Microbial community structure and metabolic property of biofilms in vermifiltration for liquid-state sludge stabilization using PLFA profiles

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  • bta

    , Babor

    h i g h l i g h t s

    lter (Vthe bioogical ainant inrmanc

    to saturated (mono:sat) PLFAs of VF biolms was higher than that of BF bio-e physiological and nutritional stress for microbial community in VF was

    (MWWTP) have been built in small towns in China due to the

    most MWWTPs in small towns can not afford to construct andmaintain conventional sludge treatment processes such as anaero-bic and aerobic digestion (Wei et al., 2003; Xing et al., 2011). Theapplication of vermiltration (a liquid-state vermiconversion) forsludge treatment has turned out to be ecologically sound, econom-ically viable and socially acceptable way to treat liquid-statesludge before dewatering (Xing et al., 2011; Zhao et al., 2010).

    Yang et al., 2013b).position processand microthe micro

    gradationorganics, compared with the microbial community develothe conventional biolters, microbial communities devin the VF, which are mainly affected by the earthworm activitiesin the lter bed, are exposed to different conditions (Liu et al.,2012). Earthworms can modify microora directly and indirectlyby three main mechanisms: (1) comminution, burrowing and cast-ing; (2) grazing; (3) dispersal (Brown, 1995). These activities maychange the substrates physico-chemical and biological statusand cause drastic shifts in the density, diversity, compositionsand activities of microbial communities in the VF biolms (Li

    Corresponding author. Tel./fax: +86 21 65984275.

    Bioresource Technology 151 (2014) 340346

    Contents lists availab

    T

    elsE-mail addresses: xingmeiyan@tongji.edu.cn, xmy5000@163.com (M. Xing).requirements of better quality water and the implementation ofstricter environment laws (Chen et al., 2008). This leads to a sharpincrease in sewage sludge production. Sludge management cost ac-counts for up to 60% of the total operation cost of MWWTPs, and

    Vermiltration refers to an organic decominvolving the interactions between earthwormsisms (Liu et al., 2012; Xing et al., 2012). Althoughisms are responsible for the bio-chemical de0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.10.075organ-organ-of theped inelopedKeywords:Sludge vermiconversionPhospholipid fatty acidMicrobial activityMicrobial community diversityMetabolic property

    relieved due to the increasing of soluble substances caused by the earthworm ingestion. Further investi-gation showed that the burrowing action of earthworms promoted the aeration condition and led to aer-obic microorganisms were predominant in VF. Those results indicated earthworms improved microbialcommunity structure and metabolic properties of biolms and thus resulted in the overall optimizationof the vermiltration system for liquid-state sludge stabilization.

    2013 Elsevier Ltd. All rights reserved.

    1. Introduction

    More and more municipal wastewater treatment plants

    Compared with the conventional biolter (BF), the treatment per-formance of liquid-state sludge by vermilter (VF) was improvedsignicantly due to the presence of earthworms (Liu et al., 2012;Available online 1 November 2013ratio of monounsaturatedlms, which indicated thMicrobial metabolic property of vermi Richer fungi diversity was featured in Earthworms relieved microbial physiol Aerobic microorganisms were predom Earthworms optimized treatment perfo

    a r t i c l e i n f o

    Article history:Received 4 September 2013Received in revised form 20 October 2013Accepted 23 October 2013F) biolms has never been reported.lms of VF than biolter (BF).nd nutritional stress in VF biolms.VF due to earthworm burrowing action.e of VF on sludge stabilization.

    a b s t r a c t

    To investigate effects of earthworms on microbial community structure and metabolic properties of bio-lms in vermiltration for liquid-state sludge stabilization, a vermilter (VF) with earthworms and a con-ventional biolter (BF) without earthworms were compared. The Shannon index of fungi in VF was 16%higher than that in BF, which indicated earthworm activities signicantly enhanced fungi diversity. TheMicrobial community structure and metain vermiltration for liquid-state sludge s

    Chunhui Zhao, Meiyan Xing , Jian Yang, Yongsen LuKey Laboratory of Yangtze River Water Environment, Ministry of Education, State Key Land Engineering, Tongji University, Shanghai 200092, China

    Bioresource

    journal homepage: www.olic property of biolmsbilization using PLFA proles

    aoyi Lvatory of Pollution Control and Resources Reuse, College of Environmental Science

    le at ScienceDirect

    echnology

    evier .com/locate /bior tech

  • membrane.

