ENVIRONMENTAL BIOTECHNOLOGY

Enhanced remediation of black liquor by activated sludgebioaugmented with a novel exogenous microorganism culture

Yu Zheng & Li-Yuan Chai & Zhi-Hui Yang &

Chong-Jian Tang & Yue-Hui Chen & Yan Shi

Received: 22 July 2012 /Revised: 18 September 2012 /Accepted: 18 September 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract Black liquor (BL) is a notoriously difficult waste-water to treat due to the economic and efficiency limitationsof physiochemical methods and intrinsic difficulties withbioremediation strategies caused by the high pH (10–13)and lignin content. This study investigated the feasibility ofa novel bioaugmentation strategy for BL treatment, whichuses a mixed microorganism culture of lignocellulose-degrading microorganisms isolated from degraded bambooslips. Black liquor treatment was assessed in terms of chem-ical oxygen demand (COD) and color removal with a se-quencing batch reactor organic loading rate of 9 kg COD/L·day under highly alkaline conditions (pH10). Resultsrevealed that bioaugmented activated sludge treatment ofBL with special mixed microorganisms significantly en-hanced the removal efficiency of COD, color, and ligninfrom the wastewater up to 64.8, 50.5, and 53.2 %, respec-tively. Gel permeation chromatography profiles showed thatthe bioaugmentation system could successfully degradehigh molecular lignin fragments in black liquor. This workconfirms bioaugmentation as a feasible alternative strategyfor enhanced biological treatment of wastewater with highlignin content and high organic load rate under stronglyalkaline conditions.

Keywords Bioaugmentation . Special mixedmicroorganisms . Black liquor . High-alkaline conditions .

Alkaline lignocellulolytic enzymes

Introduction

The pulp and paper industry generates large volumes ofhigh-lignin content wastewater, known as black liquor(BL). BL is a complex and variable mixture of multiplenon-biodegradable substances, including lignin, polysac-charides, and resinous compounds (Cardoso et al. 2009).On account of its extremely high pH (10–13) and chemicaloxygen demand (COD; 100,000–250,000 mgL−1), BL isamong the most difficult wastewaters to treat.

While multiple physicochemical methods (e.g., alkalirecovery process, acid precipitation, UV/TiO2 systems,Fenton reactions, and coagulation) have been employed totreat BL (Garg et al. 2010; Shi et al. 2011), most of thesemethods are neither technologically nor economically feasi-ble, and they have not been adopted by the majority ofsmall- and medium-scale pulp and paper mills (Chandra etal. 2011). Furthermore, the lignocellulosic compounds inBL are not degraded in these treatments. Rather, they aresimply transformed from a water-soluble state into a solidstate, which facilitates secondary treatment (Wu et al. 2005).Alternatively, biological treatments, such as a conventionalactivated sludge (AS) process involving degradation of or-ganic components using a suspended growth biomass, areenvironmentally friendly and cost-effective alternatives tophysicochemical wastewater treatments.

Biological treatments have been proven effective forCOD removal in various applications. However, biologicaltreatments for BL remediation are difficult due to the highpH and lignin content of the wastewater, which severelylimit the activity and survival of microorganisms in thesystem. Lignin, which may account for up to 30–45 % ofBL wastewater, is a three-dimensional phenylpropanoidpolymer (Hatakeyama and Hatakeyama 2010) with lowbiodegradability by many organisms. Moreover, the highpH of BL typically results in negative impacts on microor-ganism activity, in turn limiting the effectiveness of the

Y. Zheng : L.-Y. Chai (*) : Z.-H. Yang :C.-J. Tang :Y.-H. Chen :Y. ShiInstitute of Environmental Science and Engineering, School ofMetallurgical Science and Engineering, Central South University,Changsha 410083, People’s Republic of Chinae-mail: liyuan.chai@yahoo.com

L.-Y. Chai : Z.-H. Yang : C.-J. TangChinese National Engineering Research Center for Control &Treatment of Heavy Metal Pollution,Changsha 410017, China

Appl Microbiol BiotechnolDOI 10.1007/s00253-012-4453-x

biological treatment. Therefore, the development of novelbiological technologies is necessary and highly desirable foreffective and commercially viable BL treatment.

Bioaugmentation, which involves the addition of specificmicrobial cultures to a treatment system, has been shown to bean efficient method to enhance the removal of toxic orrecalcitrant compounds in a number of biotreatment systems(Limbergen et al. 1998; Quan et al. 2004; Kumar et al. 2012).Bioaugmentation cultures are specifically selected microor-ganisms capable of degrading target compounds while surviv-ing/remaining active under the conditions of the biotreatmentsystem. Bioaugmentation of AS has been proposed as a pow-erful tool to improve the wastewater treatment processes. Forexample, bioaugmentation improved the flocculation of ASand accelerated the removal and biodegradation of pollutantssuch as crude oil (Odokuma and Dickson 2004; El Fantroussiand Agathos 2005). Furthermore, the application of bioaug-mentation to municipal wastewater treatment has been widelydescribed in the literature (Stephenson and Stephenson 1992).To date, however, very few examples of successful bioaug-mentation of wastewater containing high concentrations oforganic compounds (high COD) have been reported.

