Cordi et al., 2012, 3:11
Bioremediation & Biodegradation R e s e a r carticle h A r t ic le Research
http://dx.doi.org/10.4 http://dx.do i.org/10.4172/2155-6199.100 172/2155-6199.1000169 0169
O pOpen e n A cAccess c ess
Identification of Microbiota for Activated Sludge Acclimated By B y Paper Mill Effluent Kraft E1 Bioremediation Lívia Cordi 1, Márcia Regina Assalin 2, Alexandre Nunes Ponezi 3 and Nelson Durán 1,4* 1
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, Jaguariúna, SP, Brazil ³Division of Microbial Resources, Research Centre for Chemistry, Biology and Agriculture (CPQBA), U niversidade Estadual de Campinas, P.O.Box 6171, Campinas, SP, 13081-970, Brazil 4 Center of Natural and Human Sciences, Universidade Federal do ABC, Santo André, SP, SP, Brazil 2
Abstract Conventional systems treating paper efuents and cellulose are quite efcient in the reduction of Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD). In general, there is primary stage clarication followed by secondary treatment, usually with a biological system (activated sludge). However, for the efcient degradation of some organic compounds in this efuent, adapted microorganism accomplish the treatment which are necessary resulting in efuents 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 efuent, Kraft E 1. 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 veried 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 identication of protozoa that are considered bioindicators of the activated sludge sys tem. The biomass acclimation period was 39 days. The stabilization of the sy stem was observed through constant rates of total COD (56%). The suspended solids at the beginning of the acclimation phase were from 1580 mg L -¹ to 3580 mg L-¹, 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 xed ciliated were observed, indicating a good purication capacity of the system. Within the organisms amoeba was observed and this indicates good quality to the nal efuent. Then, it is possible to correlate the biota presence with the high capacity of COD removal from the activated sludge system.
Keywords: Biomass; Wastewater treatment; Activated sludge; Microorganism; Protozoa; Biomarker
Introduction Te pulp and paper industry is considered to be a serious environmental hazard due to the multiplicity o impurities to the complexity o the chemical structures present and to the significant quantities o wastewater generated [1,2]. Tis industry sec tor discharges large volumes o brown colored effluents as a result o the different processes applied in wood and pulp bleaching, generating diverse pollutants [3,4]. Te high chemical diversity o these pollutants causes a variety o clastogenic, carcinogenic and mutagenic effects on fish and other aquatic communities in recipient water bodies [5]. Te main treatment process used at pulp and paper mill plants involves primary clarification, succeeded by secondary treatment, generally o a biological nature. Tere are numerous biological treatment systems available, the most common being the activated sludge process [6,7].
types o protozoa is related to effluent quality and plant perormance, so they can be used as bioindicators [13]. Protozoa play a secondary but important role in wastewater system purification [14-16]. Te protozoa in the activated sludge treatment process all into our main classes: amoebae, flagellates and ciliates (ree-swimming, crawling and stalked) and metazoa that are separated into rotiers, nematodes Aelosoma a [14,17]. (sub-class) and Oligotrichia such as Aelosom o apply the activated sludge process to industrial effluents, which have specific compositions, effluent characterization is needed beore treatment and the sludge needs to be acclimatized (microorganism acclimation to the effluent to be treated) [18,19]. Te aim o this work was veriying the changes o the activated sludge microorganisms
*Corresponding author: Nelson Duran, Duran, Institute Institute of Chemistry, Biological Chemistry Laboratory, Center of Natural and Human Sciences, Universidade Federal do ABC, Santo André, SP, Brazil; Tel: 55 19 3521-3149; E-mail:
[email protected]
Activated sludge system technology consists basically in the agitation o the effluent in the presence o aerobic bacteria, protozoa, metazoa and atmospheric oxygen or a sufficient period to metabolize and to flocculate a large part o the organic material [8-10].
Received December 22, 2011; Accepted Received December Accepted October October 12, 2012; Published Published October October 14, 2012
Te most common organisms that participate in the biodegradation o organic material in the biological system or effluent treatment are bacteria, protozoa and annelids [8,11,12]. Te presence o particular
Copyright: © 2012 Cordi L, et al. This is an open-a ccess article distributed under Copyright: © under the terms of the C reative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Citation: Cordi L, Assalin MR, Ponezi AN, Durán N (2012) Identication of Citation: Microbiota for Activated Sludge Acclimated By Paper Mill Efuent Kraft E1 Bioremediation. J Bioremed Biodeg 3:169. doi: 10.4172/2155-6199.1000169
Volume 3 • Issue 11 • 1000169
Citation: Cordi L, Assalin MR, Ponezi AN, Durán N (2012) Identication of Microbiota for Activated Sludge Acclimated By Paper Mill Efuent Kraft E1 Bioremediation. J Bioremed Biodeg 3:169. doi: 10.4172/2155-6199.1000169
Page 2 of 4
during acclimation period in the bioremediation o paper mill effluent rom the Kraf E1 process.