    2.3. Dehydrogenase activity

    Dehydrogenase activity has been adopted to assess the totalmicrobial activity of sludge (Liu et al., 2012). Thus the total micro-bial activity of biolms during vermiltration of liquid-state sludgewas evaluated according to the method proposed by Caravelli et al.(2004). One unit of dehydrogenase activity was dened as thecatalysis capacity required for producing 1 mg INTF per hour.

    2.4. Phospholipid fatty acid analysis

    Fatty acids were extracted from 4 g freeze-dried biolmsamples with approximate 40 ml extraction mixture containingphosphate buffer, chloroform and methanol (0.9:1:2, V:V:V)(Bossio and Scow, 1998). The lipid extraction was separatedthrough a solid phase extraction column (500 mg; 3 ml; Agilent

    echnet al., 2013). Polymerase chain reaction-denaturing gradient gelelectrophoresis (PCR-DGGE) technique has been applied to explorethe microbial community structure of biolms in VF and found thatthose biolms were featured on richer diversity in their microbialcommunity than BF biolms (Li et al., 2013; Liu et al., 2012). How-ever, due to the limitation of PCR-DGGE technique, the function ofmicrobial community such as metabolic properties has not beenfully studied (Leckie, 2005; Suzuki and Giovannoni, 1996). There-fore, a suitable technology needs to be applied to further discoverthe mechanism such as the diversity and the metabolic property ofmicrobial community in biolms during vermiltration of liquid-state sludge.

    Phospholipid fatty acids (PLFAs) is one of the most importantbiomarkers of microorganisms, which is useful for characterizingthe microbial biomass in various environments, such as agricul-tural soils and various other systems (Amir et al., 2008). The mem-brane PLFA contents and composition in living bacterial cells arerelatively unchanged under various growth conditions and PLFAsare rapidly degraded after the death of microorganisms (Amiret al., 2008). Hence, PLFAs can be used to estimate the total viablemicrobial biomass contained in a sample. Moreover, differentmicrobial communities have different PLFA compositions. ThusPLFA composition analysis can provide the information on thecompositions and overall changes in major groups, such as bacte-ria, actinomycetes and fungi. Furthermore, the analysis of charac-teristic PLFA ratio also provides more information on themetabolic properties of the microbial community structure. Forexample, the ratio of monounsaturated to saturated (mono:sat)PLFAs was used as an indicator of physiological or nutritional stressin microbial communities. This ratio is generally lower in microbialcommunities in the environment with limited organic carbon andnutrients (Gomez-Brandon et al., 2011). Generally, the PLFA ap-proach is superior to PCR-DGGE in providing a quantitative mea-sure of the microbial community structure composition (bacteria,actinomycetes and fungi) and reecting the metabolic propertiesof microbial community (White et al., 1998).

    In this study, the application of PLFA analysis presented new in-sights into microbial community structure and metabolic proper-ties of biolms in the VF. The hypothesis that earthworms in theVF could result in the overall optimization of the vermiltrationsystem for liquid-state sludge stabilization could be proposedbased on the results: (1) earthworm activities in the VF signi-cantly enhanced the microbial activity and microbial communitydiversity; (2) the ingestion of earthworm increased the solublesubstances and thereby relieved physiological or nutritional stressof microbial community; (3) the burrowing action of earthwormspromoted the aeration condition and led to aerobic microorgan-isms were predominant in the VF.

    2. Methods

    2.1. Vermilter setup and operation

    Two sets (each set has three parallel reactors) of cylindrical l-ters were set up. One set was the vermilters (Fig. 1) with an initialearthworm density of 32 g/L (fresh weight basis) as suggested byZhao et al. (2010), while the conventional biolters (BF) withoutearthworms were used as the control. Each lter (diameter of20 cm and depth of 100 cm, made of perspex) had a working vol-ume of 31.4 L and was packed with ceramsites (1020 mm indiameter). A layer of plastic ber was placed on the top of the lterbed to avoid the direct hydraulic inuence on the earthworms andensure an even inuent sludge distribution. The earthworms,

    C. Zhao et al. / Bioresource TEisenia fetida, used in this study were purchased from a farm inYancheng City, China. The inuent sludge was obtained from thesecondary sedimentation tank of a municipal WWTP in Shanghai,China. The hydraulic load of the two sets of lters was kept at4 m/d, and the organic load of the inuent sludge was maintainedwithin the range of 1.101.28 kg-VSS/(m3 d). After passing throughthe lter bed continuously, the sludge entered into a sedimenta-tion tank. These lters ran steadily for 8 months to investigatetheir treatment performances on liquid-state sludge stabilizationafter about 30-day acclimation.

