Desalination 158 (2003) 331339

Bioremediation post-photo-oxidation and coagulation for black

liquor effleunt treatment
Samia M. Helmy, Shadia El Rafie*, Montaser Y. Ghaly
Department of Microbial Chemistry and Department of Chemical Engineering, National Research Center,
Dokki, Cairo, Egypt
Tel. +20 (2) 337-1433; Fax +20 (2) 337-0931; shelrafie0000@yahoo.com
Received 17 February 2003; accepted 27 February 2003

Abstract
In an attempt to achieve water suitable for recycling from paper-mill effluent, photo-Fentons advanced oxidation
and chemical coagulation were investigated as options to remove both non-biodegradable COD and color from prebioremediated black liquor effluent. It was found that at the first bioremediation COD removal ranged from 3598%
and TSS removal ranged from 1289%. Almost 20% of the heavy metals were removed. Photo-Fentons UV advanced
oxidation and lime coagulation combination treatment achieved complete removal of COD, TSS and color. It was able
to remove 80100% COD, 70100% TSS and 77100% color. The removal of heavy metals was enhanced to reach
>80% removal. Two potential microbial species out of the eight tested strains were the most dominant species in the
three media. Diplodia oryzae and Phaerochaete chrysosporium NRRL6364 enhanced the post-physicochemical
treatment to reach optimum clean-up after 12 days of incubation at 32C. They produced a medium-grade recycled
water. Operating costs are outlined.
Keywords: Bioremediation; Advanced oxidation process; Wastewater; Recycling; Treatment

1. Introduction
Many industrial wastewaters contain high
amounts of nondegradable organics. Paper and
pulp industry effluent is one of the important
*Corresponding author.

environmental problems, which has been correlated with mutagenic and carcinogenic activity
[1,2]. Many attempts have been made to remove
black liquor by bioremediation, which is considered inexpensive clean-up technology [3,4].
Industrial biotechnology applications help the
elimination of environmentally hazardous wastes.

Presented at the European Conference on Desalination and the Environment: Fresh Water for All, Malta, 48 May 2003.
European Desalination Society, International Water Association.
0011-9164/03/$ See front matter 2003 Elsevier Science B.V. All rights reserved

332

S.M. Helmy et al. / Desalination 158 (2003) 331339

Some application methods of biotechnologies

such as Aspergillus sp. remove color of effluent
from pulp waste [5] and from kraft mill by
Phanerochaete chrysosporium [6]. Still biological treatment will not remove all organics and
the effluents of the biological purification stages
exhibit COD values that may exceed discharge
standards. Advanced oxidation processes (AOP)
using OH radicals are of great promise for COD
reduction of these effluents.
Verenish et al. [7] treated pulp and paper
effluent using a combination of photo-oxidation
coagulation treatment and enhanced the removal
efficiency almost 100%.
Hydroxyl radicals are strong and nonselective
oxidants for organic pollutants in wastewater.
The photo-Fenton method was applied successfully for highly contaminated wastewaters up to
COD 12,000 mg/L. The photo-Fenton reaction is
mainly a metal to ligand charge transfer that
produces the hydroxyl radical. In addition, it can
be compared to reactions that are also of great
importance for pollutants degradation [8,9].
The dark brown color of black liquor is
formed during the degradation processing of
lignocelluloses. Color is an indirect measurement
of the amount of lignin compounds in the effluent. The greater the amount of lignin compounds,
the darker the effluent and the greater the
tendency to produce foam [10]. Due to the
presence of various dissolved inorganic and
organic chemicals like dyes, heavy metals,
detergents, starch, etc., the effluent contains
normally high COD, heavy metals and high
alkalinity.
One of the main problems encountered in
biological wastewater treatment of paper mills is
poor settling of sludge. Moreover, bulking of
sludge is more frequent than usual.
Lime Ca(OH)2 was tested to improve settling
of sludge. Operating conditions were optimized
for the chemicals applied, their dosages and the
optimum pH. The process produced medium-

