Presented at the European Conference on Desalination and the Environment: Fresh Water for All, Malta, 4–8 May 2003.European Desalination Society, International Water Association.

0011-9164/03/$– See front matter © 2003 Elsevier Science B.V. All rights reserved

Desalination 158 (2003) 331–339

Bioremediation post-photo-oxidation and coagulation for blackliquor effleunt treatment

Samia M. Helmy, Shadia El Rafie*, Montaser Y. GhalyDepartment of Microbial Chemistry and Department of Chemical Engineering, National Research Center,

Dokki, Cairo, EgyptTel. +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-Fenton’s advanced oxidationand chemical coagulation were investigated as options to remove both non-biodegradable COD and color from pre-bioremediated black liquor effluent. It was found that at the first bioremediation COD removal ranged from 35–98%and TSS removal ranged from 12–89%. Almost 20% of the heavy metals were removed. Photo-Fenton’s UV advancedoxidation and lime coagulation combination treatment achieved complete removal of COD, TSS and color. It was ableto remove 80–100% COD, 70–100% TSS and 77–100% 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 thethree media. Diplodia oryzae and Phaerochaete chrysosporium NRRL6364 enhanced the post-physicochemicaltreatment to reach optimum clean-up after 12 days of incubation at 32°C. They produced a medium-grade recycledwater. Operating costs are outlined.

Keywords: Bioremediation; Advanced oxidation process; Wastewater; Recycling; Treatment

1. Introduction

Many industrial wastewaters contain highamounts of nondegradable organics. Paper andpulp industry effluent is one of the important

*Corresponding author.

environmental problems, which has been cor-related with mutagenic and carcinogenic activity[1,2]. Many attempts have been made to removeblack liquor by bioremediation, which is con-sidered inexpensive clean-up technology [3,4].

Industrial biotechnology applications help theelimination of environmentally hazardous wastes.

S.M. Helmy et al. / Desalination 158 (2003) 331–339332

Some application methods of biotechnologiessuch as Aspergillus sp. remove color of effluentfrom pulp waste [5] and from kraft mill byPhanerochaete chrysosporium [6]. Still bio-logical treatment will not remove all organics andthe effluents of the biological purification stagesexhibit COD values that may exceed dischargestandards. Advanced oxidation processes (AOP)using OH radicals are of great promise for CODreduction of these effluents.

Verenish et al. [7] treated pulp and papereffluent using a combination of photo-oxidationcoagulation treatment and enhanced the removalefficiency almost 100%.

Hydroxyl radicals are strong and nonselectiveoxidants for organic pollutants in wastewater.The photo-Fenton method was applied success-fully for highly contaminated wastewaters up toCOD 12,000 mg/L. The photo-Fenton reaction ismainly a metal to ligand charge transfer thatproduces the hydroxyl radical. In addition, it canbe compared to reactions that are also of greatimportance for pollutants degradation [8,9].

The dark brown color of black liquor isformed during the degradation processing oflignocelluloses. Color is an indirect measurementof the amount of lignin compounds in the efflu-ent. The greater the amount of lignin compounds,the darker the effluent and the greater thetendency to produce foam [10]. Due to thepresence of various dissolved inorganic andorganic chemicals like dyes, heavy metals,detergents, starch, etc., the effluent containsnormally high COD, heavy metals and highalkalinity.

One of the main problems encountered inbiological wastewater treatment of paper mills ispoor settling of sludge. Moreover, bulking ofsludge is more frequent than usual.

Lime Ca(OH)2 was tested to improve settlingof sludge. Operating conditions were optimizedfor the chemicals applied, their dosages and theoptimum pH. The process produced medium-

grade recycled water suitable for reuse, andoperating costs of producing this water weredetermined.

The purpose of the present study is to examinethe effect of eight different strains on remediatedblack liquor effluent, fewer than three differentsupplement nutrients as primary treatment. Thiswas followed by a photo-Fenton’s reaction andlime coagulation combination treatment as thesecondary treatment. The primary treatmentsucceeded in removing 35–100% COD, TSSremoval ranged from 4–90% and heavy metalremoval ranged from 10–56%. The sequentialoxidation using Fenton’s reagent followed bylime was very efficient for COD removal, rangingfrom 47–100%, TSS removal from 70– 100%,heavy metal removal from 10–83%,decolorization from 95–100%. Bulking by set-tling of sludge was overcome.