    2.2. Sampling and chemical analysis

    Biolm samples were collected from the lter bed in both BFand VF reactors in the depths of 12, 37, 62 and 87 cm after theexperiment completion to evaluate the microbial activities andPLFA proles of microbial communities. Samples form the BF atthe depths of 12, 37, 62 and 87 cm were, respectively designatedas B1, B2, B3 and B4, while those from the VF were designated asV1V4. The biolms on the ceramsites were rinsed into centrifugetubes with sterile water, and then centrifuged (9000 rpm) for15 min at 4 C. The dewatered biolm samples were freeze-driedand grounded through the 0.15 mm mesh for further analysis.

    Sludge characteristics such as the suspended solids (SS) and vol-atile suspended solids (VSS) were assessed according to ChineseStandard Methods. Total chemical oxygen demand (TCOD) and sol-uble COD (SCOD) were measured with a NOVA60 COD meter(Merck, Germany), and the samples for the SCOD measurementwere rstly ltered through the 0.45 lm mixed cellulose ester

    Fig. 1. Schematic diagram of the vermilter (VF, with earthworms in the lter bed).

    ology 151 (2014) 340346 341Technologies Inc., UK). The neutral lipids, glycolipids andphospholipids were eluted with chloroform, acetone and metha-nol, respectively (Frostegard and Baath, 1996). The Phospholipid

  • shows that the average VSS/SS ratio of the VF efuent sludge(VES) was decreased to 0.63 from 0.73 (the inuent sludge) afterVF treatment. The decrease of VSS/SS ratio of the BF efuent sludge(BES) was also observed, whereas the observed decrease was not assignicant as that of VES. The different sludge reduction degrees byBF and VF treatment resulted in the difference in TCOD and SCODconcentrations. As shown in Table 1, after BF and VF treatment, theSCOD concentrations in the efuent sludge were 36.3 and 68.6 mg/L, respectively. It suggested that the earthworm activities trans-formed much more insoluble substance into soluble organics. Itis well known that microbial communities preferentially select sol-uble substances as their diets (Sen and Chandra, 2009), and thisfraction could be utilized by microorganism as carbon and energysources and be related positively to microbial activities. Thus, theincrease of soluble organics might affect the microbial biomassand activity of microorganisms in the VF reactor.

    3.2. Total microbial biomass

    echnology 151 (2014) 340346fatty acid (PLFA) fraction was transesteried into fatty acid methylesters (FAMEs) through mild alkaline methanolysis reaction (Amiret al., 2010). Then FAMEs were analyzed on a Trace DSQ gas chro-matographymass spectrometer (Thermo, USA). The detailed GCMS experimental conditions were obtained according to the meth-od proposed by Dungait et al. (2008). The PLFAs were quantiedthrough the comparing the peak areas with those of an internalstandard nonadecane (19:0) peak. In order to identify the FAMEs,both the retention time and the mass spectra were compared withthe FAME standards (Bacterial Acid Methyl Esters Mix 47080-Uand SupelcoTM 37 Component FAME Mix 18919-1AMP, SigmaAldrich, USA).

    PLFA nomenclature used in this study was performed accordingto Amir et al. (2010). Total carbon atoms: double bonds, followedby the position x of the double bond from the methyl end of themolecule. The cis and trans congurations are indicated by c andt, respectively. The anteiso and iso branching are designated bythe prex a or i. Cyclopropyl fatty acids is expressed as cy.

