Deg of Phenols by Algae

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Degradation of phenols by algaeV. Klekner

a& N. Kosaric

b

aCzech. Acad. Sci. , Institute of Microbiology , Prague 4, Czechoslovakia , 14200

bDepartment of Chemical and Biochemical Engineering , The University of Western Ontario ,

London, Ontario, Canada , N6A 5B9

Published online: 17 Dec 2008.

To cite this article:V. Klekner & N. Kosaric (1992) Degradation of phenols by algae, Environmental Technology, 13:5, 493-501,

DOI: 10.1080/09593339209385176

To link to this article: http://dx.doi.org/10.1080/09593339209385176

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2/10

rwimnmtnt t Technology

Vol 13 pp 493 501

ublication

Division Selper Ltd., 1992

EGR TIONOF PHENOLS BY ALGAE

V. KLEKNER

1

AND N. KOSARIC

2

*

1

Institute of Microbiology, Czech. Acad. Sci., Prague 4, Czechoslovakia 14200

2

Department of Chemical and Biochemical Engineering, The University of Western Ontario London,

Ontario, Canada N6A 5B9

Received2 May

1991;

Accepted 19 January 1992)

STR CT

Stra insof

Chlorella

sp.,

Scenedesmus obliquus

and

Spirulina maxima

were tested for degradation

of

some phenolic compounds listed by U.S. EPA as priority pollutants. Toxins were dissolved in a

medium

(pH 7 - 7.2) without carbon source (except for testingSpirulina, in which case sodium

bicarbonate

was part of the medium at pH 9 - 9.2) and algae prepared by batch cultivation were

added.

Phenol was found to be degraded easily by all tested algae at a concentration about 1000 mg

1

-1

. 2,4-dimethylphenol was found to be converted by

Chlorella

(even at a concentration of about

1000mg1

-1

) to an isomer of dimethylbenzenediol that was in some cases accumulated in the

medium.

Depending upon biomass and toxin concentration the rate of degradation changed and an

optimum

of toxin concentration which induces degradation might exist. Complete degradation

could

be reached with biomass concentrations higher than 4 g 1

-1

. 2,4-dinitrophenol at a

concentration

of about 190 mg 1

-1

was degraded by

Scenedesmus

quickly after an adaptation period

of 5 days. 2-chlorophenol at a concentration about 200 mg 1

-1

was degraded and partly

dechlorinated

byChlorella.Biodegradation of 2,4-dichlorophenol was not proven but the condition

under

which algae can survive a higher concentration of toxin could be found. All algae tested have

a

mechanism for degradation of phenolic compounds.

Ke y wo r d s :

Biodegradation , algae, phenols , toxins, poll utan ts

INTRODUCTION can be complete to CO2, transformation to an

intermediate product, enzymatic or non-

Algae are a group of microorganisms that enzymat ic. Degradation of low phenol and

can play a role in determining the fate of toxic catechol to CO2 by some fresh-water algae ha s

compounds but this has not been widely studied, been described (5) as has been the transformation

Research in using algae for waste water of naphthalene to

1-naphthol

by cyanobacteria and

treatment has a long tradition but previous microalgae (6, 7). Ellis (5) has measured the

studies have dealt primarily with the removal of evolution of

14

CQ2 from radioactively labelled

ni trogen an d phosphorus from non-toxic phenol and catechol by fresh-water algae. The

municipal or agricultural wastes (1). transformation of naphalene to

1-naphtol

by

Algae have a potential for removal of cyanobacteria and microalgae has been also

organic compounds t ha t includes accumulation reported (6, 7). However, the mechanisms of the

and degradation (2). A number of compounds are degradation of toxic compounds by algae, and the

accumulated by algae including pesticides DDT, reason why algae perform these reactions and

al dr in , di eld rin, end rin , lin dane, mirex what is the extent of the degradation, are not

methoxychlor, toxaphene, parathion, carbaryl, understood. When considering algae for waste

and chlordan (3, 4). Mechanisms may be physical wat er tr ea tmen t it is obvious th at more

sorption as the process was usually dependent on fundamental results are needed to evaluate their

the hydrophobicity of the compound. Degradation capabil ity. Thus, we selected three common,

493


3/10

Table 1.