When applying biotreatment to high COD wastewaters,the bioaugmented species often fail to compete with theindigenous population in the system, and process inhibitionmay occur (Boon et al. 2000). Moreover, survival of theintroduced microorganisms does not ensure effective degra-dation of target compounds, due to a variety of reasons(Quan et al. 2004). Therefore, it is necessary to selectsuitable special microbial cultures and a strategy to achievesuccessful bioaugmentation to treat BL.

Recently, three lignocellulose-degrading microorgan-isms, Pandoraea B-6 (CGMCC No. 4239), ComamonasB-9 (CGMCC No. 4251), and Aspergillus F-1(CGMCCNo. 5858), were isolated from eroded bamboo slips (ancientwriting tablets) dating back to the Three Kingdoms period(220–265 A.D.) in China (Chai et al. 2010). The presence ofthese organisms in the degraded bamboo slips indicatesorganism-specific bioavailability. Therefore, it was hypoth-esized that a conventional AS process for BL treatmentcould be enhanced by bioaugmentation with a mixed culturecomposed of the bamboo slip microorganisms. However, noinformation regarding the lignin-degradation abilities of thebamboo slip microorganisms in a BL biotreatment systemexisted. The conditions in a BL treatment system are farmore complex than those of pure cultures, due to the pres-ence of complex components in the system.

This study examined the performance of bioaugmenta-tion with the three microorganism strains isolated fromeroded bamboo slips in long-term (up to 60 days) biotreat-ment of BL. Moreover, while recent reports on AS bioaug-mentation have been conducted in a sequencing batchreactor (SBR) (Yu and Mohn 2001), bioaugmentation in a

SBR for the treatment of BL under the highly alkalineconditions presented by the wastewater have not yet beenreported. Therefore, the aim of the present study was toinvestigate the feasibility of an AS bioaugmentation with amixed culture of the bamboo slip microorganisms for thetreatment of BL in an SBR. The roles of the introduced andindigenous cultures in the biotreatment of BL were also stud-ied by analyzing changes in the molecular weight distributionsof lignin extracts from BL. The results present basic informa-tion about the effectiveness of the novel treatment strategy, aswell as preliminary optimal operating conditions for the treat-ment of BL in anAS bioaugmentation process in a SBRwith aunique bioaugmentation culture.

Materials and methods

Wastewater composition

Black liquor was obtained from a pulp and paper mill inHunan Province, China. Reed had been used as the rawmaterial and pulped by a soda process. The black liquor wasstored at 4 °C until use in analysis. Further, the BL wasanalyzed for total solid, total suspended solid, volatile fattyacids, biochemical oxygen demand (BOD), COD, ammonianitrogen, phosphate and sulfate as per APHA standard meth-ods (APHA 1998). Metals were determined by inductivecoupled plasma atomic emission spectroscopy. Cellulose andhemicellulose contents were analyzed using the methods de-scribed by Jönsson et al (2008). Lignin content was analyzedusing the method described by Gindl et al (2000).

Microorganisms and growth conditions

In our previous study, one of the lignin-degrading bacteria,Comamonas B-9, was found capable of degrading Kraftlignin with COD concentrations less than 6,000 mgL−1 asthe sole carbon and energy source (Chen et al. 2012). Theother fungus, Aspergillus F-1, was capable of metabolizinglignocellulose at concentrations as high as 4,000 mgL−1 asthe sole carbon and energy source. All of these newlyisolated microorganisms were found capable of survivingin diluted BL with tap water with COD as high as12,000 mgL−1 under high pH (10–13) conditions.

The PandoraeaB-6,ComamonasB-9, and Aspergillus F-1were routinely maintained on BL-mineral salt medium (BL-MSM, pH9.0) agar plates at 4 °C as a preculture. The BL-MSM consisted of (grams per liter) 1.0 KHPO4, 1.0 K2HPO4,0.5 (NH4)2SO4, 0.05 MgSO4, 0.01 CaCl2, and 2 % BL (v/v).For the inoculum preparation, single colonies of each strainwere withdrawn from the preculture and inoculated in 300 mLBL-MSM. Each strain was cultivated on rotary shaker at 30 °C and 120 rpm for 5 days, respectively. The cells of the two

Appl Microbiol Biotechnol

bacteria and the mycelia of the fungus were then collected bycentrifugation, respectively. The collections were washedthree times with sterile water and then used as inoculum forbiotreatment of BL.