grade recycled water suitable for reuse, and

operating costs of producing this water were
determined.
The purpose of the present study is to examine
the effect of eight different strains on remediated
black liquor effluent, fewer than three different
supplement nutrients as primary treatment. This
was followed by a photo-Fentons reaction and
lime coagulation combination treatment as the
secondary treatment. The primary treatment
succeeded in removing 35100% COD, TSS
removal ranged from 490% and heavy metal
removal ranged from 1056%. The sequential
oxidation using Fentons reagent followed by
lime was very efficient for COD removal, ranging
from 47100%, TSS removal from 70 100%,
heavy metal removal from 1083%,
decolorization from 95100%. Bulking by settling of sludge was overcome.
2. Experimental
2.1. Materials
2.1.1. Organism
Different strains of bacteria, yeast and fungi
used for examination were obtained from the
Microbial Chemistry Unit, National Research
Center, and the Department of Agricultural
Microbiology Faculty of Agriculture, Cairo
University, Egypt, except for two strains of
white-rot fungi that were obtained from the
Department of Agriculture, Agriculture Research
Services, Peoria, IL, USA. Many attempts have
been made for decolorization of unsterilized
black liquor effluent by different studied strains
to find the most vigorous one.
2.1.2. Materials used in photochemical and
combination coagulation treatment
Authentic wastewater was obtained from a
water disposal site of Racta Paper Industries in
Alexandria. Hydrogen peroxide, Fentons rea-

S.M. Helmy et al. / Desalination 158 (2003) 331339

gent, calcium hydroxide and sulfuric acid (all

p.a.) were obtained from Merck and were used as
received. The solvent was determined by an
atomic absorption spectrophotometer, model
2380 (Perkins-Elmer) with a double beam and
background correction. The pH was measured by
a pH-meter (955, Fisher). For determination of
the COD, a spectrophotometer (DR2000, Hach)
was used. A UV lamp, model EMITA VP.60,
180 W, was used as the irradiation source. A
UV/VIS spectrophotometer model (559A,
Perkins-Elmer) was used to determine color
concentration in mg/L.
2.2. Methods
2.2.1. Culture and conditions for primary
treatment
The effluent samples were filtered through
ordinary filter paper and were supplemented with
optimal carbon and nitrogen sources. To
determine the influence of supplemented nutrition on reducing color of effluent, 1 ml of heavy
activated spore suspensions were cultivated separately in three different media (M1, M2 and M3)
without modified pH (pH of M1=4.84, M2 =4.72
and M3 = 6.74). Each flask (250 cm3) contained
50 cm3 sterilized media (M1,M2 or M3) with
25 cm3 of unsterilized effluent. The initial pHs
after adding the unsterilized effluent to each
medium were: M1 = 8.62, M2 = 7 and M3 =
7.48.The different media (in g/l) consisted of M1
= 1g yeast and 1 g glucose, M2 = 1.5 g NH4H2PO4
and 2 g glucose and M3 = 1 g KH2PO4, 4 g
Na2HPO4, 0.2 g NaCl, 0.2 g MgSO4 .7H2O and
0.2% (w/v) yeast extract (Difco) and 0.05 g
CaCl2. Eight strains were tested on the three
media mentioned above for their potential to
remove the dark brown color of unsterilized
effluent under sterilized conditions. The three
different media were incubated at 32C for 12
days under static conditions.

333

2.2.2. Secondary treatment: photo-Fentons

oxidation and lime coagulation combination
The authentic effluent after microbial treatment was centrifuged, then acidified, with sulfuric acid till pH = 3; then the photo-Fenton
reaction was followed. Fentons reagent was
added as 0.3 g ferrous sulfate and 5 ml H2O2 to 50
ml of the biologically pre-treated effluent
samples. Mixing was provided using a magnetic
stirrer in the presence of UV radiation, 180 W, for
half an hour. The addition of the first portion of
hydrogen peroxide and simultaneous start of UVirradiation marked the starting time of the
experiment. Lime was added in the form of a 10%
suspension until the desired pH of 5.5 was
reached. The lime suspension was dosed into
samples and a jar test was used. The jar test
procedure included a 1-min high shear mixing of
coagulant in the sample followed by 4 min of
slow mixing, and then 10 min of settling. The
clarity of the supernatant of each sample was
assessed visually, and for accuracy samples of the
supernatant were removed by pipette and then
analyzed for residual heavy metals, COD values,
total suspended solids and color removal. All
analysis was carried out following the procedures
outlined in the APHA [12].

3. Results and discussion

3.1. Effect of nutrient supplement on growth,
color, COD and total suspended solids reduction
The characteristics of the authentic black
liquor sample is present in Table 1. Eight different strains were examined for their potential to
remove the dark brown color of the effluent under
sterilized conditions. To find out the most
efficient strain on three different nutrients, M1,
M2 or M3 were supplemented singly to the original waste.