Composition of solutions used for preparation of the cultivation media.

Fe solution

Trace metal solution I

Trace metal solution II

NajjEDTA 189 mg, F eC l

3

.6 H

2

O 244 mg

H3BQ3 2.86 g, MnCl

2

1.81 g, ZnSO

4

.7 H

2

O, 0.22 g,

CuSO

4

.5 H

2

O 0.079 g, (NH4)

6

M o

7

024.2H

2

0 0.038 g

N H

4

V O

3

23 mg, NiSO

4

.7H

2

O 48 mg, CaWO

4

20 mg,

C o S O

4

.7 H

2

O 4 0 mg

widely used algae, and tested them as to whether

or not they have the capability to degrade higher

c o n c e n t r a t i o n o f t o x i c p h e n o l s . A s t h e

degradation of toxic compounds reported (5, 6, 7)

had been slow, we used large concentrations of

a lga l biomass in order to acce lera te the

degradation. Such large concentrations could be

used in a reactor with immobilized algae on a

solid support. Results of our investigations are

repor ted he re .

MATERIALS AND METHODS

Stra ins of

Chlorella sp.

and

Scenedesmus

obliquus

were obtained from Prof. J. de la Noue

(Universite Laval, Quebec).

Spirulina maxima

was from Lake Texcoco (Mexico).

Chlorella

an d

Scenedesmus

were grown in the medium

proposed by Borow itzka and Borowitzka (8) for

f resh water a lgae . The medium was s l ight ly

modif ied by addi t ion of some t race meta ls .

Composition of the medium was (in 1 1 of wa ter):

KNO3 1 g, K

2

H PO

4

0.2 g, Mg SO

4

.7 H

2

O 0.2 g, CaSO

4

saturated solution 20 ml, Fe solution 10 ml, Trace

me tal solution I 1 ml, and Trace m etal solution II

I ml (Table 1). Initial pH was in the range of 7 -

7.2 and was not controlled during cultivation.

Spirulina

wa s cultivated on med ia containing (in

I 1 of water) NaHCO

3

10 g, Na

2

H PO

4

.12H

2

O 1 g,

KNO

3

1.5 g, K

2

SO

4

1 g, MgS O

4

.7 H

2

O 0.02 g, CaCl

2

0.04 g, NaCl 1.5 g, Fe solution 10 ml, Trace metal

solution I 1 ml, and Trace metal solution II 1 ml.

Initial pH was adjusted between 9 and 9.2.

Cul t iva t ion was ca r r i ed ou t a t room

tem pe rat ur e in 10 1 flasks fil led w ith 7 1 of

medium. Flasks were i l luminated with 8 wide

spectrum tubes (GE F40/GS/W8, 40 W) from one

side at a distance of 15 cm. Mixing was ensured

by a magnet ic s t i r re r . Air (0.5 W M ) enr iched

(for cultivation of

Chlorella

and

Scenedesmus)

with 2 - 3 % of carbon dioxide was distributed by

means of porous ce ramic tube . Af te r 4

{Chlorella)

or 5

.Scenedesmus)

or 7

Spirulina)

days , which corresponded to ear ly s ta t ionary

growth phases , the ce l ls were harves ted by

centrifugation (5000 g), resusp ende d in distil led

water and used in exper iments . The biomass

concentration was determined as the weight after

drying a su spensio n of th e cells for 24 h a t 100C.