Acclimation of activated sludge

The raw AS, used as the endogenous seed culture, wasprocured from the secondary clarifier of the same mill.The AS was acclimated to the synthetic wastewater withan initial COD of 2,400 mgL−1 in a 2.4 L working volumecolumn type reactor. The final pH was adjusted to 9.0 usingHCl. The composition of synthetic wastewater was as fol-lows (in grams per liter): 1.0 KHPO4, 1.0 K2HPO4, 0.5(NH4)2SO4, 0.05 MgSO4, 0.01 CaCl2 with 1 mLL-1 traceelement solution, 1.0 glucose, 0.2 carbamide, and variableCOD concentration of BL from 800–4,600 mgL-1, accord-ing to the experimental plan. When a constant COD removalof approximately 65 % was achieved at a retention time of4 days, part of the supernatant was removed and replacedwith an equal volume of fresh synthetic wastewater. Thisprocess was repeated until no glucose was added, allowingBL with a COD concentration of 4,600 mgL−1 to serve asthe sole carbon source in the reactor. The whole process wascompleted in 60 days, and the mixed liquor suspended solid(MLSS) content in the reactor was maintained at 4–5 gL−1.The acclimated AS was harvested by decanting, washedtwice with phosphate buffered saline (PBS (in grams perliter): 8 NaCl, 0.2 KCl, 1.44 Na2HPO4, 0.2 NaH2PO4, pH7.0), and stored as a glycerol culture at −70 °C until use. Theacclimated AS served as indigenous microorganisms in thebioaugmentation experiments.

Optimization of inoculum concentration in bioaugmentationexperiments

In order to find the optimum inoculum concentration, bio-augmentation experiments were first performed in fiveErlenmeyer flasks (1,000 mL) with 600 mL working vol-ume for 7 days. An aliquot of 20 g (wet weight) AS with3.5 g/L MLSS was introduced into each flask with a diluted(10 % v/v) BL-supplemented medium (COD, ∼12,000 mgL−1). Four of the flasks were bioaugmented with 5, 10, 15,and 20 % (the dry weight of the supplemented exogenousculture to the dry weight of indigenous culture) mixedexogenous microorganisms, and the ratio of PandoraeaB-6, Comamonas B-9, and Aspergillus F-1 was 2:1:2 (inweight). The other reactor was not supplemented withmixed special microorganisms and served as a control.The final biomass added to each flask amounted to4.5 gL−1 MLSS. The effect of inoculum concentrationwas optimized to obtain a maximum reduction in CODcontent.

Adsorption evaluation in bioaugmentation experiments

This study was carried out to verify whether the observedbiodegradation of lignin in BL was due to biological or non-biological activity. The living and heat killed AS with orwithout 15 % mixed exogenous microorganisms consortiumwere added into a 250-mL Erlenmeyer flask containing50 mL of 10 % (v/v) BL as wastewater medium. The flaskswere placed on rotary shaker (120 rpm) at 30 °C. Sampleswere withdrawn at respective time points and then centri-fuged at 10,000 rpm for 10 min. The supernatants wereanalyzed for lignin content.

Sequencing batch reactor design

Simulative operation of SBRs was facilitated by plexiglasswith a working volume of 2 L and ports for effluent andsludge wastage. Air was provided using a glass diffuserconnected to an air pump. Temperature and dissolvedoxygen (DO) were approximately T030±2 °C andDO02±0.5 mg/L. The effluent was withdrawn at theend of the settling period, and sludge was wasted at theend of the two-cycle time.

Evaluation of bioaugmentation in the SBR system

This study was conducted through parallel experiments.Simulated SBRs with the same structure and volume asdescribed above were used to evaluate the efficiency oftwo systems: (1) the conventional AS process (AS system)as a control and (2) bioaugmented sludge system (Bio-system) with added exogenous mixed bamboo slip micro-organisms. The AS was added as seed source to the bothsystems. In the AS system, only AS was added to determinewhether or not the presence of mixed special microorgan-isms would influence biotreatment efficiency. Feed water ofthe parallel Bio-system reactor contained mixed specialmicroorganisms. Every experiment was conducted untilthe system reached a steady state, yielding the same CODin the effluent for three consecutive days. Average timeelapsed for each experiment was about 1 to 2 weeks.Throughout the experiment, no other carbon or nutrient sub-stance was added to any reactor. Samples were taken atspecific time intervals, centrifuged, filtered, and stored at 4 °C.

Long-term operation of Bio- and AS systems under stepwiseincreased OLR

On the basis of results obtained from the experiments out-lined above, a lab-scale Bio-system treatment of BL wascarried out at an optimum inoculum concentration of 15 %and pH of 10 with stepwise increases in the organic load rate(OLR) for a long-term operation of 60 days. The AS system

Appl Microbiol Biotechnol

was operated under similar conditions and served as a con-trol. To prevent inhibition from toxic compounds, such asresin and lignin, at reactor start-up, and to promote gradualbiomass acclimation, the BL was diluted and fed to the Bio-system with a gradually increasing OLR from 5 to 11 gCOD/L·day with the hydraulic retention time (HRT) in-creasing from 4 to 7 days. For the AS system, the OLRwas increased gradually from 5 to 8 g COD/L·day with theHRT increasing from 4 to 7 days.

Analytical methods

Samples were withdrawn at specific time intervals andcentrifuged at 8,000×g for 20 min to remove biomass fromthe liquid phase for later analysis. Clear supernatants wereanalyzed for their COD, color, and lignin content. Beforemeasurement, the samples were diluted 500 times withdistilled water.

