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Volume 3 Issue 11 1000169

J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Research Article Open Access
Cordi et al., 2012, 3:11
Research article Open Access
Bioremediation & Biodegradation
Keywords: Biomass; Wastewater treatment; Activated sludge;
Microorganism; Protozoa; Biomarker
Te pulp and paper industry is considered to be a serious
environmental hazard due to the multiplicity of impurities to the
complexity of the chemical structures present and to the signifcant
quantities of wastewater generated [1,2]. Tis industry sector discharges
large volumes of brown colored efuents as a result of the diferent
processes applied in wood and pulp bleaching, generating diverse
pollutants [3,4]. Te high chemical diversity of these pollutants causes
a variety of clastogenic, carcinogenic and mutagenic efects on fsh and
other aquatic communities in recipient water bodies [5].
Te main treatment process used at pulp and paper mill plants
involves primary clarifcation, succeeded by secondary treatment,
generally of a biological nature. Tere are numerous biological
treatment systems available, the most common being the activated
sludge process [6,7].
Activated sludge system technology consists basically in the
agitation of the efuent in the presence of aerobic bacteria, protozoa,
metazoa and atmospheric oxygen for a sufcient period to metabolize
and to focculate a large part of the organic material [8-10].
Te most common organisms that participate in the biodegradation
of organic material in the biological system for efuent treatment are
bacteria, protozoa and annelids [8,11,12]. Te presence of particular
types of protozoa is related to efuent quality and plant performance,
so they can be used as bioindicators [13]. Protozoa play a secondary but
important role in wastewater system purifcation [14-16].
Te protozoa in the activated sludge treatment process fall into four
main classes: amoebae, fagellates and ciliates (free-swimming, crawling
and stalked) and metazoa that are separated into rotifers, nematodes
(sub-class) and Oligotrichia such as Aelosoma [14,17].
To apply the activated sludge process to industrial efuents, which
have specifc compositions, efuent characterization is needed before
treatment and the sludge needs to be acclimatized (microorganism
acclimation to the efuent to be treated) [18,19]. Te aim of this work
was verifying the changes of the activated sludge microorganisms
*Corresponding author: Nelson Duran, Institute of Chemistry, Biological
Chemistry Laboratory, Center of Natural and Human Sciences, Universidade
Federal do ABC, Santo Andr, SP, Brazil; Tel: 55 19 3521-3149; E-mail:
Received December 22, 2011; Accepted October 12, 2012; Published October
14, 2012
Citation: Cordi L, Assalin MR, Ponezi AN, Durn N (2012) Identifcation of
Microbiota for Activated Sludge Acclimated By Paper Mill Effuent Kraft E1
Bioremediation. J Bioremed Biodeg 3:169. doi:10.4172/2155-6199.1000169
Copyright: 2012 Cordi L, et al. This is an open-a ccess article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Identification of Microbiota for Activated Sludge Acclimated By Paper Mill
Effluent Kraft E
Lvia Cordi
, Mrcia Regina Assalin
, Alexandre Nunes Ponezi
and Nelson Durn
Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, P.O.Box 6154, 13083-970, Campinas-SP, SP, Brazil
EMBRAPA (Environment), Rodovia SP 340 km 127.5, Tanquinho Velho, P.O.Box 69, 13820-000, Jaguarina, SP, Brazil
Division of Microbial Resources, Research Centre for Chemistry, Biology and Agriculture (CPQBA), Universidade Estadual de Campinas, P.O.Box 6171, Campinas, SP,
13081-970, Brazil
Center of Natural and Human Sciences, Universidade Federal do ABC, Santo Andr, SP, Brazil
Conventional systems treating paper effuents and cellulose are quite effcient in the reduction of Chemical
Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD). In general, there is primary stage clarifcation
followed by secondary treatment, usually with a biological system (activated sludge). However, for the effcient
degradation of some organic compounds in this effuent, adapted microorganism accomplish the treatment which
are necessary resulting in effuents with low concentrations of recalcitrant compounds. There are two methods for
microorganism acclimation: genes cloning or natural.
In this work, the original biomass collected from a domestic sewage treatment plant was adapted for application
in the bioremediation of paper mill effuent, Kraft E
. So, the aim of this work was verifying the correlation of protozoa
organisms and physical-chemical parameters during the acclimation period.