Batch experiments were carried out in 250

ml Er lenmayer f lasks with 50 ml of l iquid

plugged with foam plugs. Toxins were dissolved

in a sterile medium (pH 7.1 - 7.3) without a

carbon source, except for testing

Spirulina,

in

which case sodium bicarbonate was part of the

medium (at pH 9 - 9.2). Suspensions of algae

were added to the flasks. No other precautions

were made to keep algae axenic but the purity of

cultures was checked microscopically during the

experiments and no contamination was observed.

Flasks were shaken on a Lab-Line Orbit Shaker at

100 rpm and were illuminated with 2 x 40 W broad

spect rum tubes from a i m d i s tance . B iomass

c o n c e n t r at i o n w a s n o t d e t e r mi n e d d u r i n g

the exper iments . Thus , a l l concentra t ions of

biomass reported here are initial concentrations

ca lcula ted f rom the concentra t ion of added

suspension. For each experiment a control f lask

without algae was run in parallel.

Phenol (Sigma) , 2,4-dimethylphenol (2,4-

DMP, Aldr ich) , 2,4-dichlorophenol (2,4-DCP,

Sigma), 2-chlorophenol (2-CP, Supelco), and 2,4-

dinitrophenol (2,4-DNP, Aldrich) used as test

compounds we re of ana ly t i ca l g rade or

c h r o m a t o g r a p h y s t a n d a r d s g r a d e s . 2 , 4 -

d ime thy lphenol , 2 ,4-d ich lorophenol , and 2-

chlorophenol were determined by GC (Varian

3400,

FID, 1% SP-1240-DA on 100/120 Supelcoport

2 mm ID x 6' glass, N 30 ml m in

1

. , t empe ra ture

of injector 180C, column: 1 min. 70C then to

180C at 15C min'

1

, and hold 5 min., detector

230C) with phenol as an inte rna l s tandard.

Phenol and 2,4-dinitrophenol were determined by

HPLC (Wate r s HPLC pump 501 , Rheodyne

49 4


4/10

injector 7161 with 20 ul loop, Waters Absorbance

detector 440 at 254 nm, Waters column

Bondapack C

18>

mobile phase 25% methanol, 0.1%

acetic acid in water). Samples were centrifuged

(2000 g, 10 min.) and filtered (0.4 urn) before

analysis. The values reported here are the

averages of two or more analyses of a sample

taken from the flask.

GC/MS analysis was performed on GC HP

5890 (HP-S capillary column 25 um x 25 m,

temperature of injector 180C, column: 80C 1

min then to 225 C at 10C min'

1

hold 5 min) with

ms VG70S-250 as a detector.

To detect compounds on/in cells, 10 ml of

sample suspension was centrifuged (2000 g),

biomass was extracted with 3 ml of chloroform

for 30 min at room temperature and the extract

was analyzed by GC. The toxic compound was

always analyzed in the cells when its presence in

the test solution was detected. However, this was

not monitored regularly but extraction was used

in order to detect the toxic compound in the cells,

in case when the toxic compound was depleted

from the test solution, and these results are

reported.

Concentration of chloride ions was

determined according to Bergmann and Sanik

(9). Samples from a control flask served as

blank samples and a difference was measured.

Calibration was done with analytical grade NaCl.

starting the degradation. Spirulina seemed to

proceed with degradation after a much shorter

adaptation period. When phenol was added to the

final concentration of about 3000 mg 1

1

, both

algae tested ChlorellaandScenedesmus) turned

brown

and no rapid degradat ion occur red.

aoo

put

on liebt

O - cells at light

9

- cells in dark

-

l

-e-

10 15 20 25 30

time (days)

Figure 1. Changes in phenol con centra tion

with Chlorellacells (3.4 g 1

1

) at light

and in dark.

RESULTS

Pheno l

Phenol

was found to be degraded easily by all

tested

alga e. Phenol was clearly biodegraded by

algae

because i l luminat ion and presence of l ive

cells were necessary for degradation to proceed.