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S.M. Helmy et al. / Desalination 158 (2003) 331339

Table 1
Specifications of effluent sample before treatment
Parameter

Value

pH
COD, mg/L
TS, mg/L
DS, mg/L
TSS, mg/L
Color
Heavy metals, mg/L:
Cr
Cu
Fe
Ni
Pb

8.77
1790
1690
1080
59.4
Dark brown
0.140
0.344
1.960
0.152
0.11

3.1.1. Glucose and yeast (M1 )

The effects of this content on COD reduction
are presented in Table 2. The addition of glucose
(1 g/L) and yeast extract (1 g/L) growth factor
help to stimulate the vigorous activity of some
strains. In general, the best-characterized values
of COD removal were induced firstly by fungi
strains, followed by S. carlsprgenisis > bacteria
strains > yeast strains (ranging from 98.5% to
46.84%). This result was found less important
during the experiment. It declined according to
potential capacity by different strains such as
Diplodia oryzae > P. chrysosporium NRRL6364
> P. chrysporium NRRL6359 > Candida tropicalis > E. coli > Helminthsporium turcicum >
Saccharomyces carlsprgenisis >Candida pseudotropicalies). These results show similar trends as
reported by earlier workers [13,14]. Many studies
showed that degradation of xenobiotic compounds by P.chrysosporium was enhanced under
nutrient limited conditions [15]. However, high
concentrations of carbon or nitrogen were also
suggested to induce the peroxidases enzymes
production in other white rot fungal strains and
thus can positively affect the degradation rates
[16]. Some of the common substructures of lignin

resemble the chemical structure of many persistent organic compounds contaminating the
environment; consequently, the fungi can effectively degrade a pollutant [17]. From Table 2 we
obtain similar results in COD reduction, firstly by
P. chrysosorium NRRL 6364 (92.82%) and then
P. chrysosporium NRRL 6359 (79.64%). While
Diplodia oryzae is the most efficient strain
(98.50%), C. pseudotropicalies has a poor reduction of COD (46.84%).
3.1.2. Ammonia dihydrogen phosphate and
glucose (M2 )
This compound was more efficient than the
previous medium (M1). All strains displayed their
susceptibility for reduction value of COD.
Results show that the removal value of COD
ranged between (44.8397.13%) for 12 days of
incubation at 32C. P. chrysosporium NRRL
6364 showed a greater reduction in COD
(97.13%) than P. chrysosporium NRRL 6359
(85.06%), which played an important part of
pollutant removal [17]. Helminthsporium turcicum and Diplodia oryzae have a similar action
on COD (91.3091.38%), followed by Candida
tropicalis (87.76%). E. coli and yeast strains
recorded less efficient COD reduction except for
C. tropicalis (87.76%), as noted in Table 2.
3.1.3. Mineral salt solution (M3 )
This medium was less efficient than the
previous M1 and M2, as shown in Table 2. The
fluctuation shown in COD% removal ranged
from 34.4889.65% confirmed by Ian [13],
Kannan et al. [14], Katayama et al. [15] and Kaal
et al [16). COD reduction values descend
according to vital activity of different strains such
as Helminthsporium turcicum > Diplodia oryzae
> C. tropicalis > P. chrysosporium NRRL 6364
> S. calsprgenisis > P. chrysosporium NRRL
6359 > C. pseudotropicalies > E. coli).
Helminthsporium turcicum and Diplodia oryzae
are more efficient strains than Candida tropicalis

Table 2
Percentage of COD, TSS and color removal resulting from bioremediation of black liquor by eight strains in three different media (M1, M2 and M3)
followed by photo-Fetons oxidation and lime coagulation
Name of
organism

Primary treatment as bioremediation (% removal) Secondary treatment as photo-Fentons oxidation and lime coagulation
(% removal)
M2

M3

M1

M2

M3

COD TSS

COD

TSS

COD

TSS

COD

TSS

Color

COD

TSS

Color

COD

TSS

Color

E. coli

57.47 12.00

49.14

34.48

38.00

67.24

88.00

59.50

92.53

100

100

46.55

62.00

64.55

Candida
pseudotopicalies

46.84 39.60

44.83

44.67

46.55

66.93

82.90

60.40

75.00

95.80

55.33

75.00

100

99.34

100

Candida tropicali

58.62 62.93

87.76

53.06

85.06

75.80

100

86.5

95.98

46.94

90.00

95.06

100

30.00

Saccharomyces
carlsprgenisis

47.70 75.06

45.97

74.93

70.11

100

99.19

100

100

100

100

93.50

100

45.00

Diplodia oryzae

98.50 89.70

91.30

83.23

89.08

41.34

100

100

100

100

100

85.25

100

58.66

70.00

Helminthsporium
turcicum

56.32 33.73

91.38

4.00

89.65

92.36

98.53

77.00

100

99.46

81.00

100

100

100.00

P. chrysosporium
NRRL6359

79.64 12.13

85.06

15.60

62.360

99.30

71.20

67.50

100

84.40

100

93.30

97.73

77.5055

P. chrysosporium
NRRL6364

92.82 28.53

97.13

22.66

75.86

100

87.60

83

100

77.34

100

85.06

100

55.00

S. M. Helmy et al. / Desalination 158 (2003) 331339

M1

Note: No change in color was sensed after primary treatment.