The pH of samples was measured with a pH electrode(Model PHS-3E; Leici, China). For the measurement oflignin content, the supernatant (1 mL) was adjusted to pH8.0 by adding 0.1 molL−1 NaOH or 0.30 molL−1 HCl, theabsorbance was measured at 280 nm on a spectrophotome-ter. The absorbance value was transformed into lignin con-centration (milligrams per liter) (Gindl et al. 2000).Supernatant color was assessed spectrophotometrically at465 nm (A465) with the absorbance value transformed intocolor units (CU) (Yang et al. 2010). MLSSs were measuredperiodically using standard methods (APHA 1998).

Enzyme activity analysis

The activities of various enzymes in the Bio- and ASsystems were determined in the supernatant obtained bycentrifugation of samples at 5,000×g for 10 min at 4 °C.Before measurement, the samples were diluted 500 timeswith distilled water. One unit of enzyme activity wasexpressed as the amount of enzyme required for an increaseof 1.0 absorbance unit per minute (units per milliliter).

Cellulase (carboxymethylcellulase, CMCase) and xyla-nase activities were determined by measuring sugars re-leased from an appropriate substrate, as described byLopez et al. (2007). Laccase activity was determined bymeasuring the oxidation of 2,2′-azino-bis-3-ethylbenzthia-zoline-6-sulfonic acid (Nakagawa et al. 2010). Lignin per-oxidase (LiP) was determined by the peroxide-dependentoxidation of 2 mM veratryl alcohol to veratraldehyde in25 mM tartrate buffer, pH2.5 with 0.4 mM H2O2 (Orthet al. 1993). Manganese peroxidase (MnP) was assayedby oxidation of Mn2+ to Mn3+ and formation of theMn3+–tartrate complex from 0.1 mM MnSO4 in0.1 mM sodium tartrate buffer, pH5.0 with 0.1 mMH2O2 (Camarero et al. 1999).

Molecular weight distribution of BL treated by ASand Bio-systems

Supernatants in the AS and Bio-systems were analyzedusing gel permeation chromatography (GPC) with a high-performance liquid chromatography (HPLC model 244;Waters Associates, USA) equipped with Sephadex LH-60gel columns (pore size, 50, 100, 103, and104Å; length,300 mm each; with inner diameter of 7.8 mm) and comparedto the untreated BL. The acetylated samples were dissolvedin tetrahydrofuran at a concentration of 2 mgmL−1 andstored for 24 h at 5 °C to avoid variations in molecularweight (Mw). Subsequently, 20 μL of each sample wasinjected into the GPC. Tetrahydrofurane was used as aneluent at a flow rate of 1 mL/min at room temperature.Measurements were made on a UV detector at 280 nm(Beckman-Coulter, USA). For calibration, polystyrene sam-ples with various Mw were used. The Mw were calculatedfrom the experimental data using normal GPC calculationprocedures.

Statistical analysis

All experiments were conducted in triplicate under iden-tical conditions, and the results were presented as theaverage value of the three data sets. In figures, errorbars are not shown.

Results

Characterization of black liquor

The detected characteristics of BL are shown in Table 1. Theresults indicated that BL had high amounts of COD, color,lignin and related compounds, and pH. The lignin content inthe BL sample was much higher than those of previouslyreported BL wastewaters, due to the soda process used forpulping reed (Wallberg et al. 2005). In addition, the BLsample contained significant amounts of ionic nutrients,such as ammonia and phosphates, which have been reportedas essential for microorganism growth (Goodwin et al.1990). On account of its composition, the BL wastewatersample was determined to be of complex character withlow biodegradability (BOD/COD<0.5) and very highpH (13.1–13.6).

Effect of inoculum concentration on COD removal efficiencyin Bio-system reactors

Appropriate inoculum concentration is crucial to successfulbioaugmentation. Diluted (10 % v/v) BL was fed into the ASand Bio-system reactors at an initial COD concentration of

Appl Microbiol Biotechnol

∼23,000 mg/L. Bioaugmentation levels significantly affect-ed biotreatment efficiency (Fig. 1). For the first 2 days, onlya slight COD removal about 2.8 to 5.7 % was observed forthe AS system. With the addition of the exogenous micro-bial culture to the Bio-system, the COD removal increasedwith increases in inoculum proportion from 5 to 15 %. Forthe Bio-system reactors augmented with 10 and 15 % of theexogenous culture, obvious COD removals of 21.8 and

25.7 % were observed on the second days, while less en-hanced COD removal of 13.9 % was observed for thereactor augmented with 5 %. However, when the inoculumconcentration in the Bio-system reactor increased to 20 %,although COD removal was apparent on the first day, CODremoval had declined by day5.

Effect of initial pH on COD and lignin removal efficienciesin Bio-system reactors

As BL is highly alkaline in nature, a pH range (7–13) wasinvestigated in the Bio-system as an assessment of its suit-ability for BL remediation compared with the AS system.The effect of different initial pH values on various parame-ters (Fig. 2) showed that BL could be successfully treatedwith the Bio-system over the pH range from 9–12. TheCOD removal efficiencies of the Bio-system at pH of 9and 12 were up to 65.3 and 44.1 %, respectively, while theremoval of COD at pH of 7 and 8 only to 23.2 and 38.6 %,indicating that bioaugmentation using the special exogenousmixed culture can improve biotreatment of BL under highlyalkaline conditions. The lignin content of samples indicatedthat the microorganism culture present in the Bio-systemreactor also demonstrated a much stronger lignin degrada-tion capacity than that of the endogenous AS culture atoptimal pH of 10. The lignin removal for AS system wasup to only 12.1 %, while that in the Bio-system was as highas 55.2 %.

