rate was 1.0 ml min1 . The glucose concentration was determined using the glucose oxidase/peroxidase
enzymatic assay reagent (Glucose C-test Wako; Wako Jyunyaku
Kogyo Co. Ltd., Osaka, Japan). All data in this study were the
mean values and standard deviations corresponding to threetime experiment.
3. Results and discussion
3.1. Removal of copper by using three mosses
Among the bryophytes, mosses comprise approximately
10,000 or more species [13]. For testing the removal ability
of copper by moss, P. patens, P. formosum, and S. cataractae, were used. P. patens is a most extensively studied moss
species at the molecular level [14]. This moss was used as a
control in this study because it was not metal tolerant. Polytrichum spp. is known as a fire moss and withstand even in
harsh conditions, which rapidly colonize the surface of ground
after fire [15]. Therefore, P. formosum seems to have the ability of metal tolerance and absorbance. S. cataractae is known
as a copper moss and has the ability of copper tolerance and
absorbance [5]. However, few papers on the liquid culture of
mosses, for removing metals have been reported. Table 1 shows
the comparison of residual copper ion concentrations among P.
patens, P. formosum, and S. cataractae, in the liquid culture
using multiple well plates after 3 weeks. In the cultivation of

F. Kobayashi et al. / Biochemical Engineering Journal 28 (2006) 295298

297

Table 1
Comparison of residual copper ion concentrations among three kinds of mosses
Initial copper ion (mg l1 )
concenration

Residual copper ion concentration (mg 11 )

P. patens

P. formosum

S. cataractae

20
40
60
80
100

19.7 0.4
39.9 0.2
59.8 0.6
79.8 0.5
99.6 0.5

18.4 1.1
39.2 0.5
59.8 0.2
79.1 0.7
99.8 0.5

1.5 0.7
3.1 1.1
4.6 1.5
5.5 2.0
7.5 1.5

P. patens and P. formosum, the copper ion concentrations did

not decrease at all. Beyond the initial copper ion concentration of 60 mg l1 no decrease of copper ion concentration was
observed and the intensity of green color in well plates decreased
with the incubation time because cell death occurred due to toxicity of more 60 mg l1 copper ion concentration. On the other
hand, S. cataractae could remove copper efficiently in the initial
copper ion concentration by 100 mg l1 . The amount of copper
removed increased with the initial copper ion concentration and
the removal ratio, i.e. the residual copper ion concentration to the
initial copper ion concentration, was corresponded to about 0.93
regardless of the initial copper ion concentration. Though little
is known about the mechanism of copper removal system by S.
cataractae, a large amount of copper seems to be accumulated
in the cell walls of cortex [6]. Though the data is not shown, this
cultivation was continued for 5 weeks. All mosses died after 3
weeks and the copper ion concentrations at 5 weeks were almost
the same as those at 3 weeks as shown in Table 1. These results
suggest that the copper uptake was not reversible and physical
adsorption on the surface of mosses occurred hardly.
3.2. The treatment of copper using the suspended
cultivation system owing air
Though S. cataractae could remove a large amount of copper
in the tissue cultivation using multiple well plates, the removal
ratio obtained seems to be not the maximum value because this
cultivation was not carried out with efficient supply of carbon
dioxide and agitation. Therefore, the suspend cultivation system
flowing air was attempted for the efficient treatment of copper as
shown in Fig. 1. Fig. 2 shows the time courses of dry weight of S.
cataractae, glucose concentration, and copper ion concentration
in a suspended cultivation system flowing air. The initial copper
ion concentration was 100 mg l1 . The copper ion concentration decreased gradually with the increase of cultivation time
and reached almost zero at 19 d. The dry weight of S. cataractae
concentration increased slowly from about 5 to 6.5 g l1 as the
glucose concentration decreased. One hundred milligram per
liter copper ion was removed completely but the time required
for complete removal was very long, i.e. 19 d. Kondo et al. [16]
investigated the removal of mercury by a moss, Pohlia exuosa, and suggested that the moss must grow rapidly even after
absorbing mercury in order to act as an efficient adsorbent.
The breakage of the protonema filaments of mosses prevents
the development of the gametophytes, i.e. the cell growth rate of
the gametophyte is low than that of the protonema, and enhances

Fig. 2. Time courses of dry weight of S. cataractae, glucose concentration, and

copper ion concentration using a suspended cultivation system flowing air.

Fig. 3. Treatment of copper using a suspended cultivation system flowing air

coupled with mechanical disruption of the protonema filaments of S. cataractae
by a homogenizer. The mechanical disruption was carried out every 12 h.

the formation of a new apical cell starting a cell division [17].