    The sum of all the identied PLFAs was used to estimate the to-tal viable microbial biomass (Zelles, 1999). Certain PLFAs wereused as biomarkers to determine the presence and abundance ofspecic microbial groups (Joergensen and Wichern, 2008). Thesum of PLFAs characteristic of Gram-positive (i15:0, a15:0, i16:0and i17:0) and Gram-negative bacteria (16:1x9c, 17:1x9c,cy17:0, 18:1x9t, cy19:0) was selected to represent bacterial PLFAs(Amir et al., 2008; Zornoza et al., 2009). The PLFAs of 10Me16:0and 10Me18:0 were selected as the indicators of actinomycete bio-markers (Schmitt et al., 2010). The PLFAs (18:3x3, 18:1x9c,18:2x6, 20:5x3 and 20:1x9) were used as fungal biomarkers(Madan et al., 2002; Zelles, 1997).

    2.5. Data analysis

    The Shannon index was calculated to represent the diversity ofthe microbial community structure based on the number of identi-ed PLFAs (Zornoza et al., 2009). The Shannon index for the iden-tied PLFAs (HPLFA) was calculated as follows:

    HPLFA XR

    i1pi ln pi 1

    where, pi is the relative abundance of each PLFA in the total sum; Ris the number of the identied PLFAs.

    All assays were conducted in triplicate and the results were ex-pressed as mean standard deviation. The analysis of variance(ANOVA) was used to test the signicance of the assays. Ifp < 0.05, the results were considered to be statistically signicant.A principal component analysis of PLFA data was used to assessthe inuences of earthworm presence and the depth of lter beddepth on the microbial community structures of BF and VF bio-lms. All the statistical analysis was performed with the softwareSPSS 17.0.

    3. Results and discussion

    3.1. Treatment performance

    Both the BF and VF reactors were operated steadily withoutclogging during the experimental period and their performanceson sludge reduction were presented in Fig. 2.

    As shown in Fig. 2a, the VSS reduction of sludge in VF was49.9 2.8%, which was 14.5% higher than that in BF. It means thatthe presence of earthworms in the VF signicantly enhanced

    342 C. Zhao et al. / Bioresource Tsludge reduction. The degree of the sludge stabilization can be as-sessed by the VSS/SS ratio. The lower VSS/SS ratio represents thehigher degradation degree of organics (Zhao et al., 2010). Fig. 2bThe total microbial biomass of the biolms could be indicatedby total PLFAs. As the above has shown that the depth of the lteris 100 cm and different microbial biomass quantities in thebiolms might correspond to certain depth. Thus, their microbialbiomass as a function of the depth distribution of biolms in thetwo lters was investigated and shown in Fig. 3.

    As shown in Fig. 3, the total PLFA concentrations of these twolters were decreased with the deepening of the depth. Withinthe investigated depth of the VF and BF biolms, total PLFA con-centrations in VF and BF biolms were correspondingly decreasedfrom 1750 to 1401 lg g1-VSS and 2271 to 1609 lg g1-VSS,respectively. In the ltration systems, when the inuent sludgepassed through the lter beds, the available organics weredecreased with the gradual organic degradation. Accordingly, thetotal microbial biomass distribution along the depth followed thesame trend. As shown in Fig. 3, total PLFA concentrations in VFbiolms were signicantly lower than those in BF biolms, indicat-ing that earthworm activities led to the decrease in microbial bio-mass of biolms for the earthworms accelerated the consumptionof resources for the microbes (Dominguez et al., 2004). Addition-ally, the selective ingestion of microorganisms by earthwormmight be another reason for the decrease of microbial biomass(Thimm et al., 1998). Gomez-Brandon et al. (2011) reported thatthe decrease in the viable microbial biomass (indicated by totalPLFAs) by earthworm activities was approximately 1.3 times ofFig. 2. Performance data of the BF and the VF reactors: (a) the VSS reduction ofsludge; (b) the ratios of VSS/SS in the inuent and efuent sludge.

  • distribution. Different letters indicate statistical differences from each other

    echnthat in the control without earthworms during the vermicompo-

    (p < 0.05).Fig. 3. Variation of microbial biomass in the BF and VF biolms versus the depthTable 1Concentrations of TCOD and SCOD in the inuent, BF and VF efuent sludge.

    Samples Average SCOD (mg/L) Average TCOD (mg/L) SCOD/TCOD

    Inuent 65.4 7.4 411 11.1 0.16 0.08BF efuent 36.3 4.1 279 9.4 0.13 0.05VF efuent 68.6 10.0 210 8.9 0.32 0.09

    C. Zhao et al. / Bioresource Tsting of pig slurry. They also observed that the decrease in theabundance of characteristic bacterial and fungal PLFAs in the castsof earthworms (Gomez-Brandon et al., 2010).