Effect of adsorption on lignin removal efficiencyin bioaugmentation experiments

To confirm that the effective lignin degradation in BL oc-curred by biological activity rather than an adsorption mech-anism, an abiotic degradation study was carried out (Fig. 3).

Table 1 Characteristics of untreated black liquor

Characteristics BL

pH 13.1–13.6

Color (CU) 524,300–675,000

COD (mgL−1) 180,000–230,000

BOD (mgL−1) 12,000–16,000

Lignin (gL−1) 139.41–154.97

Cellulose (gL−1) 79.22–88.69

Hemicellulose (gL−1) 35.43–39.66

Total solids (mgL−1) 42,000–51,000

Total suspended solids (mgL−1) 11,000–13,000

Volatile fatty acids (mgL−1) 1,700–2,200

Ammonia nitrogen (mgL−1) 800–1,050

Phosphates (mgL−1) 600–700

Sulphates (mgL−1) 800–1,000

Heavy metals

Cr (mgL−1) 0.126–0.140

Cu (mgL−1) 0.312–0.337

Fe (mgL−1) 1.794–1.915

Pb (mgL−1) 0.103–0.117

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

Time (d)

CO

D r

emov

al (

%)

control5%10%15%20%

Fig. 1 Effect of inoculum concentration on the systems start-up andremoval efficiency in COD. Control system (solid square), system with5 % (solid circle), 10 % (open triangle), 15 % (open star), and 20 %(open diamond) special mixed culture. The values are mean of threereplicates

7 8 9 10 11 12 130

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rem

oval

(%

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initial pH

reduction in lignin(Bio-system) reduction in lignin (AS-system)

reduction in COD (Bio-system)reduction in COD (AS-system)

Fig. 2 Effect of different initial pH values on COD removal in a Bio-system and an AS system at influent COD concentration of 23,000 mgL−1. The values are mean of three replicates

Appl Microbiol Biotechnol

Results showed that the final fractions of lignin degradationby adsorption were negligible, accounting for only 3.6 and1.2 % in Bio- and AS system, respectively. These resultsclearly indicated that the lignin degradation in BL was dueto biological mechanisms in the Bio-system.

Effect of OLR on COD removal efficiency in long-termoperation

The effect of OLR on COD removal efficiency was exam-ined over a long-term (60 days) operation of the bioaug-mented SBR reactor. The results of long-term operation ofthe AS and Bio-systems are shown in Table 2 and Fig. 4.The AS system performed very differently from the Bio-system. The Bio-system was found to yield better CODremoval efficiency as a result of the addition of the mixedspecial microorganisms. In the AS system, biomass concen-trations gradually decreased to below 2 g/L (day36) when

OLR was increased to 6 g COD/L·day. Considering the lowremoval rates of COD and the low biomass concentrationsin the AS reactor, the system was shut down on day36(Fig. 5). When OLR was increased, the COD removalefficiencies decreased in both systems, except for OLR upto 6 g COD/L·day for the Bio-system. The decrease inefficiency was more pronounced in the AS system, as com-pared to the Bio-system. When OLR was increased from 5to 8 g COD/L·day, COD removal efficiency decreased from54.6 to 27.8 % in the AS system and from 78.3 to 64.3 % inthe Bio-system. In the AS reactor biomass, inhibition beganat OLR >6 g COD/L·day. This is in contrast to the Bio-system, for which even the OLR of 9–10 g COD/L·dayallowed for efficient removal in the stable range of 46–53 %. An increase in the OLR to 11 g COD/L·day led to

1 2 3 4 5 6 70

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60

Lig

nin

redu

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(a)

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(b)Fig. 3 Degradation andadsorption process during blackliquor biotreatment by Bio-system, compared with AS-system as a control under opti-mized condition. Bio-system(a) and AS system (b). Thevalues are mean of threereplicates

Table 2 Average removal parameters and the final pH in the Bio-system and AS system during the operation

OLR (g/L·days) COD (%) Lignin (%) Color (%) Final pH

Bio-system

5 74.8 55.9 58.4 8.4

6 78.3 56.1 60.2 8.7

7 75.7 54.3 55.4 8.4

8 64.3 52.8 52.6 8.8

9 64.8 53.2 50.5 9.2

10 53.2 46.3 42.5 9.4

11 37.9 29.6 28.7 9.6

AS system

5 54.6 47.3 38.2 8.8

6 49.4 35.7 31.5 9.1

7 32.1 22.4 19.7 9.6

8 27.8 19.6 17.2 9.6

The values are mean of three replicates

0 10 20 30 40 50 60 700

10

20

30

40

50

60

70

80

90

BioaugmentedAS

CO

D r

emov

al(%

)