The acclimation of the biomass was verifed mainly through the reduction of COD. The monitoring of biomass
growth was followed by measurement of suspended solids and volatile suspended solids. Parameters such as
pH, Dissolved Oxygen (DO) and temperature were also monitored daily. The biological study was accomplished
microscopically through the identifcation of protozoa that are considered bioindicators of the activated sludge system.
The biomass acclimation period was 39 days. The stabilization of the system was observed through constant rates of
total COD (56%). The suspended solids at the beginning of the acclimation phase were from 1580 mg L
to 3580 mg
, indicating biomass growth. A small acclimation period was necessary to observe the diversity of protozoa, mainly
free ciliates, but nematodes were also present, indicating poor sludge sediment. At the end of the acclimation phase
predominantly rotifers, but some free ciliates, ciliated communities and fxed ciliated were observed, indicating a
good purifcation capacity of the system. Within the organisms amoeba was observed and this indicates good quality
to the fnal effuent. Then, it is possible to correlate the biota presence with the high capacity of COD removal from
the activated sludge system.
Citation: Cordi L, Assalin MR, Ponezi AN, Durn N (2012) Identifcation of Microbiota for Activated Sludge Acclimated By Paper Mill Effuent Kraft E1
Bioremediation. J Bioremed Biodeg 3:169. doi:10.4172/2155-6199.1000169
Volume 3 Issue 11 1000169
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Page 2 of 4
during acclimation period in the bioremediation of paper mill efuent
from the Kraf E
Materials and Methods
Paper mill efuent
Wastewater was obtained from a bleach Kraf mill located in the
Campinas city region (So Paulo State, Brazil). Tis mill process uses
only Eucalyptus grandis wood. Sample efuent was obtained afer the
frst alkaline extraction stage (E
). Te aqueous samples were stored at
4C in bottle fasks and used without prior fltration.
Acclimation of activated sludge to kraf E
paper efuent
Te acclimation of the activated sludge biomass from the Samambaia
Sewage Treatment Plant, in a suburb of eastern region of Campinas, SP,
Brazil, was studied for 39 days by the natural selection method. Te
biomass acclimation was followed by COD reduction from the efuent.
Te biomass growth was observed by Suspended Solids (SS) and
Volatile Suspended Solids (VSS). Te activated sludge was collected and
acclimated in a reactor (Figure 1) by specifc conditions using a Kraf
efuent as feed to microorganisms presents in the biological system.
Activated sludge system
An aerobic reactor (2.5 L) was operated as a continuous system
and continuously fed with Kraf E
efuent (at pH 7) by means of a
peristaltic pump. Te Hydraulic Retention Time (HRT) was maintained
at 12 h. Te sludge was periodically recycled, and excess sludge which
grew during the aeration stage was withdrawn from the bottom to
obtain Solids Retention Times (SRT) in 10 days. Te dissolved oxygen
concentration was maintained above 5 mg L
by an aquarium-type air
pump with sintered-sand difusers at the bottom of the reactor [16].
Te schema of activated sludge reactor used in this study was presented
in fgure 1.
Analytical control
pH Determination: Te pH was measured using an Orion model
EA 940 pH meter from Cole-Parmer, with a combined glass electrode.
Determination of total suspended solids: For analysis of
suspended solids an aliquot of 10 mL of sludge from the bioreactors was
fltered using GF-C Whatman flter papers that were previously dried
and weighed (retention sizes of 1 mm). Afer fltration and drying for 2 h
in an oven (~100C), the flter paper containing the solids was removed
at room temperature and again weighed. From the mass diference the
amount of suspended solid was calculated.
Determination of volatile suspended solids: A 100 mL aliquot
of sludge coming from the bioreactor was fltered using a previously
weighed nitrocellulose flter (pore size 0.2 mm). Te flter plus solids
was dried at 105C during 2 h. Afer this, it was lef in a desiccator for 1
h and again weighed. Te mass of the flter with solid was determined
and by diference the amount of suspended solid.
Determination of Chemical Oxygen Demand (COD): A COD
was measured using the procedure of the Standard Methods for the
Examination of Water and Wastewater of the American Society of Civil
Engineers (1992) [20].