Phenol

was not degraded in the dark by l iving

cells

of Chlorella (Fi g. 1) . De ad cells of

Chlorella (steri l ized for 20 min at 121C, Fig. 2)

on

the l ight d id not degrade phenol e i ther .

Neverthe less ,

the degradation of phenol is not a

necessity for algae to survive harsh condit ions as

Chlorella

cells were quite viable even after 23

days

spent in the presence of phenol during the

exper iment .

When the cells were taken from the

dark to l ight they star ted to degrade phenol (Fig.

1) . pH of the medium decreased very s l ight ly

(0.1)

du ring th e e xperim ents. All three species of

algae (Fig. 3) degrad ed abo ut 1000 mg 1

1

of phenol

in

le ss tha n 6 days af ter degrading about 400

mg 1

1

in 6 days. Chlorella and Scenedesmus

needed an adaptation period of a few days before

I0OO

80 0

= 600

2

O - without algae

- live algae

V dead algae

12 U 16

time

Figure 2. Com parison of live

Chlorella

cells (6.3

removal of phenol.

and dead

g I '

1

) for

49 5


5/10

3000

2500

2000

1500

A - without biomaas

A - Spirulina 4.4 g/1

O

-

Scne


6/10

-

- without algae

- Chlorella S A g/l

10 20 30

40

50

time days)

Symbols are the same as

10 15 20 25 30 35

time (days)

Figure 6. Influence of different am ount of

Chlorella on biodgradation of 2,4-

dimethyphenol.

Figure 7. Influence of the initial concentration

on the degradation of 2,4-dimethyl-

phenol byC hlorella(1.7 g H ) .

2.0

o

E

1.5

a

v

- 1.0

o

o

c

Initial concentration of

2.4 -dimethylpheno l

O - 2S0 mg/1

- 650 mg/1

V - 980 mg/1

> - 1110 m g/l l

1000

900

800

700

600

500

400

300C

2 J

ol

O . D - without

-

^7 - Spirulinn

\ 4.4 g/1

7 V

Scenedesmus

\ 4.a g/ i


7/10

of toxin were applied the conversion and

degradation efficiency differed substantially

(Figs 7, 8). There seems to be an optimum

concentration of toxin that can induce conversion

but is not yet inhibiting. In the case of low

concentration (250 mg/1) the adaptation period

was not much longer and the amount actually

degraded lower , as re la t ively h igher

accumulation of DMBD was found (Fig. 8). Much

higher concentrations of biomass (Fig. 9) did not

accelerate degradation in the case of Chlorella

and the time needed was similar to that with a

lower amount of cells. This may be because of

limitation of the light supply caused by very

opaque suspension. On the other hand, no

intermediate product of degradation was found in

the solution during the experiment and

degradation was complete as no toxin was found

in the chloroform extract of the cells. Spirulina

degraded about 150 mg/1 of toxin without any

adaptation in a few days (Fig. 9). Scenedesmus

degraded the toxin faster than Chlorella(Fig.9).

After subsequent addition of toxin to the final

concentration of about 900 gm/1, cells of

Scenedemus were slowly turning brown and an

intermediate product recorded on GC. The

compound was the same isomer of

dimethylbenzenediol as in case of Chlorella

(confirmed by GC/MS). Other volatile compounds

were also found in these cells of Scenedesmus

(Pig. 10).

2,4-dinitrophenol

Scenedesmuswas found to degrade about 190

mg I

1

of 2,4-DNP and it degraded or converted all

compounds (Fig. 11) because no toxin was

detected in the chloroform extract of the cells.

Also, no product was detected in the medium by

GC but in this case a derivative of catechol might

not be detected by our GC arrangement. The

degradation was very fast with an adaptation

period of about 5 days. This concentration of

toxin was not changed in the presence of

lia

in m

WJ

CH

2

0H

J

I . I l l ,l,

I l l U l

Figu re 10. MS of produ cts found in the medium with

Scenedesmus

cells and 2,4-dim ethylphen ol. The

structures of compounds depicted are highly tentative and were not confirmed by any

other ana lys is .