Time (d)

Fig. 4 Comparison of effect of OLR on the COD removal efficiencyin AS and Bio-systems. COD removal by the AS system (solid circle)and Bio-system (solid square). I (0–9 days): 5 g COD/L·day; II (10–17 days): 6 g COD/L·day; III (18–25 days): 7 g COD/L·day; IV (26–34 days): 8 g COD/L·day; V (35–44 days): 9 g COD/L·day; VI (45–54 days): 10 g COD/L·day; VII (55–64 days): 11 g COD/L·day. Thevalues are mean of three replicates

Appl Microbiol Biotechnol

removal efficiency of 29.6 % for COD. However, when theOLR was increased to 13 g COD/L·day, detectable levels oflignin degradation were not observed in the Bio-system,which indicated the OLR limit of the bioaugmented reactor.

Lignin and color removal efficiency

At the beginning of culture, the lignin degradation rate ofthe AS system was obviously slower than that of the Bio-system (Table 2). When the OLR increased from 5 to 8 gCOD/L·day, lignin removal efficiency varied between 52.8and 55.9 % and between 19.6 and 47.3 % in the Bio- and ASsystems, respectively. Color removal efficiency in the ASsystem varied between 19.7 and 38.2 %, whereas it was52.6–60.2 % in the Bio-system (Table 2). When the OLRwas 9–10 g COD/L·day, the removal efficiency in the Bio-system was always in the stable range of 46–53 %, and evenwhen the OLR was increased to 11 g COD/L·day, theremoval efficiency for color was 28.7 %.

Relationship between MLSS concentration and OLR

The variation in MLSS concentration at different OLRs isdepicted in Fig. 5. In the AS reactor, biomass inhibition ofOLR started above 6 g COD/L·day. No inhibition occurredat OLR of 5 g COD/L·day, indicating that biomass could beadapted to the toxic compounds in BL (as mentioned above)and that the stable operation could be attributed to theincrease of acclimated AS concentration from 4.5 to 6.2 g/L.

Meanwhile, bioaugmentation demonstrated no inhibitoryeffect at OLR from 5–10 g/L·day, and the total biomass

concentration remained relatively stable at an average of7.0 to 5.2 g/L in the Bio-system, respectively. When theOLR was increased to 10–11 g/L·day, the MLSS concentra-tion decreased sharply to 3.2 g/L, with concurrent declinesin COD and color removal efficiencies.

Comparison of enzyme activities in AS and Bio-systems

To obtain additional insights into the advantages of bioaug-mentation on the treatment of BL, the activities of variousenzymes were determined for systems at steady-state undervarious OLR. The presence of diverse lignocellulolytic en-zyme systems was observed during experiments, whichinvolved degradation of lignocellulosic materials and phe-nolics in BL under highly alkaline conditions. Figure 6shows significant differences in enzyme activities betweenthe two systems. The AS system showed high levels ofcellulase (0.80–0.84 UmL−1), xylanase (0.54–0.57 U

0 10 20 30 40 50 60 70

2

4

6

8

Time (d)

ML

SS (

g/L

)

Bioaugmented

AS

Fig. 5 Comparison of variation of MLSS concentration in AS andBio-systems. MLSS concentration in AS system (solid circle) and inBio-system (solid square). I (0–9 days): 5 g COD/L·day; II (10–17 days): 6 g COD/L·day; III (18–25 days): 7 g COD/L·day; IV (26–34 days): 8 g COD/L·day; V (35–44 days): 9 g COD/L·day; VI (45–54 days): 10 g COD/L·day; VII (55–64 days): 11 g COD/L·day. Thevalues are mean of three replicates

0.0

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1.0

CellulaseXylanaseLacMnp

Enz

yme

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ivit

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ml-1

) E

nzym

e A

ctiv

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(U m

l-1)

5 6 7 8

a

0.0

0.4

0.8

1.2

1.6

2.0CellulaseXylanaseLacMnpLip

OLR (g /l d)

OLR (g /l d)

5 6 7 8 9 10 11

b

Fig. 6 Comparison of enzyme activities in AS (a) and Bio-systems(b). The values are mean of three replicates

Appl Microbiol Biotechnol

mL−1), the ligninolytic enzyme of laccase (0.48–0.31 UmL−1), and MnP (0.21–0.18 UmL−1) when OLR rangedfrom 5 to 6 g COD/L·day; however, LiP activity was notobserved (Fig. 6a). In contrast, the Bio-system clearlyexhibited lignocellulolytic enzyme activities, with the high-est activities occurring for an OLR of 6 g COD/L·day. In theBio-system, the activities of cellulose, xylanase, laccase,MnP, and Lip stabilized at 0.83, 0.56, 1.71, 0.54, and0.49 UmL−1, respectively. The enzyme activities graduallydecreased with increasing OLR. The enzyme profile of theBio-system was in agreement with the Bio-system resultsthat demonstrated significant color and COD removals (upto 78.3 and 60.2 %, respectively), indicating that high lig-ninolytic ability could be easily correlated with high degra-dation of lignin (removal of 56.1 %).