Biological control
Qualitative analysis of protozoa and metazoa was carried out by
optical microscopy (Olimpus Vanox microscopy, model AH2) and
these organisms were divided into Philo Protozoa, protozoan classes
such as sarcodine, ciliata, mastigophora and metazoa, such as rotifers,
nematode, tardigrade and anelide [8].
Results and Discussion
Prior to this experiment in a continuous activated sludge reactor,
the biomass from domestic wastewater was acclimated to the substrate
of interest (Kraf E
efuent) for 39 days to permit the growth of
the microorganisms which can use the efuent as a carbon source.
Acclimation is widely used to maximize the removal efciency of specifc
compounds that are difcult to biodegrade. Te steady-state condition
was verifed when the average of the consecutive measurements showed
a constant rate of COD removal [7].
Te acclimation process was stopped when the total COD removal
rate was constant, around 56%. Te operating parameters of activated
sludge reactor were present in the table 1. Te variation of the operating
parameters observed in this study was according to the literature [21].
Te oxygen dissolved concentration in the activated sludge reactor
was maintained using an aquarium-type air pump with sintered-sand
difusers that presented a good aeration to homogenize the aerobic
phase permitting the focks formation and stabilization, avoiding Pin
Floc that indicate bad settleability conditions, which causes the biomass
loss in the reactor system [22]. It is important to maintain the DO at the
minimum about 2 mg L
Te biomass concentration has an important efect on treatment
performance. Previous studies had shown that the minimum of Mixed
Liquor Suspended Solids (MLSS) in the activated sludge process for
paper mill efuent treatment is 2000-2500 mg L
[1]. Tsang et al. [2]
showed that a higher concentration of MLSS promoted activated sludge
Figure 1: Activated sludge reactor system applied to effuent treatment.
Operating Parameters Value
Temperature (C) 24-28
pH 7.0-8.0
COD (mg L-) 5.5-6.5
TSS (mg L-) 1580-3340
VSS (mg L-) 1300-2790
DO (mg L-) 4.5-5.5
Table 1: Operating parameters of activated sludge reactor during acclimation
Citation: Cordi L, Assalin MR, Ponezi AN, Durn N (2012) Identifcation of Microbiota for Activated Sludge Acclimated By Paper Mill Effuent Kraft E1
Bioremediation. J Bioremed Biodeg 3:169. doi:10.4172/2155-6199.1000169
Volume 3 Issue 11 1000169
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Page 3 of 4
performance in organic parameters removal, however the improvement
of COD removal efciency from increasing MLSS was limited within a
certain range.
Te SS increased value during the frst 14 days indicated the
biomass growing, forming more organisms that are able to use the
efuent studied as carbon source. Afer this period the activated sludge
biomass had been acclimated to bioremediation efuent process.
Te Total Suspended Solids (TSS), Volatile Suspended Solids (SSV)
and total COD removal changes are presented in the (Figure 2 and 3)
Other parameter applied to determine the biomass acclimation
was the reduction of the COD charge of the efuent. Afer 10 days of
acclimation occurred the COD stabilization that did not presented
more variation of this parameter. In the period between day 9 and 11
it was possible to observe an increasing COD value reduction caused
by the signifcant biomass growing, resulting in the best value of COD
removal in 56%. Afer acclimation period the COD removal was
stabilized around 45%.
Te evaluation of protozoa and metazoa organisms was done
at the initial and the fnal periods of acclimation. Te frequencies of
organisms present at both periods of acclimation were presented in the
table 2.
Te frequency of protozoa and metazoa present in this work was
represented in magnitude org/mL [23]. Low frequency was considered
when the microbiota was under 10 and high frequency when it was
observed, the order above of 10
In initial period of acclimation nematodes were observed indicating
the poor sedimentability of the biological system, but at the end of the
period they were not observed.
Te rotifer populations increased signifcantly, also free and sessile
ciliates, indicating the good efciency the efuent treated. Ciliated
protozoa produce clear efuents treated of good quality because of
their ability to feed on bacteria and suspended particles and to induce
focculation [24].
At the initial period of acclimation Aspidisca costata, Tracheophyllum
sp. (free swimming ciliate), Vorticella sp., Opercularia sp. (sessile ciliate),
Tecamebas (sarcodine), Rotaria citrinus and Philodinavus paradoxus
(rotifer) were observed.