49 8


8/10

O - Uhout algae -

- Chlorella

8.2g/1

Scenedesmus

4.8g/1

10 20 30 40

time (days)

5

Figure 11. Changes in 2,4-dinitrophenol

concentration.

Chlorella even if algae remained greenand

alive.Lowconcentration(70 mg I

1

)seemedto be

degraded

by

Chlorella very quickly after

an

adaptation periodofabout20days (not shown).

2-chlorophenol

2-chlorophenol

is

rather semivolatile

and

was relatively quickly evaporated from the

medium without algae. However, the compound

disappeared from

the

medium with Chlorella

in

22 days (Fig.12).Therewas notoxin left in the

solution

and

also none

was

found

in the

chloroform extract. Degradation

was not as

fast

as in case ofphenol butfaster tha n with2,4-

dimethylphenol. Scenedesmus seems not to

degrade this compound.

In the next experiment the amount of

chloride ions released during the processwas

estimated (Fig. 13). 2-CP was converted and

par t l y dech lo r ina t ed . The e x t e n t of

dechlorination may be approximately estimated

at 30%ofutilized 2-chlorophenol.

2,4-dichlorophenol

The concentration of 2,4-DCP decreased

relatively fast regardless of the presence of

algae. This

may be due to

evaporation

or

photodecomposition. 2,4-DCP is known to be

easily photodegradedbut in ourcasenoproductof

such degradation

was

found

by

GC. Neve rtheless,

the rate of2,4-DCP disappearance washigherin

4

^ 300

25

o

1 200

o

5

u

~

100

5

0 -

without algae

Chlorella 8.2 g/1 -

Scenedesmus

4.8 g/1

\

:

^ i

A i

5 10 15 20 25 JO

time (days)

8 12 16 20 24

time (days)

Figure

12.

Changes in

concentration.

2-chlorophenol Figure

13.

Degradation of 2-chlorophenol with

Chlorella

(6.3 g I*

1

) and

release

of

chloride ions.

499


9/10

3

c

Q .

0

o

o

1

*

e i

80

7 0

6 0

(

50

*0

30

20

10

C

o

k

r is v_^ n

1 \ \

1 \ \

t f

10 20

time

without algae

- Chlorella

8.2 g/1

- Scenedesmus

4.8 g/1

-

1

v\

t 1

30 *0 5C

(days)

Figure

14.

C h a n g e s

in

2,4-dichlorophenol

c o n c e n t r a t i o n .

the presence of cells (Fig. 14). A sharp decrease

at the beginning of the experiment may indicate

sorption

by the

cells because

the

process stopped

a n d

the

c o n c e n t r a t i o n

of

t o x in r e m a i n e d

unchanged

for the

next

10

days

and

because

a

very

high concent ra t ion

of

cells

was

used

in the

exper imen t . Never the l ess ,

th e

algae remained

green and alive in this experiment which was

verified

by

cult ivation

of a

sample taken from

flasks. Higher concent ra t ions

of

toxin tested

previously were fatally toxic

to

algae.

Clearly, thebiodgradation ofthis compound

w as not proven but the condition under which

algae

can

survive

a

higher concentration

of

toxin

could

be

found.

DISCUSSION

All tested a lgae have

a

mechanism

for

degr ada t ion

of

phenolic com pounds. This

has

been p r ev ious ly r epor t ed

for

pheno l

and

catechol by El l i s (5). How ever, Ell is applied

concentration about two orders lower than th at

used

in our

study which were close

to

le thal

concentration.

The

first step

of

degradation

may

be

an

oxidation

of the

second carbon

on the

benzene ring

to

form

a

derivative

of

catechol.

All

tested phenolic compounds were proven

to be

a t t acked by il lum inate d algae cells, converted

and/or degraded except for 2,4-dichlorophenol.