Effect of bioaugmentation on the molecular weightdistribution of lignin in BL

The effect of biological treatment with an OLR of 6 g COD/L·day on the molecular weight distribution of lignin in BLwas studied by GPC techniques (Table 3 and Fig. 7). GPCresults clearly demonstrated that high Mw lignin fragmentsin BL were more biodegradable by the Bio-system in com-parison to the AS system. This observation is in accordancewith the COD and color removal and enzyme-profile resultsfor the Bio-system.

As shown in Table 3, lignin in untreated BL had a highMw (ranging between 2,770 and 12,000). With treatment inthe Bio-system, the fraction of high Mw lignin (Mw>12,000) decreased from 13.1 to 5.5 %, while in the ASsystem, the fraction only decreased to 12.4 %. The medi-um–high Mw lignin fraction (6,520<Mw<12,000) de-creased from 19.7 to 11.9 % in the Bio-system, and theconcentration of the aromatics increased from 54.6 to76.2 %.

Discussion

Efficient and economical treatment of BL is difficult, due tolimitations of physicochemical methods and intrinsic diffi-culties with bioremediation strategies. The present study

confirmed that the Bio-system could produce better removalefficiencies, owing to the addition of the mixed specialexogenous microorganisms that played an important rolein improving COD removal and maintaining stable removalefficiencies.

For the treatment of BL, the addition of 15 % exogenousinoculum was found to be the optimal concentration thatsignificantly improved the performance of the biotreatmentprocess. However, when inoculum concentration increasedabove the optimal amount (to 20 %), the overall CODremoval declined. It is hypothesized that the addition ofexcessive inoculum may affect the indigenous microbialcommunity, thereby resulting in an unbalanced system(Bouchez et al. 2000). The multi-species microbial commu-nity present in the endogenous AS system also demonstratedthat the start-up time of the Bio-system was accelerated bythe introduction of the appropriate inoculum concentration.A similar result was reported by Quan et al. (2004), whodemonstrated shorter start-up times for an SBR systemremoving 2,4-dichlorophenol with the addition of an immo-bilized mixed culture.

One of the difficulties of BL bioremediation is its highlyalkaline nature; therefore, the study of the effect of initial pHon the Bio-system compared to the AS system was testedover a wide pH range, from 7 to 13. The result was inter-esting as the AS system had a poor capability of remediatingBL in the whole pH range of 7–13, while the Bio-systemwas found capable of effective treating BL in the pH rangefrom 9–11. The superior performance of the Bio-system incomparison to the AS system can be explained as a largeportion of the various populations in the Bio-system secret-ing significant lignocellulolytic enzymes under high pHconditions. Previous studies have reported that a pH of 8.5or higher severely inhibits lignin-degrading microorganismgrowth and enzyme secretion (Grover et al. 1999; Singhaland Thakur 2009). Limited research has focused on micro-organisms capable of treating BL at pH>10 (Yang et al.2008, 2010; Wu et al. 2005). Yang et al. (2010) reported thatHalomonas sp. Y2 was efficient in lowering pH and color ofBL with a pH of 11.0 and COD of 139,300 mg/L. Theyfound after 48 h that Y2 decreased the pH from 11.0 to 7.4,and reduced color and COD by 52.5 and 6.7 %, respectively.They also reported that the polysaccharides in the BL were

Table 3 Characteristics of bio-treatment on the molecularweight distribution of lignin BLby AS and Bio-system at OLRof 6 g COD/L·day

Molecular weightdistribution

Amount of fraction (%)

Mw <2,770 2,770–6,520 6,520–12,000 >12,000

Control 54.6 12.6 19.7 13.1

AS 58.9 9.4 19.3 12.4

Bioaugmentation 76.3 6.3 11.9 5.5

Appl Microbiol Biotechnol

utilized for cells growth and transformed into organic acidsresulting in deceased in pH and COD values. However, noother published data on bioaugmented activated sludgetreatment of BL with special mixed microorganisms athigher pH (>10) are available.

The present study demonstrated that high removal ofCOD, color, and lignin by 64.8, 50.5, and 53.2 % at OLRof 9 g COD/L·day was attained under high-alkaline condi-tions (pH10) in the novel Bio-system, which is superior tothe capabilities of other systems previously reported in theliterature. For instance, Grover et al. (1999) reported thatwith biotreatment of BL using an anaerobic baffled reactor,the reactor showed stable operation at an OLR of 6 g COD/L·day with COD removal up to 54 %. They also demon-strated that BL is toxic to anaerobic treatment at high con-centration and needs to be diluted to a subtoxic level. Vidalet al. (2001) noted that the COD removal ranged between 30and 50 % while the OLR was increasing from 1.2 to 3.3 gCOD/L·day during anaerobic sludge blanket operation at a10 % dilution of BL with tap water. It was also reported thatlignin removal during anaerobic wastewater treatment waspoor. Compared to the above reports, the treatment processidentified in the present study (Bio-system) had effectivelyreduced COD and color. Thus, the newly developed Bio-system achieved effective treatment of BL with a highbiodegradability rate with OLRs from 5–9 g COD/L·day,indicating that a remarkably higher OLR can be managed bythe Bio-system.