According to this results it is possible to understand the attempts
to relate the physico-chemical parameters of efuent or the activated
sludge to the species of protozoa present, generating rapid analysis
observing the microbiota instead of doing the physical-chemical
analysis [24].
Te more signifcant protozoa and metazoan organisms present at
the end of acclimation period were presented in the table 3 ( Figure 4).
Te protozoa and metazoa presence indicates that adaptive biomass
presents good quality for biological system treatment. Also, the presence
of Tecameba indicates the good quality of the treated efuent. Te
presence of fagellates in a low frequency indicates a poor concentration
of organic soluble nutrients according to COD removal results. Te
high frequency of ciliate indicated the reduction of bacterial presence
confrming the organic matter consumption by the primary depredator
0 5 10 15 20

Figure 2: Total suspended solids and volatile suspended solids changes during
the acclimation phase (measurements in triplicate; average values are shown.
SD ~2%).
0 2 4 6 8 10 12


Figure 3: Total COD removal from the effuent during the acclimation phase
(measurements in triplicate; average values are shown. SD ~2%).
a - Vorticella alba
(x 400)
b - Euplotes patela.
(x 400)
c Aspidisca costata
(x 400)
d - Rhabditis sp.
(x 200)
e - Philodinavus paradoxus
(x 200)
f - Aeolosoma sp
(x 200)
Figure 4: a, b and c show some Protozoa observed in the acclimation period
and Picture d, e and f show some Metazoa that are observed in the same period.
Protozoa and Metazoa Initial Final
(free swimming and sessile) Low frequency High frequency
Sarcodine NI * High frequency
Flagellate High frequency Low frequency
Rotifers Low frequency High frequency
Nematodes Low frequency NI *
Oligotrichia NI * NI *
*N.I. = Not identifed
Table 2: Protozoa and Metazoa frequency in initial and fnal biomass acclimation
Citation: Cordi L, Assalin MR, Ponezi AN, Durn N (2012) Identifcation of Microbiota for Activated Sludge Acclimated By Paper Mill Effuent Kraft E1
Bioremediation. J Bioremed Biodeg 3:169. doi:10.4172/2155-6199.1000169
Volume 3 Issue 11 1000169
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Page 4 of 4
Trough natural adaptation it was possible to select the organisms
able to degrade pulp and paper efuent, giving a signifcant COD
removal. Tis phase had a period of 39 days wherein it was possible
to observe biomass growth and reactor stabilization by the SS and VSS
Te predominant protozoa afer the acclimation phase were
rotifers, a large quantity of free ciliate, pedunculate colonial ciliates
and isolated ciliate, indicating an excellent capacity of decontamination
of the biological treatment system used. Besides that, the presence of
Tecameba indicated the excellent quality of the fnal treated efuent.
In this way, it was possible to correlate the found microbiota with
operating parameter of activated sludge reactor during the acclimation
The authors would like to thank the fnancial support from National Research
Council (Conselho Nacional de Pesquisa - CNPq) and Foundation in Support
of Research and Technology of the State of So Paulo (Fundao de Apoio a
Pesquisa e Tecnologia do Estado de So Paulo - FAPESP).
1. Kansal SK, Singh M, Sud D (2008) Effuent quality at kraft/soda agro-based
paper mills and its treatment using a heterogeneous photocatalytic system.
Desalination 228: 183-190.
2. Tsang YF, Hua FL, Chua H, Sin SN, Wang YJ (2007) Optimization of biological
treatment of papermill effuent in a sequencing batch reactor. Biochem Engg J
34: 193-199.
3. Durn N, Esposito E, Innocentini-Mei LH, Canhos VP (1994) A new alternative
process for Kraft E
effuent treatment. Biodegradation 5: 13-19.
4. Moraes SG, Durn N, Freire RS (2006) Remediation of Kraft E
and black liquor
effuents by biological and chemical processes. Environ Chem Lett 4: 87-91.
5. Freire RS, Kubota LT, Durn N (2001) Remediation and toxicity removal from
Kraft E
paper mill effuent by ozonization. Environ Technol 22: 897-904.
6. Thompson G, Swain J, Kay M, Forster CF (2001) The treatment of pulp and
paper mill effuent: a review. Bioresour Technol 77: 275-286.