2. Experimental2.1. Materials

2.1.1. OrganismDifferent strains of bacteria, yeast and fungi

used for examination were obtained from theMicrobial Chemistry Unit, National ResearchCenter, and the Department of AgriculturalMicrobiology Faculty of Agriculture, CairoUniversity, Egypt, except for two strains ofwhite-rot fungi that were obtained from theDepartment of Agriculture, Agriculture ResearchServices, Peoria, IL, USA. Many attempts havebeen made for decolorization of unsterilizedblack liquor effluent by different studied strainsto find the most vigorous one.

2.1.2. Materials used in photochemical andcombination coagulation treatment

Authentic wastewater was obtained from awater disposal site of Racta Paper Industries inAlexandria. Hydrogen peroxide, Fenton’s rea-

S.M. Helmy et al. / Desalination 158 (2003) 331–339 333

gent, calcium hydroxide and sulfuric acid (allp.a.) were obtained from Merck and were used asreceived. The solvent was determined by anatomic absorption spectrophotometer, model2380 (Perkins-Elmer) with a double beam andbackground correction. The pH was measured bya pH-meter (955, Fisher). For determination ofthe COD, a spectrophotometer (DR2000, Hach)was used. A UV lamp, model EMITA VP.60,180 W, was used as the irradiation source. AUV/VIS spectrophotometer model (559A,Perkins-Elmer) was used to determine colorconcentration in mg/L.

2.2. Methods

2.2.1. Culture and conditions for primarytreatment

The effluent samples were filtered throughordinary filter paper and were supplemented withoptimal carbon and nitrogen sources. Todetermine the influence of supplemented nutri-tion on reducing color of effluent, 1 ml of heavyactivated spore suspensions were cultivated sepa-rately in three different media (M1, M2 and M3)without modified pH (pH of M1=4.84, M2 =4.72and M3 = 6.74). Each flask (250 cm3) contained50 cm3 sterilized media (M1,M2 or M3) with25 cm3 of unsterilized effluent. The initial pHsafter adding the unsterilized effluent to eachmedium 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 NH4H2PO4and 2 g glucose and M3 = 1 g KH2PO4, 4 gNa2HPO4, 0.2 g NaCl, 0.2 g MgSO4 .7H2O and0.2% (w/v) yeast extract (Difco) and 0.05 gCaCl2. Eight strains were tested on the threemedia mentioned above for their potential toremove the dark brown color of unsterilizedeffluent under sterilized conditions. The threedifferent media were incubated at 32°C for 12days under static conditions.

2.2.2. Secondary treatment: photo-Fenton’soxidation and lime coagulation combination

The authentic effluent after microbial treat-ment was centrifuged, then acidified, with sul-furic acid till pH = 3; then the photo-Fentonreaction was followed. Fenton’s reagent wasadded as 0.3 g ferrous sulfate and 5 ml H2O2 to 50ml of the biologically pre-treated effluentsamples. Mixing was provided using a magneticstirrer in the presence of UV radiation, 180 W, forhalf an hour. The addition of the first portion ofhydrogen peroxide and simultaneous start of UV-irradiation marked the starting time of theexperiment. Lime was added in the form of a 10%suspension until the desired pH of 5.5 wasreached. The lime suspension was dosed intosamples and a jar test was used. The jar testprocedure included a 1-min high shear mixing ofcoagulant in the sample followed by 4 min ofslow mixing, and then 10 min of settling. Theclarity of the supernatant of each sample wasassessed visually, and for accuracy samples of thesupernatant were removed by pipette and thenanalyzed for residual heavy metals, COD values,total suspended solids and color removal. Allanalysis was carried out following the proceduresoutlined in the APHA [12].

3. Results and discussion3.1. Effect of nutrient supplement on growth,color, COD and total suspended solids reduction

The characteristics of the authentic blackliquor sample is present in Table 1. Eight dif-ferent strains were examined for their potential toremove the dark brown color of the effluent understerilized conditions. To find out the mostefficient strain on three different nutrients, M1,M2 or M3 were supplemented singly to the ori-ginal waste.