335

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S.M. Helmy et al. / Desalination 158 (2003) 331339

in COD reduction 89.6589.08%, 85.06%

respectively). While P. chrysosporium NRRL
6364 (75.86%) and P. chrysosporium NRRL
6359 (62.36%) had less reduction values of COD
than M2 and M1. C. pseudotropicalis recorded
somewhat the result in COD reduction on media,
M1, M3 and M2 (46.8446.55% and 44.83%,
respectively. This medium was most efficient in
the case of Saccharomysis carlsprgenisis
(70.11%, while in M1 = 47.70% and M2 = 45.97%
COD reduction, respectively). From Table 2 the
highest COD removals were obtained by supplement nutrients in the sequence M1 > M2 > M3.
TSS percent removals were found less spectacular during experimental study on different
media by different strains, as shown in Table 2.
Diplodia oryzae is the most efficient strain in
TSS removal for media M1 and M2 (89.70
83.23%, respectively). Also S. carlsprgenisis and
C. tropicalis record the best results for media M1
and M2. Medium M3 is not available for TSS
removal except for C. pseudotropicalis (66.93%)
and Diplodia oryzae (41.34%).

3.2. Secondary treatment using photo-Fentons

oxidation and lime coagulation combination
An inoculums volume from each culture was
used for experimental determination of COD,
total suspended solids, color removal and some
heavy metals such as Cr, Cu, Fe, Ni and Pb.
Photochemical coagulation combination treatment methods proceeded as mentioned above in
Section 2.2.2. COD values, total suspended solids
and color removal were determined in 24 samples
and three control samples, each eight samples and
a control one in a different media M1or M2 or M3,
before and after photochemical coagulation
combination. The heavy metals in selected
samples under optimum conditions were determined before and after photochemical coagulation combination and in another control sample.

3.2.1. Effect of photo-Fentons reaction and

lime coagulation combination on COD% removal
It was observed that the photo-Fenton/UV/
lime coagulation combination achieved 90 100%
COD removal in 75% of the tested samples, while
the rest of the tested samples (25%) showed
46.585% COD removal as shown in Table 2. It
was noted that the pre-remediation of black liquor
effluent indicated the fluctuations in COD%
removal depending on the strain efficiency and
media of inoculation. Heinzle et al. [18] used a
combination of biological and chemical (ozone)
treatment for pulp and paper industry wastewater
and reached complete removal of pollutants.
Similar results achieved by the present study were
samples biologically pre-treated with medium M2
and combined with oxidation and coagulation
reached 100% COD removal [7] and treated pulp
and paper mill effluents using a combination of
coagulation and catalytic wet oxidation treatment.
The coagula-tion using Fe2(SO4)3 removed 50%
of the COD and the oxidation enhanced the
removal effi-ciency almost 100%. They also
added that dissolved organic matter could be
easily removed biologically. Freire et al. studied
remediation and toxicity removal from paper-mill
effluent by ozonation and UV irradiation. They
concluded that a treatment reaction with time of
90 min was the most effective for decoloration
(45%). These results agree with our results that
confirmed that 30 min was the most effective for
COD, TSS and color 100% removal using a
photooxidation and lime coagulation
combination.
3.2.2. Effect of photo-Fentons reaction and
lime coagulation on TSS % removal
The reaction with lime was applied as
mentioned in Section 2.2.2. Table 2 shows that
the lime combination coagulation removed TSS
by 90100% in two-thirds of the total samples
while 4684% TSS removal was achieved in the

S.M. Helmy et al. / Desalination 158 (2003) 331339

rest of the samples. Murphy et al. [19] used

advanced oxidation treatment using hydrogen
peroxide and ultraviolet-radiation of pulp and
paper mill wastewater. Olecksy and Jekel [20]
showed the same results that the biological
treatment of black liquor is generally less
selective and they used aluminum precipitation;
it was very effective and agrees with our results.
Liu [21] used a treatment method for black liquor
of polymer complexed with lime. He added that
these flocculants with post-coagulation were
better for purifying water than any single or
mechanical mixture. These methods confirmed