From above reasons, the intermittent mechanical disruption of
the protonema filaments by a homogenizer seems to be effective
for increasing the growth rate of moss. Fig. 3 shows the treatment
of copper in the suspended cultivation system flowing air coupled with intermittent mechanical disruption of the protonema
filaments of S. cataractae by a homogenizer. The protonema filaments were disrupted every 12 h using the homogenizer. The glucose concentration decreased rapidly and reached almost zero at
3 d. This means that S. cataractae could remove an organic component in wastewater. The dry weight of S. cataractae increased
rapidly from about 5 to 10.7 g l1 with the decrease of glucose
concentration. The rapid glucose consumption and cell growth
were observed in comparison with the result as shown in Fig. 2.
The copper ion concentration decreased abruptly and reached
zero at 9 d. It was confirmed that the mechanical disruption of
the protonema filaments of S. cataractae by a homogenizer not
only enhanced the growth rate of mosses but also shortened the
time required for removing copper.
4. Conclusions
The treatment of copper using the mosses and the suspended
cultivation system flowing air coupled with intermittent mechanical disruption was investigated experimentally. S. cataractae

298

F. Kobayashi et al. / Biochemical Engineering Journal 28 (2006) 295298

could remove copper rather than P. patens and P. formosum. One

hundred milligram per liter copper ion was removed completely
by the suspended cultivation system flowing air for 19 d. Furthermore, the intermittent mechanical disruption of the protonema
filaments by a homogenizer could decrease the time required for
removing copper up to 9 d.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Young
Scientists (B): 17710060 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References
[1] A.H. Scagg, Biotechnology for Engineers, John Wiley and Sons, New
York, USA, 1988.
[2] G. Schwedt, The Essential Guide to Environmental Chemistry, John
Wiley and Sons, New York, USA, 2001.
[3] J.W. Bates, Mineral nutrition, substratum ecology, and pollution, in:
A.J. Shaw, B. Goffinet (Eds.), Bryophyte Biology, Cambridge University
Press, Cambridge, UK, 2000, pp. 248311.
[4] D.H. Brown, Mineral nutrition, in: A.J.E. Smith (Ed.), Bryophyte Ecology, Chapman and Hall, London, UK, 1982, pp. 383444.
[5] K. Satake, A copper moss Scopelophila cataractae and copper (1)distribution of Scopelophila cataractae in the world, Proc. Bryol. Soc.
Jpn. 5 (1990) 4953.
[6] T. Oda, T. Honjyo, Characterization of copper, lead, and zinc in metal
tolerant mosses, Atrichum undulatum (Hedw.) P. Beauv., Scopelophila
cataractae (Mitt) Broth, and Pohlia bulbifera (Warnst) Warnst. in heavy
metals pollution areas, J. Phytogeogr. Taxon. 43 (1995) 91102.

[7] N.W. Ashton, D.J. Cove, The isolation and preliminary characterization of auxotrophic and analogue resistant mutants in the moss
Physcomitrella patens, Mol. Gen. Genet. 154 (1977) 8795.
[8] T. Nishiyama, Y. Hiwatashi, K. Sakakibara, M. Kato, M. Hasebe, Tagged
mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle
mutagenesis, DNA Res. 7 (2000) 917.
[9] M.L. Sargent, A guide to the axenic cultureing of a spectrum of
bryophytes, in: J.M. Glime (Ed.), Methods in Bryology, Hattori Bot.
Lab, Nichinan, Japan, 1988, pp. 1724.
[10] P.K. Boyd, J. Hall, D.J. Cove, An airlift fermenter for culture of the
moss Physcomitrella patens, in: J.M. Glime (Ed.), Methods in Bryology,
Hattori Bot. Lab, Nichinan, Japan, 1988, pp. 4145.
[11] S. Ichinoki, M. Yamazaki, Simultaneous determination of nickel, lead,
zinc, and copper in citrus leaves and rice flour by liquid chromatography
with hexamethylenedithiocabamate extraction, Anal. Chem. 57 (1985)
22192222.
[12] S. Ichinnoki, N. Hongo, M. Yamazaki, Multielement analysis by highperformance liquid chromatography following solvent extraction with
acelacetone, Anal. Chem. 60 (1988) 21042110.
[13] W.R. Buck, B. Goffnet, Morphology and classification of mosses, in:
A.J. Shaw, B. Goffinet (Eds.), Bryophyte Biology, Cambridge University
Press, Cambridge, UK, 2000, pp. 71123.
[14] D. Cove, Molecular genetic studies of moss species, in: A.J. Shaw, B.
Goffinet (Eds.), Bryophyte Biology, Cambridge University Press, Cambridge, UK, 2000, pp. 182198.
[15] K.P. ONeill, Bryophytes in the global carbon budget, in: A.J. Shaw, B.
Goffinet (Eds.), Bryophyte Biology, Cambridge University Press, Cambridge, UK, 2000, pp. 344368.
[16] M. Kondo, M. Fukuda, M. Azuma, H. Ooshima, J. Kato, Removal of
mercury ion by the moss Pohlia exuosa, J. Ferment. Bioeng. 86 (1998)
197201.
[17] E.L. Decker, R. Reski, The moss bioreactor, Curr. Opin. Plant Biol. 7
(2004) 166170.