S.M. Helmy et al. / Desalination 158 (2003) 331–339334

Table 1Specifications of effluent sample before treatment

Parameter Value

pHCOD, mg/LTS, mg/LDS, mg/LTSS, mg/LColorHeavy metals, mg/L: Cr Cu Fe Ni Pb

8.7717901690108059.4Dark brown

0.1400.3441.9600.1520.11

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 factorhelp to stimulate the vigorous activity of somestrains. In general, the best-characterized valuesof COD removal were induced firstly by fungistrains, followed by S. carlsprgenisis > bacteriastrains > yeast strains (ranging from 98.5% to46.84%). This result was found less importantduring the experiment. It declined according topotential capacity by different strains such asDiplodia oryzae > P. chrysosporium NRRL6364> P. chrysporium NRRL6359 > Candida tropi-calis > E. coli > Helminthsporium turcicum >Saccharomyces carlsprgenisis >Candida pseudo-tropicalies). These results show similar trends asreported by earlier workers [13,14]. Many studiesshowed that degradation of xenobiotic com-pounds by P.chrysosporium was enhanced undernutrient limited conditions [15]. However, highconcentrations of carbon or nitrogen were alsosuggested to induce the peroxidases enzymesproduction in other white rot fungal strains andthus can positively affect the degradation rates[16]. Some of the common substructures of lignin

resemble the chemical structure of many per-sistent organic compounds contaminating theenvironment; consequently, the fungi can effec-tively degrade a pollutant [17]. From Table 2 weobtain similar results in COD reduction, firstly byP. chrysosorium NRRL 6364 (92.82%) and thenP. chrysosporium NRRL 6359 (79.64%). WhileDiplodia oryzae is the most efficient strain(98.50%), C. pseudotropicalies has a poor reduc-tion of COD (46.84%).

3.1.2. Ammonia dihydrogen phosphate andglucose (M2 )

This compound was more efficient than theprevious medium (M1). All strains displayed theirsusceptibility for reduction value of COD.Results show that the removal value of CODranged between (44.83–97.13%) for 12 days ofincubation at 32°C. P. chrysosporium NRRL6364 showed a greater reduction in COD(97.13%) than P. chrysosporium NRRL 6359(85.06%), which played an important part ofpollutant removal [17]. Helminthsporium tur-cicum and Diplodia oryzae have a similar actionon COD (91.30–91.38%), followed by Candidatropicalis (87.76%). E. coli and yeast strainsrecorded less efficient COD reduction except forC. 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. Thefluctuation shown in COD% removal rangedfrom 34.48–89.65% confirmed by Ian [13],Kannan et al. [14], Katayama et al. [15] and Kaalet al [16). COD reduction values descendaccording to vital activity of different strains suchas Helminthsporium turcicum > Diplodia oryzae> C. tropicalis > P. chrysosporium NRRL 6364> S. calsprgenisis > P. chrysosporium NRRL6359 > C. pseudotropicalies > E. coli).Helminthsporium turcicum and Diplodia oryzaeare more efficient strains than Candida tropicalis

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Table 2Percentage 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-Feton’s oxidation and lime coagulation

Name oforganism

Primary treatment as bioremediation (% removal) Secondary treatment as photo-Fenton’s oxidation and lime coagulation(% removal)