Further insights into the advantages of the Bio-system inthe treatment of BL can be gained from comparing theenzyme activities from the AS only and Bio-systems atsteady state under varying OLR. In the Bio-system, thepresence of diverse lignocellulolytic enzyme systems wasobserved during experiments, which were clearly capable of

degrading lignocellulosic materials and phenolics in BLunder highly alkaline conditions. In contrast, the AS systemshowed high levels of the cellulase, xylanase, and laccasewhen the OLR ranged between 5 and 6 g COD/L·day;however, LiP activity was not observed. The absence ofLiP in the AS system demonstrates its importance in thedepolymerization of lignin (Stewart et al. 1992; Sayadi andEllouz 1995; Chatzisymeon et al. 2009), as the AS systempresented less lignin removal efficiency. These alkaline-tolerant lignocellulolytic enzymes are unique, as most ligno-cellulolytic enzymes reported to date have activities in theacidic range (Alessandro et al. 1999, 2000). Similar resultswere reported by Mishra and Thakur (2010), who found thatxylananse production was induced by 10 % BL with Bacil-lus sp., and the enzyme exhibited high activity (221 U/mg)at pH8. Yang et al. (2010) reported that Halomonas sp.Y2was efficient in biotreatment of alkaline BL, and they ob-served the strain could produce CMCase and xylanase whenthe strain was cultured in basal liquid medium with CMC-Na and xylan but not given the value of the enzyme activity.Peňalva and Arst (2002) also reported that xylanases andcellulases were regulated by pH. At the present time, fewreports are available regarding the ligninolytic enzymesproduced by these microorganisms in Bio-system and theirrole in BL biotreatment; however, future studies will explorethis in detail. In addition, it is noteworthy that the lignocel-lulolytic enzymes of the Bio-system exhibited high activityand good stability under alkaline conditions, making theBio-system a potent candidate for direct treatment of thealkaline BL and avoiding the cost-incurring and time-consuming steps in pH readjustment.

The degradation of lignin by the Bio- and AS systemswas also assessed by comparing the Mw distribution oflignin in the untreated and treated wastewater. Wastewaters,such as BL and bleaching effluents, in which lignin canaccount for up to 50 % of the COD, are generally only50 % biodegradable or less by conventional biological treat-ment (Sierra-Alvarez et al. 1991). However, the presentresults clearly demonstrated that high Mw lignin fragmentsin BL were biodegradable by the Bio-system. Compared tothe results from the Bio-system, the profile of the AS systemwas shifted to a smaller Mw region and the change in theMw distribution (Mw<6,520) was only observed withdecreases from 12.6 to 9.4 %. This result was likely attrib-utable to the fact that lignin in the AS system could be partlydegraded to some smaller lignin aromatic compounds,resulting in an increase of the low molecular weight lignin.

Furthermore, the present study has demonstrated the syn-ergistic potential of bioaugmentation with endogenous co-culture (bacterial and fungal cultures) for the biotreatment ofBL. Similar results were observed by Stanley et al. (1999)when benzo[α]pyrene (BaP) was incubated with fungi and aBaP-degrading bacterial community. However, there is

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Fig. 7 Characteristics of biotreatment on the molecular weight distri-bution of lignin in BL by AS and Bio-system compared with untreatedcontrol when OLR is 6 g COD/L·day

Appl Microbiol Biotechnol

scarce information outlining the synergistic effects of BLbiotreatment with co-cultures. Further studies should becarried out to elucidate the interaction mechanisms at theinterface of co-cultured bacterium and fungi using efficientapproaches, to characterize the physicochemical and bio-chemical properties of enzymes, and to analyze the exis-tence and variations of exogenous microorganisms in theBio-system in order to obtain more knowledge about thelignocellulolytic degradation system.

This study is the first to report on bioaugmentation of ASwith unique lignocellulose-degrading microorganisms isolatedfrom degraded bamboo slips in an SBR as a novel strategy forBL treatment. The study demonstrated successful treatment ofBL with an activated sludge system bioaugmented with alignin-degrading exogenous mixed culture isolated from an-cient bamboo slips. The bioaugmented activated sludge systemwas shown to successfully degrade lignin, removing COD andcolor under high OLR and very alkaline conditions for a long-term operation of up to 60 days. Significant advantages of thebioaugmentation system for treatment of BL are (a) achieve-ment of a rapid start-up time with the Bio-system and (b) ahigher stability of biotreatment performance compared to theAS system. Moreover, the AS system failed to treat BL withOLR>8 g COD/L·day, whereas the system augmented with15 % special mixed microorganisms could cope with BLefficiently under OLR as high as 10 g COD/L·day. Exogenousbioaugmentation of activated sludge with a unique microor-ganism culture is a feasible alternative strategy for enhancedbiological treatment of wastewater with high lignin content andhigh OLR under strong alkaline conditions.

Acknowledgments This work was supported financially by the ChinaNational Funds for Distinguished Young Scientists (50925417), the KeyProject of National Natural Science Foundation of China (50800301).

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