Materials and Methods
and weighed (retention sizes o 1 mm). Afer filtration and drying or 2 h in an oven (~100°C), the filter paper containing the solids was removed at room temperature and again weighed. From the mass difference the amount o suspended solid was calculated.
Paper mill effluent
Determination o volatile suspended solids: A 100 mL aliquot
Wastewater was obtained rom a bleach Kraf mill located in the Campinas city region (São Paulo State, Brazil). Tis mill process uses only Eucalyptus grandis wood. Sample effluent was obtained afer the first alkaline extraction stage (E1). Te aqueous samples were stored at 4°C in bottle flasks and used without prior filtration.
Acclimation o activated sludge to kraf E1 paper effluent Te acclimation o the activated sludge biomass rom the Samambaia Sewage reatment Plant, in a suburb o eastern region o Campinas, SP, Brazil, was studied or 39 days by the natural selection method. Te biomass acclimation was ollowed by COD reduction rom the effluent. 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 specific conditions using a Kraf E1 effluent as eed to microorganisms presents in the biological system.
Activated sludge system An aerobic reactor (2.5 L) was operated as a continuous system and continuously ed with Kraf E 1 effluent (at pH 7) by means o a peristaltic pump. Te Hydraulic Retention ime (HR) was maintained at 12 h. Te sludge was periodically recycled, and excess sludge which grew during the aeration stage was withdrawn rom the bottom to obtain Solids Retention imes (SR) in 10 days. Te dissolved oxygen concentration was maintained above 5 mg L–1 by an aquarium-type air pump with sintered-sand diffusers at the bottom o the reactor [16]. Te schema o activated sludge reactor used in this study was presented in figure 1.
Analytical control pH Determination: Te pH was measured using an Orion model
EA 940 pH meter rom Cole-Parmer, with a combined glass electrode. Determination o total suspended solids: For analysis o
suspended solids an aliquot o 10 mL o sludge rom the bioreactors was filtered using GF-C Whatman filter papers that were previously dried
Feed
Effluent
o sludge coming rom the bioreactor was filtered using a previously weighed nitrocellulose filter (pore size 0.2 mm). Te filter plus solids was dried at 105°C during 2 h. Afer this, it was lef in a desiccator or 1 h and again weighed. Te mass o the filter with solid was determined and by difference the amount o suspended solid. Determination o Chemical Oxygen Demand (COD): A COD was measured using the procedure o the Standard Methods or the Examination o Water and Wastewater o the American Society o Civil Engineers (1992) [20].
Biological control Qualitative analysis o 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 rotiers, nematode, tardigrade and anelide [8].
Results and Discussion Prior to this experiment in a continuous activated sludge reactor, the biomass rom domestic wastewater was acclimated to the substrate o interest (Kraf E1 effluent) or 39 days to permit the growth o the microorganisms which can use the effluent as a carbon source. Acclimation is widely used to maximize the removal efficiency o specific compounds that are difficult to biodegrade. Te steady-state condition was verified when the average o the consecutive measurements showed a constant rate o COD removal [7]. Te acclimation process was stopped when the total COD removal rate was constant, around 56%. Te operating parameters o activated sludge reactor were present in the table 1. Te variation o 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 diffusers that presented a good aeration to homogenize the aerobic phase permitting the flocks ormation 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 -¹. Te biomass concentration has an important effect on treatment perormance. Previous studies had shown that the minimum o Mixed Liquor Suspended Solids (MLSS) in the activated sludge process or paper mill effluent treatment is 2000-2500 mg L–1 [1]. sang et al. [2] showed that a higher concentration o MLSS promoted activated sludge Operating Parameters
Recirculation
purge
Figure 1: Activated sludge reactor system applied to efuent treatment.
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Value
Temperature (°C)
24-28
pH
7.0-8.0
COD (mg L-¹)
5.5-6.5
TSS (mg L-¹)
1580-3340
VSS (mg L-¹)
1300-2790
DO (mg L-¹)
4.5-5.5
Table 1: Operating parameters of activated sludge reactor during acclimation period.