7. Assalin MR, Dos Santos Almeida E, Durn N (2009) Combined system of
activated sludge and ozonation for the treatment of kraft E
effuent. Int J
Environ Res Public Health 6: 1145-1154.
8. CETESB (2000) Activated sludge microbiology (Microbiologia de lodos
ativados). Editora da CETESB, So Paulo.
9. von Sperling M (1995) Biological principles of used water treatment- Introduction
to water quality and sewage treatment. Minas Gerais, ABES, Brazil.
10. Reali MAP (1999) General view of the treatment and fnal deposition of the
biological sludge from treatment stations. Rio de Janeiro, ABES, Brazil.
11. Cordi L, Almeida ES, Assalin MR, Durn N (2007) Bulking on the activated
sludge process applied to the cheese whey effuent treatment: Characterization
and use of chemical focculants to improve settling. Engenharia Ambiental:
Pesquisa e Tecnologia 4: 26-37.
12. Assalin MR, Fabrin-Neto JB, Durn N, Haun M (2007) Toxicity assay in Kraft E

effuent treated by ozone: Algae growth inhibition and cytotoxicity in V79 cells.
Ozone-Science and Engineering 29: 47-53.
13. Gerardi MH (1986) An operator s guide to protozoa and their role in the
activated sludge process. Public Works 90-92: 44-47.
14. Curds CR (1982) The ecology and role of Protozoa in aerobic sewage treatment
process. Annu Rev Microbiol 36: 27-46.
15. Madoni P (1994) Microfauna Biomass in Activated Sludge and Bioflm. Water
Sci Technol 29: 63-66.
16. Cordi L, Assalin MR, Diez MC, Durn N (2008) Assembly, start and operation
of an activated sludge reactor for the industrial effuents treatment: physico
chemical and biological parameters. Engenharia Ambiental: Pesquisa e
Tecnologia 5: 97-115.
17. Amaral AL, da Motta M, Pons MN, Vivier H, Roche N, et al. (2004) Survey of
Protozoa and Metazoa populations in wastewater treatment plants by image
analysis and discriminant analysis. Environmetrics 15: 381-390.
18. Reginatto V, Amante ER, Gerhardy K, Kunst S, Duran N (2009) Biodegradation
and ecotoxicological assessment of pectin production wastewater. J Environ
Sci (China) 21: 1613-1619.
19. Buitrn G, Gonzlez A (1996) Characterization of the microorganisms from an
acclimated activated sludgedegradingphenoliccompounds. Water Sci Technol
34: 289-294.
20. Assalin MR, De Moraes SG, Queiroz SC, Ferracini VL, Durn N (2010)
Studies on degradation of glyphosate by several oxidative chemical processes:
ozonation, photolysis and heterogeneous photocatalysis. J Environ Sci Health
B 45: 89-94.
21. Glymph T (2005) Wastewater Microbiology: A Handbook for Operators.
American Water Works Association, Colorado.
22. Wanner J (1994) Activated Sludge: Bulking and Foaming Control. Taylor &
Francis, Oxford.
23. Vazollerr RF, Garcia MAR, Garcia AD, Neto JC (1989) Activated Sludge
Microbiolgy. CETESB-Companhia de Tecnologia de Saneamento Ambiental 1:
24. Ratsak CH, Maarsen KA, Kooijman SALM (1996) Effects of Protozoa on
carbon mineralization in activated sludge. Water Research 30: 1-12.
Pseudoplepharisma crassum, Aspidisca costata, Eu-
plotes patella, Vorticella sp., Blepharisma lateratum,
Euplotes moebiusi, Chilodonella cracullata, Opercu-
laria sp., Podophyra fxa, Tracheophyllum pusillum,
Paramecium sp., Oxytrichia fallax, Coleps hirtus,
Colpoda cucullus, Blepharisma lateratum, Podophyra
mollis, Pseudoplepharisma crassum
Sarcodine Tecameba
Mastigophora Peranema trichophorum
Rotifer Philodinavus paradoxus,Rotaria citrinus
Nematodes Rhabditis sp.
Oligotrichia Aesoloma hemprichi
Table 3: Mainly Protozoa and Metazoa organisms presented after biomass
acclimation period
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