    3.3. Total microbial activity

    Despite the lower microbial biomass in the VF biolms was ob-served, the results of dehydrogenase activity variation (Fig. 4)showed that the presence of earthworms enhanced the totalmicrobial activity in the VF biolms compared with the BFbiolms.

    At different investigated lter depths, the dehydrogenase activ-ity in VF biolms was always higher than that in the BF biolms(the maximum increase level was 0.58 mgh1g1-VSS). In the VFsystem, the improved aerobic condition caused by the burrowingaction of earthworms provided a favorable microenvironment foraerobic microorganisms, thus increasing the microbial activity.Additionally, the released mucus and urine into the lter bed byearthworms was also responsible for the higher microbial activityin VF reactor (Liu et al., 2012). Hence, despite the microbial bio-mass was decreased, the presence of earthworms played an impor-tant role in enhancing the microbial activity and improving organicmatter degradation during vermiltration of liquid-state sludge.

    3.4. Microbial community structure

    3.4.1. PLFA prolesPLFA proles can provide the insights into the microbial com-

    munity structure because that a relative abundance of characteris-tic PLFAs is considerably different among specic groups ofmicroorganisms (Li et al., 2010). PLFA proles of the BF and VF bio-lms at different depths were investigated in this study and shownin Fig. 5. It was found that there were 16 identied PLFAs in VFbiolms at every investigated depth (V1V4), and the correspond-ing numbers in B1B4 were 14, 14, 13 and 12, respectively. The10Me18:0 (PLFA of actinomycetes) and cy17:0 (PLFA of bacteria)were only detected in the VF biolms. The loss of cy19:0 (PLFAof bacteria) was observed in both B3 and B4. 20:1x9c, as thebiomarker of fungi, was not detected in B4. However, the concen-trations of most microbial groups in VF biolms were lower thanthose in BF. These results were consistent with the above observa-tion that the microbial biomass of biolm in VF was lower thanthat in BF biolms.

    The data of PLFA proles (the 16 identied PLFAs) were furtheranalyzed with the principal component analysis (PCA) to assessoverall differences in the microbial community structures of BFand VF biolms of the two lters at different depths as well asthe inuence of the earthworm presence and the different depthsof lter bed on microbial community structures. The rst principalcomponent (PC1) and the second principal component (PC2)explained 52.1% and 27.7% of total data variability, respectively.As shown in the score plot of PC1 versus PC2 (Fig. 6a), biolm

    Fig. 4. Variations of dehydrogenase activity in the BF and VF biolms versus depthdistribution. Different letters indicate statistical differences from each other(p < 0.05).

    ology 151 (2014) 340346 343samples could be clearly distinguished from each other based onsampling depth (12, 37, 62 and 87 cm) and the presence of earth-worms. Biolm samples from VF were clustered on the positiveside of PC1, while samples from BF were distributed on the nega-tive side of PC1. Thus, the PC1 reected the effect of earthwormactivity on the variation of PLFA proles. Both BF and VF biolmssampled along the depth showed a positive relationship withPC2. Therefore, the PC2 mainly reected the variation betweenPLFA proles and depths of the lter beds. Additionally, the bio-lms sampled along the depth showed a negative relationship withPC2 due to the type and concentration of PLFAs were decreasedwith the deepening of the lter bed. The factor loadings of the 16identied PLFAs (Fig. 6b) were divided into three domains (IIII).Both Domain I (i16:0, cy17:0, 10Me18:0, cy19:0 20:1x9c and20:5x3) and Domain II (16:1x7c, i17:0, 18:1x7c and 18:1x9c)of the PLFAs were mainly clustered on the positive side of PC1and PC2, respectively, and Domain III (a15:0, i15:0, 10Me16:0and 16:1w7t) was in negative relationship with PC1 and in moder-ately positive relationship with PC2. As the above has shown(Fig. 6a) biolm samples from VF reactor were mainly clusteredon the positive side of PC1, indicating a correlative relationshipwith Domain I of PLFA proles. The results suggested that earth-worm activities could benet the microorganisms containing thosetypes of PLFAs (Domain I) and thus modied the structure of themicrobial communities in VF reactor.