S u b s t i t u e n t s on benzene r ing can i n c r e a s e

toxici ty of compounds and they in f luence

degradabi l i ty . This

is a

wel l -known fact

for

bac te r i a

(11) and it is

r e a s o n a b l e

to

expect

s imi lar re la t ions

for

other microorganisms

as

well. Phenol

was

degraded easily, methyl

or

nitro

groups

are not

probably

as

toxic

as

chlorines.

2-

chlorophenol was st i l l deg raded at l e a s t by

Chlorella but no clear conclusioncan bemadeon

2,4-dichlorophenol. Scenedesmus seemed not to

degrade chlorinated phenols

in our

exper iments

but,

on the

other hand,

it

easily degraded 2,4-DNP.

The toxicity

of

chlorinated phenols

was

described

to increase with

the

number

of

chlorine atoms

in

a molecule

(12) and our

resul t s

are in

agreement

as a lgae could survive only re la t ively low

concentra t ion of 2,4-DCP. In any case when

compared

to

bacteria

the

degradation

by

algae

was

much slower.

Dialkylated compounds

are

know n

to

slow

down and even block deg rada t ion path wa ys

(13) . In case of a lgae and 2 ,4 -DMP the

s u b s t a n t i a l a m o u n t

of

i n t e r m e d i a t e p r o d u c t

( d i m e t h y l b e n z e n e d i o l )

can be

a c c u m u l a t e d

suggest ing that

the

next step

in

degradat ion

is

much slower.

As

nothing

is

known about enzymes

and pathways involved

in

this process inside

algae cellsone mayspeculate tha t there are more

different pathways and some of them may be

completely blocked

by an

intermediate product.

Mixture

of

both algae

or

algae

and

bacter ia

m ay be a reas ona ble choice for i n d u s t r i a l

purposes

as

different algae

can act

differently

with different compounds.

One may

even expect

a

symbios i s

of

d i f f er en t m ic r oor g an i sm s

in

degradation

of a

toxin

as

intermediate products

may

be

available free

in the

solution.

T h e s o r p t i o n

of

n o n - p o l a r a r o m a t i c

compounds

by

Selenastrum were est imated

by

(14)

who

found relatively high value s

of

so-called

bioconcetration factor (BCF)t h a t is defined as a

rat io between concentration in/on biomass and

in the solution. Values ofBCF , rangin g from 3000

to 55000, were correlated with n-octanol-water

par t i t ion coef f ic ient . Consider ing these data

majority

of

tes ted phenolic compounds should

have been eliminated from solution

by

sorption.

In fact,

no

profound so rption took place

in our

experiments. Very rough guessof BCFvalu e from

our data would be 100-200. However, we were

using substantial ly higher concentrations than

in

sorption experiments

and

this

may

contr ibute

to

above-mentioned difference.

In a

longer period

of time evaporation

may

have contr ibuted m ore

to

the decrease

in

concentra t ion

of

toxins t han

s o r p t i o n .

500


10/10

In order to more fully evaluate the potential well as othe r pollu tants should be teste d becau se

of usin g algae for biod grad ation of specific so-called co-oxidation may contribute to faster

indu strial toxins, more investigation is needed, degradation, e .g. phenol can improve degradation

Oth er algae ma y posses different pro perties and of chlorina ted phenols in bacterial cultu res (15).

should a lso be inves t iga ted. Other pr ior i ty

po llu tan ts sho uld also be add ed to th e lis t for ACKNOWLEDGEMENTS

testing. More experiments are needed to find the

way of degradation and especially the natu re of This research was partly supporte d by a

th e end pro du cts. To find the conditions and/or gr an t from ISCT to

Prof.

N. Kosaric. We thank

reac tor a r rang em ents fur ther exper iment ing is Dr . Alex Young (Univers i ty of Toronto) for

also neces sary. M ixture of different phenols as performing GC/MS spec tra.

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Deg of Phenols by Algae