M1 M2 M3 M1 M2 M3

COD TSS COD TSS COD TSS COD TSS Color COD TSS Color COD TSS Color

E. coli

Candidapseudotopicalies

Candida tropicali

Saccharomycescarlsprgenisis

Diplodia oryzae

Helminthsporiumturcicum

P. chrysosporiumNRRL6359

P. chrysosporiumNRRL6364

57.47

46.84

58.62

47.70

98.50

56.32

79.64

92.82

12.00

39.60

62.93

75.06

89.70

33.73

12.13

28.53

49.14

44.83

87.76

45.97

91.30

91.38

85.06

97.13

0

44.67

53.06

74.93

83.23

4.00

15.60

22.66

34.48

46.55

85.06

70.11

89.08

89.65

62.360

75.86

38.00

66.93

0

0

41.34

0

0

0

67.24

82.90

75.80

100

100

92.36

99.30

100

88.00

60.40

100

99.19

100

98.53

71.20

87.60

59.50

75.00

86.5

100

100

77.00

67.50

83

92.53

95.80

95.98

100

100

100

100

100

100

55.33

46.94

100

100

99.46

84.40

77.34

100

75.00

90.00

100

85.25

81.00

100

100

46.55

100

95.06

93.50

100

100

93.30

85.06

62.00

99.34

100

100

58.66

100

97.73

100

64.55

100

30.00

45.00

70.00

100.00

77.5055

55.00

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

S.M. Helmy et al. / Desalination 158 (2003) 331–339336

in COD reduction 89.65–89.08%, 85.06%respectively). While P. chrysosporium NRRL6364 (75.86%) and P. chrysosporium NRRL6359 (62.36%) had less reduction values of CODthan M2 and M1. C. pseudotropicalis recordedsomewhat the result in COD reduction on media,M1, M3 and M2 (46.84–46.55% and 44.83%,respectively. This medium was most efficient inthe case of Saccharomysis carlsprgenisis(70.11%, while in M1 = 47.70% and M2 = 45.97%COD reduction, respectively). From Table 2 thehighest COD removals were obtained by supple-ment nutrients in the sequence M1 > M2 > M3.TSS percent removals were found less spec-tacular during experimental study on differentmedia by different strains, as shown in Table 2.Diplodia oryzae is the most efficient strain inTSS removal for media M1 and M2 (89.70–83.23%, respectively). Also S. carlsprgenisis andC. tropicalis record the best results for media M1and M2. Medium M3 is not available for TSSremoval except for C. pseudotropicalis (66.93%)and Diplodia oryzae (41.34%).

3.2. Secondary treatment using photo-Fenton’soxidation and lime coagulation combination

An inoculums volume from each culture wasused for experimental determination of COD,total suspended solids, color removal and someheavy metals such as Cr, Cu, Fe, Ni and Pb.Photochemical coagulation combination treat-ment methods proceeded as mentioned above inSection 2.2.2. COD values, total suspended solidsand color removal were determined in 24 samplesand three control samples, each eight samples anda control one in a different media M1or M2 or M3,before and after photochemical coagulationcombination. The heavy metals in selectedsamples under optimum conditions were deter-mined before and after photochemical coagula-tion combination and in another control sample.

3.2.1. Effect of photo-Fenton’s reaction andlime 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, whilethe rest of the tested samples (25%) showed46.5–85% COD removal as shown in Table 2. Itwas noted that the pre-remediation of black liquoreffluent indicated the fluctuations in COD%removal depending on the strain efficiency andmedia of inoculation. Heinzle et al. [18] used acombination of biological and chemical (ozone)treatment for pulp and paper industry wastewaterand reached complete removal of pollutants.Similar results achieved by the present study weresamples biologically pre-treated with medium M2and combined with oxidation and coagulationreached 100% COD removal [7] and treated pulpand paper mill effluents using a combination ofcoagulation and catalytic wet oxidation treatment.The coagula-tion using Fe2(SO4)3 removed 50%of the COD and the oxidation enhanced theremoval effi-ciency almost 100%. They alsoadded that dissolved organic matter could beeasily removed biologically. Freire et al. studiedremediation and toxicity removal from paper-milleffluent by ozonation and UV irradiation. Theyconcluded that a treatment reaction with time of90 min was the most effective for decoloration(45%). These results agree with our results thatconfirmed that 30 min was the most effective forCOD, TSS and color 100% removal using aphotooxidation and lime coagulationcombination.

3.2.2. Effect of photo-Fenton’s reaction andlime coagulation on TSS % removal

The reaction with lime was applied asmentioned in Section 2.2.2. Table 2 shows thatthe lime combination coagulation removed TSSby 90–100% in two-thirds of the total sampleswhile 46–84% TSS removal was achieved in the

S.M. Helmy et al. / Desalination 158 (2003) 331–339 337

rest of the samples. Murphy et al. [19] usedadvanced oxidation treatment using hydrogenperoxide and ultraviolet-radiation of pulp andpaper mill wastewater. Olecksy and Jekel [20]showed the same results that the biologicaltreatment of black liquor is generally lessselective and they used aluminum precipitation;it was very effective and agrees with our results.Liu [21] used a treatment method for black liquorof polymer complexed with lime. He added thatthese flocculants with post-coagulation werebetter for purifying water than any single ormechanical mixture. These methods confirmed

that the secondary treatment combination used inour study can give purified water that can be usedas medium-grade recycled water.