  • echn344 C. Zhao et al. / Bioresource T3.4.2. Microbial community diversityThe Shannon index for the identied PLFAs (HPLFA) was used to

    calculate the degree of microbial diversity in the biolms of thetwo lters and the high value of HPLFA corresponded to the highdiversity and complex system. As shown in Table 2, the totalHPLFA in VF biolms ranging from 3.69 to 3.79 (with the averageof 3.77), was higher than those in BF biolms (with the averageof 3.49). The higher Shannon index indicated that the populationof microbial community structure in VF reactor had been diversi-ed by the earthworm activities. This could be further supportedby HPLFA of Fungi, which was 16% higher in VF than that in BF. Thedifference of bacteria HPLFA and actinomycetes HPLFA between VF

    Fig. 5. PLFA proles of BF and VF biolms at different depths of 12 c

    Fig. 6. Principal component analysis of 16 identied PLFAs at different sampling depths oVF biolms. (a) the score plot of PC1 versus PC2; (b) the factor loadings of the 16 identology 151 (2014) 340346and BF biolms was not signicant (p > 0.05). These results re-vealed that the enrichment of fungi was the main reason forthe intensication of microbial community diversity in the VFreactor. The effect of earthworms on fungi might be caused byeither physicochemical modication of substrate or dispersionof propagules (Lavelle et al., 1997). The increase of soluble organ-ics might contribute to suitable growth conditions for fungi. Airaet al. (2007) also reported that earthworm activity enhanced thefungi populations through creating favorable conditions for fungigrowth and that the dispersion of spores caused by the burrowingaction of earthworms might play an important role in fungigrowth.

    m (B1, V1), 37 cm (B2, V2), 62 cm (B3, V3) and 87 cm (B4, V4).

    f 12 cm (B1, V1), 37 cm (B2, V2), 62 cm (B3, V3) and 87 cm (B4, V4) from the BF andied PLFAs.

  • indicating that the aerobic conditions had been improved by earth-

    had a higher efciency under aeration condition, thus the presence

    earthworms. Those results suggested that the improved microbial

    Table 2The Shannon diversity index for total PLFAs and the specic microbial groups (bacteria, fu

    Depth (cm) Total HPLFA Bacteria HPLFA

    BF VF BF VF

    12 3.50 3.69 2.13 2.0737 3.54 3.75 2.11 2.1462 3.48 3.84 2.03 2.1587 3.44 3.79 2.00 2.21Average 3.49 3.77 2.07 2.14

    C. Zhao et al. / Bioresource Techn3.4.3. Metabolic property of the microbial community structureThe PLFA proles provide not only the information on the

    microbial community structure but also the information on themetabolic status of microorganisms. The ratios of characteristicPLFAs have been used to explore the metabolic properties of themicrobial community structures (Li et al., 2010). The ratio ofmonounsaturated to saturated (mono:sat) PLFAs was an indicatorof physiological or nutritional stress in microbial communities.The ratio is generally low when organic carbon and/or nutrientsare not enough for microorganisms (Bossio and Scow, 1998;Gomez-Brandon et al., 2011). As shown in Table 3, the ratios ofmonounsaturated to saturated (mono:sat) PLFAs were decreasedwith the depth in the BF and VF reactors. And the varations ofratios indicated the decrease of available organics and the lack ofthe diet for microorganisms when the inuent sludge passedthrough the lters. Moreover, the higher mono:sat PLFA ratioswas found in the VF biolms than that in the BF biolms, revealingthat microbial communities in VF might have been less affected bylimited resource because of the earthworm activities. As shown inTable 1, the SCOD/TCOD ratio in inuent and BF and VF efuentswere 0.16, 0.13 and 0.32, respectively, indicating that the earth-worms in the VF reactor transformed more insoluble organics intosoluble organics. Microbial communities preferentially utilizedsoluble substances as their diet, and the increasing soluble organicswere used as the carbon and energy sources of microorganisms inVF biolms to enhance the microbial activities. Additionally,mucus and excretory substances such as urea and ammonia pro-duced by earthworms in the VF also formed a readily assimilablepool of nutrients for microorganisms (Dominguez et al., 2004).