337

that the secondary treatment combination used in

our study can give purified water that can be used
as medium-grade recycled water.
3.2.3. Effect of photo-Fentons reaction and
lime coagulation on color removal
Table (2) shows that 80% of the samples in M1
achieved color removal >80100%, while in M2
90% of samples recorded 80100% removal and
50% of treated samples in M3 illustrates 77100.
Cikurel et al. [22] illustrated that waste-water
after tertiary treatment can be used in

Table 3
Determination of percentage of heavy metal removal for three effective strains in three different media (M1, M2 and M3)
after primary and secondary treatment
Element
name

Element
initial conc.,
mg/L

Medium
used

Organism name
and media used

Element %
removal after
primary
treatment

Element %
removal after
secondary
treatment

Allowable
limits in
Law 4 to the
year 1992

Pb

0.11

M1
M2

Diplodia oryzae
P. chrysosporium
NRRL6364
Helminthsporium turcicum

0.00
>10

9.09
>10

0.1

9.09

>10

Diplodia oryzae
P. chrysosporium
NRRL6364
Helminthsporium turcicum

13.57
11.5

19.28
12.14

9.46

26.61

Diplodia oryzae
P. chrysosporium
NRRL6364
Helminthsporium turcicum

13.66
32.41

83.14
63.95

24.56

82.55

Diplodia oryzae
P. chrysosporium
NRRL6364
Helminthsporium turcicum

10.71
20.73

64.114
60.167

13.55

25.99

Diplodia oryzae
P. chrysosporium
NRRL6364
Helminthsporium turcicum

55.26
28.75

65.79
65.79

21.053

67.76

M3
Cr

0.14

M1
M2
M3

Cu

0.344

M1
M2
M3

Fe

1.196

M1
M2
M3

Ni

0.152

M1
M2
M3

0.05

0.1

0.05

0.1

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S.M. Helmy et al. / Desalination 158 (2003) 331339

agriculture for irrigation without causing dripper

clogging, and it can be used in industry for
cooling towers and in some cases as process
water (e.g., textile, paper) after color and
suspended soilds removal and disinfection.
3.2.4. Effect of remediation, photo-Fentons
reaction and lime combination treatment on
heavy metals removal
Table 3 shows some heavy metals such as Pb,
Cr, Cu, Fe and Ni that are often present in black
liquor effluent and indicates their percentage of
removal. Coagulation is an easy and cheap
method to remove such clean-up pollutants. For
Pb only 10% removal was achieved by both the
biological and chemical treatment, although it
achieved the allowable detection limits. Diplodia
oryzae in M1 did not respond with Pb elimination. The lime combination treatment showed
1030% Cr removal. This chemical coagulation
combination increased Cr% removal twice that
achieved by the biological pre-treatment. Cu
showed the highest % removal in the tested
metals and recorded 83% removal after chemical
coagulation. P. chrysosporium NRRL6364 in the
M2 medium was capable of decreasing Cu by
32.4% after the biological treatment. Similar
trends were obtained by Dundee [23]. Ni showed
2070% removal. The biological treatment
showed 2055% removal where Diplodia oryzae

removed 55% and the chemical coagulation

removed 6579% of Ni.
Bismas et al. studied a number of heavy
metals in textile-mill effluent using coagulation
treatment before biological treatment to remove
heavy metals and achieved considerable removal,
>90%. It was observed that the photo-Fentons
coagulation treatment was most effective for
removing COD, TSS, heavy metals and color,
although considerable amounts were removed by
initial biological pre-treatment, especially D.
oryzae and P. chrysosporium NRRL6364, which
enhanced the chemical treatment to reach
optimum clean-up removal and achieved 100%
completion.
4. Preliminary economic evaluation study
A preliminary cost estimation was calculated
according to the prices of raw materials necessary
for the treatment of 1 L of black liquor effluent.
The costs are given in Table 4. Treatment of one
liter cost 0.20029 L.E. Rupert and Hubert [24]
studied different advanced oxidation processes
for wastewater treatment and concluded that the
photo-Fenton system was the most effective compared to Ti2/UV, Fe2+/H2 O2/UV and Fe2+/O3 /UV.
They added a rough cost estimation which proved
to be far cheaper than other available advanced
oxidation processes, namely ozonization.

Table 4
Relationship between optimum dose used for treatment of 1 L of wastewater and its relative cost (in L.E.)
Materials

Price

Optimum dose/L

Cost for 1 L treatment

Ferrous sulfate, kg
Hydrogen peroxide 10%, L
Sulfuric acid, L
Calcium hydroxide, kg
Total cost of materials

3.9
1.75
3.20
0.80

6g
100 ml
2 ml
5g

0.02340
0.17200
0.00064
0.00425
0.20029

S.M. Helmy et al. / Desalination 158 (2003) 331339

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