3.2.3. Effect of photo-Fenton’s reaction andlime coagulation on color removal

Table (2) shows that 80% of the samples in M1achieved color removal >80–100%, while in M290% of samples recorded 80–100% removal and50% of treated samples in M3 illustrates 77–100.Cikurel et al. [22] illustrated that waste-waterafter tertiary treatment can be used in

Table 3Determination of percentage of heavy metal removal for three effective strains in three different media (M1, M2 and M3)

after primary and secondary treatment

Elementname

Elementinitial conc.,mg/L

Mediumused

Organism nameand media used

Element %removal afterprimarytreatment

Element %removal aftersecondarytreatment

Allowablelimits inLaw 4 to theyear 1992

Pb 0.11 M1

M2

M3

Diplodia oryzaeP. chrysosporiumNRRL6364Helminthsporium turcicum

0.00>10

9.09

9.09>10

>10

0.1

Cr 0.14 M1

M2

M3

Diplodia oryzaeP. chrysosporiumNRRL6364Helminthsporium turcicum

13.5711.5

9.46

19.2812.14

26.61

0.05

Cu 0.344 M1

M2

M3

Diplodia oryzaeP. chrysosporiumNRRL6364Helminthsporium turcicum

13.6632.41

24.56

83.1463.95

82.55

0.1

Fe 1.196 M1

M2

M3

Diplodia oryzaeP. chrysosporiumNRRL6364Helminthsporium turcicum

10.7120.73

13.55

64.11460.167

25.99

0.05

Ni 0.152 M1

M2

M3

Diplodia oryzaeP. chrysosporiumNRRL6364Helminthsporium turcicum

55.2628.75

21.053

65.7965.79

67.76

0.1

S.M. Helmy et al. / Desalination 158 (2003) 331–339338

agriculture for irrigation without causing dripperclogging, and it can be used in industry forcooling towers and in some cases as processwater (e.g., textile, paper) after color andsuspended soilds removal and disinfection.

3.2.4. Effect of remediation, photo-Fenton’sreaction and lime combination treatment onheavy metals removal

Table 3 shows some heavy metals such as Pb,Cr, Cu, Fe and Ni that are often present in blackliquor effluent and indicates their percentage ofremoval. Coagulation is an easy and cheapmethod to remove such clean-up pollutants. ForPb only 10% removal was achieved by both thebiological and chemical treatment, although itachieved the allowable detection limits. Diplodiaoryzae in M1 did not respond with Pb elimi-nation. The lime combination treatment showed10–30% Cr removal. This chemical coagulationcombination increased Cr% removal twice thatachieved by the biological pre-treatment. Cushowed the highest % removal in the testedmetals and recorded 83% removal after chemicalcoagulation. P. chrysosporium NRRL6364 in theM2 medium was capable of decreasing Cu by32.4% after the biological treatment. Similartrends were obtained by Dundee [23]. Ni showed20–70% removal. The biological treatmentshowed 20–55% removal where Diplodia oryzae

removed 55% and the chemical coagulationremoved 65–79% of Ni.

Bismas et al. studied a number of heavymetals in textile-mill effluent using coagulationtreatment before biological treatment to removeheavy metals and achieved considerable removal,>90%. It was observed that the photo-Fenton’scoagulation treatment was most effective forremoving COD, TSS, heavy metals and color,although considerable amounts were removed byinitial biological pre-treatment, especially D.oryzae and P. chrysosporium NRRL6364, whichenhanced the chemical treatment to reachoptimum clean-up removal and achieved 100%completion.

4. Preliminary economic evaluation study

A preliminary cost estimation was calculatedaccording to the prices of raw materials necessaryfor the treatment of 1 L of black liquor effluent.The costs are given in Table 4. Treatment of oneliter cost 0.20029 L.E. Rupert and Hubert [24]studied different advanced oxidation processesfor wastewater treatment and concluded that thephoto-Fenton system was the most effective com-pared to Ti2/UV, Fe2+/H2 O2/UV and Fe2+/O3 /UV.They added a rough cost estimation which provedto be far cheaper than other available advancedoxidation processes, namely ozonization.

Table 4Relationship 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, kgHydrogen peroxide 10%, LSulfuric acid, LCalcium hydroxide, kgTotal cost of materials

3.91.753.200.80

6 g100 ml2 ml5 g

0.023400.172000.000640.004250.20029

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