    The monounsaturated PLFAs such as 16:1x7, 16:1x9 and18:1x9 could be identied as the PLFAs of aerobic bacteria, andthe branched PLFAs such as a15:0, i15:0, i16:0 and i17:0 wereidentied as the PLFAs of anaerobic bacteria (Keith-Roach et al.,2002; Li et al., 2010). The ratio of aerobic to anaerobic PLFAs exceed1.00 represents the dominance of aerobic microorganisms (Li et al.,2010). As shown in Table 3, the ratios of aerobic to anaerobic PLFAsin BF were decreased from 1.13 to 0.67 with the depth, indicatingthat the anaerobic microorganisms were predominant in the bot-tom of BF. The phenomenon was caused by the insufcient venti-lation in the lower layers in the BF. However, in the VF reactor,the ratios of aerobic to anaerobic PLFAs always exceeded 1.00,Table 3Ratios of monounsaturated to saturated (mono:sat) PLFAs and monounsaturated tobranched bacteria PLFAs (mono:bran).

    Depth (cm) mono:sat PLFA ratio mono:bran PLFA ratio

    BF VF BF VF

    12 1.44 0.03a 1.83 0.01a 1.13 0.06a 1.46 0.02a

    37 1.41 0.14ad 1.70 0.01b 1.09 0.06a 1.28 0.04b

    62 1.26 0.01bd 1.45 0.04c 0.90 0.03b 1.07 0.01c

    87 1.09 0.10c 1.42 0.06c 0.67 0.02c 1.01 0.01c

    Average 1.30 0.08 1.60 0.15 0.90 0.10 1.20 0.16

    Different letters in the same column indicate statistical differences from each other(p < 0.05).community diversity and metabolic properties of biolms causedby earthworms brought about the overall optimization of the ver-miltration system for liquid-state sludge stabilization.

    Acknowledgement

    This work was partially supported by the National Natural Sci-ence Foundation of China (NSFC, No. 51109161), the PhD ProgramsFoundation of Ministry of Education of China (20110072120029),the Fundamental Research Funds for The Central Universities(0400219187), the Open Analysis Fund for Large Apparatus andEquipments of Tongji University (No. 2012055), the National SparkProgram of China (2010GA680004).and distribution of earthworms improved aeration condition in theVF and thereby enhanced organic degradation during liquid-statesludge stabilization (Liu et al., 2012).

    4. Conclusion

    Earthworms signicantly enhanced the microbial activity andmicrobial community diversity in the VF biolms. The ratio ofmonounsaturated to saturated (mono:sat) PLFAs revealed thatthe physiological and nutritional stress of microbial communityin the VF was relieved due to the increasing of soluble substancescaused by the ingestion of earthworm. Further investigationdemonstrated that more organics was decomposed owing to theimproved aeration of the lter bed by the burrowing action ofworms. Yang et al. (2013a) also reported that sufcient oxygen andimproved aerobic conditions for burrowing action of the earth-worms favor the micro-condition for aerobic microorganisms inthe VF systems. In addition, the ratio of aerobic to anaerobic PLFAswas the highest (1.46) in the depth of 12 cm. This result could beattributed to the burrowing action of Eisenia fetida used in thisstudy, which was the epigeic earthworms and mainly dwelled onthe top of the lter bed to signicantly promote the aeration ofthe lter bed. It is well known that the degradation of organicngi and actinomycetes) of both BF and VF biolms at different depths.

    Fungi HPLFA Actinomycetes HPLFA

    BF VF BF VF

    1.03 1.21 0.34 0.411.08 1.26 0.35 0.351.07 1.27 0.38 0.421.06 1.20 0.38 0.381.06 1.23 0.36 0.39

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    Microbial community structure and metabolic property of biofilms in vermifiltration for liquid-state sludge stabilization using PLFA profiles1 Introduction2 Methods2.1 Vermifilter setup and operation2.2 Sampling and chemical analysis2.3 Dehydrogenase activity2.4 Phospholipid fatty acid analysis2.5 Data analysis

    3 Results and discussion3.1 Treatment performance3.2 Total microbial biomass3.3 Total microbial activity3.4 Microbial community structure3.4.1 PLFA profiles3.4.2 Microbial community diversity3.4.3 Metabolic property of the microbial community structure

    4 ConclusionAcknowledgementReferences

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