Volume 3 • Issue 11 • 1000169
Citation: Cordi L, Assalin MR, Ponezi AN, Durán N (2012) Identication of Microbiota for Activated Sludge Acclimated By Paper Mill Efuent Kraft E1 Bioremediation. J Bioremed Biodeg 3:169. doi: 10.4172/2155-6199.1000169
Page 3 of 4
perormance in organic parameters removal, however the improvement o COD removal efficiency rom increasing MLSS was limited within a certain range. Te SS increased value during the first 14 days indicated the biomass growing, orming more organisms that are able to use the effluent studied as carbon source. Afer this period the activated sludge biomass had been acclimated to bioremediation effluent process. Te otal Suspended Solids (SS), Volatile Suspended Solids (SSV) and total COD removal changes are presented in the (Figure 2 and 3) respectively. Other parameter applied to determine the biomass acclimation was the reduction o the COD charge o the effluent. Afer 10 days o acclimation occurred the COD stabilization that did not presented more variation o this parameter. In the period between day 9 and 11 it was possible to observe an increasing COD value reduction caused by the significant biomass growing, resulting in the best value o COD removal in 56%. Afer acclimation period the COD removal was stabilized around 45%. Te evaluation o protozoa and metazoa organisms was done at the initial and the final periods o acclimation. Te requencies o organisms present at both periods o acclimation were presented in the table 2. Te requency o protozoa and metazoa present in this work was represented in magnitude org/mL [23]. Low requency was considered
6000
when the microbiota was under 10³ and high requency when it was observed, the order above o 104. In initial period o acclimation nematodes were observed indicating the poor sedimentability o the biological system, but at the end o the period they were not observed. Te rotier populations increased significantly, also ree and sessile ciliates, indicating the good efficiency the effluent treated. Ciliated protozoa produce clear effluents treated o good quality because o their ability to eed on bacteria and suspended particles and to induce flocculation [24]. At the initial period o acclimation Aspidisca costata, Tracheophyllum sp. (ree swimming ciliate), Vorticella sp., Opercularia sp. (sessile ciliate), Tecamebas (sarcodine), Rotaria citrinus and Philodinavus paradoxus (rotier) were observed. According to this results it is possible to understand the attempts to relate the physico-chemical parameters o effluent or the activated sludge to the species o protozoa present, generating rapid analysis observing the microbiota instead o doing the physical-chemical analysis [24]. Te more significant protozoa and metazoan organisms present at the end o acclimation period were presented in the table 3 ( Figure 4). Te protozoa and metazoa presence indicates t hat adaptive biomass presents good quality or biological system treatment. Also, the presence o ecameba indicates the good quality o the treated effluent. Te presence o flagellates in a low requency indicates a poor concentration o organic soluble nutrients according to COD removal results. Te high requency o ciliate indicated the reduction o bacterial presence confirming the organic matter consumption by the primary depredator organisms.
4000
1 L g m 2000 SS
SSV
0 0
5
10
15
20
days
a - Vorticella alba
b - Euplotes patela.
c – Aspidisca costata
(x 400)
(x 400)
(x 400)
d - Rhabditis sp.
e - Philodinavus paradoxus
f - Aeolosoma sp
(x 200)
(x 200)
(x 200)
Figure 2: Total suspended solids and volatile suspended solids changes during the acclimation phase (measurements in triplicate; average values are shown. SD ~2%).
80
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. 60
n o c u d e r O Q D %
Protozoa and Metazoa
t
40
20
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 *
Ciliate
0 0
Initial
2
4
6
8
10
12
days
Figure 3: Total COD removal from the efuent during the acclimation phase (measurements in triplicate; average values are shown. SD ~2%).
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
*N.I. = Not identifed
Table 2: Protozoa and Metazoa frequency in initial and nal biomass acclimation period
Volume 3 • Issue 11 • 1000169
Citation: Cordi L, Assalin MR, Ponezi AN, Durán N (2012) Identication of Microbiota for Activated Sludge Acclimated By Paper Mill Efuent Kraft E1 Bioremediation. J Bioremed Biodeg 3:169. doi: 10.4172/2155-6199.1000169
Page 4 of 4
Ciliate Protozoa
Sarcodine
Pseudoplepharisma crassum, Aspidisca costata, Eu plotes patella, Vorticella sp., Blepharisma lateratum, Euplotes moebiusi, Chilodonella cracullata, Opercularia sp., Podophyra xa, Tracheophyllum pusillum, Paramecium sp., Oxytrichia fallax, Coleps hirtus, Colpoda cucullus, Blepharisma lateratum, Podophyra mollis, Pseudoplepharisma crassum Tecameba
Mastigophora Peranema trichophorum Rotifer Metazoa Nematodes Oligotrichia
Philodinavus paradoxus,Rotaria citrinus Rhabditis sp. Aesoloma hemprichi
Table 3: Mainly Protozoa and Metazoa organisms presented after biomass acclimation period
Conclusion Trough natural adaptation it was possible to select the organisms able to degrade pulp and paper effluent, giving a significant COD removal. Tis phase had a period o 39 days wherein it was possible to observe biomass growth and reactor stabilization by the SS and VSS parameters. Te predominant protozoa afer the acclimation phase were rotiers, a large quantity o ree ciliate, pedunculate colonial ciliates and isolated ciliate, indicating an excellent capacity o decontamination o the biological treatment system used. Besides that, the presence o ecameba indicated the excellent quality o the final treated effluent. In this way, it was possible to correlate the ound microbiota with operating parameter o activated sludge reactor during the acclimation period.
11. Cordi L, Almeida ES, Assalin MR, Durán N (2007) Bulking on the activated sludge process applied to the cheese whey efuent treatment: Characterization and use of chemical occulants to improve settling. Engenharia Ambiental: Pesquisa e Tecnologia 4: 26-37. 12. Assalin MR, Fabrin-Neto JB, Durán N, Haun M (2007) Toxicity assay in Kraft E -1 efuent 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 Biolm. Water Sci Technol 29: 63-66. 16. Cordi L, Assalin MR, Diez MC, Durán N (2008) Assembly, start and operation of an activated sludge reactor for the industrial efuents 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. Buitrón G, González A (1996) Characterization of the microorganisms from an acclimated activated sludgedegradingphenoliccompounds. Water Sci Technol 34: 289-294. 20. Assalin MR, De Moraes SG, Q ueiroz SC, Ferracini V L, Durán N (2010) Studies on degradation of glyphosate by several oxidative chemical processes: ozonation, photolysis and heterogeneous photocatalysis. J Environ Sci H ealth B 45: 89-94. 21. Glymph T (2005) Wastewater Microbiology: A Handbook for Operators. American Water Works Association, Colorado.
Acknowledgement
22. Wanner J (1994) Activated Sludge: Bulking and Foaming Control. Taylor & Francis, Oxford.
The authors would like to thank the nancial support from National Research Council (Conselho Nacional de Pesquisa - CNPq) and Foundation in Support of Research and Technology of the State of São Paulo (Fundação de Apoio a Pesquisa e Tecnologia do Estado de São Paulo - FAPESP).
23. Vazollerr RF, Garcia MAR, G arcia AD, Neto JC (1989) Activated Sludge Microbiolgy. CETES B-Companhia de Tecnologia de Saneamento Ambiental 1: 1-19.
References
24. Ratsak CH, Maarsen KA, Kooijman SALM (1996) Effects of Protozoa on carbon mineralization in activated sludge. Water Research 30: 1-12.
1. Kansal SK, Singh M, Sud D (2008) Efuent 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 efuent in a sequencing batch reactor. Biochem Engg J 34: 193-199. 3. Durán N, Esposito E, Innocentini-Mei LH, Canhos VP (1994) A new alternative process for Kraft E 1 efuent treatment. Biodegradation 5: 13-19. 4. Moraes SG, Durán N, Freire RS (2006) Remediation of Kraft E 1 and black liquor efuents by biological and chemical processes. Environ Chem Lett 4: 87-91. 5. Freire RS, Kubota LT, Durán N (2001) Remediation and toxicity removal from Kraft E 1 paper mill efuent by ozonization. Environ Technol 22: 897-904. 6. Thompson G, Swain J, Kay M, Forster CF (2001) The treatment of pulp and paper mill efuent: a review. Bioresour Technol 77: 275-286. 7. Assalin MR, Dos Santos Almeida E, Durán N (2009) Combined system of activated sludge and ozonation for the treatment of kraft E 1 efuent. Int J Environ Res Public Health 6: 1145-1154. 8. CETESB (2000) Activated sludge microbiology (Microbiologia de lodos ativados). Editora da CETESB, São Paulo. 9. von Sperling M (1995) Biological principles of used water treatment- Introduction to water quality and sewage treatment. Minas G erais, ABES, Brazil. 10. Reali MAP (1999) General view of the treatment and nal deposition of the biological sludge from treatment stations. Rio de Janeiro, ABES, Brazil.
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Submit your next manuscript and get advantages o OMICS Group submissions Unique features:
• • •
User friendly/feasible website-translation of your paper to 50 world’s leading languages Audio Version of published paper Digital articles to share and explore
Special features:
• • • • • • • •
200 Open Access Journals 15,000 editorial team 21 days rapid review process Quality and quick editorial, review and publication processing Indexing at PubMed (partial), Scopus, DOAJ, EBSCO, Index Copernicus and Google Scholar etc Sharing Option: Social Networking Enabled Authors, Reviewers and Editors rewarded with online Scientic Credits Better discount for your subsequent articles
Submit your manuscript at: http://www.omicsonline.org/submission
Volume 3 • Issue 11 • 1000169
