Bioresource Technology 151 (2014) 340–346
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Bioresource Technology
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Microbial community structure and metabolic property of biofilmsin vermifiltration for liquid-state sludge stabilization using PLFA profiles
0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.10.075
⇑ Corresponding author. Tel./fax: +86 21 65984275.E-mail addresses: [email protected], [email protected] (M. Xing).
Chunhui Zhao, Meiyan Xing ⇑, Jian Yang, Yongsen Lu, Baoyi LvKey Laboratory of Yangtze River Water Environment, Ministry of Education, State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Scienceand Engineering, Tongji University, Shanghai 200092, China
h i g h l i g h t s
�Microbial metabolic property of vermifilter (VF) biofilms has never been reported.� Richer fungi diversity was featured in the biofilms of VF than biofilter (BF).� Earthworms relieved microbial physiological and nutritional stress in VF biofilms.� Aerobic microorganisms were predominant in VF due to earthworm burrowing action.� Earthworms optimized treatment performance of VF on sludge stabilization.
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 2013Available online 1 November 2013
Keywords:Sludge vermiconversionPhospholipid fatty acidMicrobial activityMicrobial community diversityMetabolic property
a b s t r a c t
To investigate effects of earthworms on microbial community structure and metabolic properties of bio-films in vermifiltration for liquid-state sludge stabilization, a vermifilter (VF) with earthworms and a con-ventional biofilter (BF) without earthworms were compared. The Shannon index of fungi in VF was 16%higher than that in BF, which indicated earthworm activities significantly enhanced fungi diversity. Theratio of monounsaturated to saturated (mono:sat) PLFAs of VF biofilms was higher than that of BF bio-films, which indicated the physiological and nutritional stress for microbial community in VF wasrelieved 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 biofilms and thus resulted in the overall optimizationof the vermifiltration system for liquid-state sludge stabilization.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
More and more municipal wastewater treatment plants(MWWTP) have been built in small towns in China due to therequirements 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, andmost 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 vermifiltration (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).
Compared with the conventional biofilter (BF), the treatment per-formance of liquid-state sludge by vermifilter (VF) was improvedsignificantly due to the presence of earthworms (Liu et al., 2012;Yang et al., 2013b).
Vermifiltration refers to an organic decomposition processinvolving the interactions between earthworms and microorgan-isms (Liu et al., 2012; Xing et al., 2012). Although the microorgan-isms are responsible for the bio-chemical degradation of theorganics, compared with the microbial community developed inthe conventional biofilters, microbial communities developedin the VF, which are mainly affected by the earthworm activitiesin the filter bed, are exposed to different conditions (Liu et al.,2012). Earthworms can modify microflora directly and indirectlyby three main mechanisms: (1) comminution, burrowing and cast-ing; (2) grazing; (3) dispersal (Brown, 1995). These activities maychange the substrate’s physico-chemical and biological statusand cause drastic shifts in the density, diversity, compositionsand activities of microbial communities in the VF biofilms (Li
Fig. 1. Schematic diagram of the vermifilter (VF, with earthworms in the filter bed).
C. Zhao et al. / Bioresource Technology 151 (2014) 340–346 341
et al., 2013). Polymerase chain reaction-denaturing gradient gelelectrophoresis (PCR-DGGE) technique has been applied to explorethe microbial community structure of biofilms in VF and found thatthose biofilms were featured on richer diversity in their microbialcommunity than BF biofilms (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 biofilms during vermifiltration 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 reflecting 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 biofilms in the VF. The hypothesis that earthworms in theVF could result in the overall optimization of the vermifiltrationsystem for liquid-state sludge stabilization could be proposedbased on the results: (1) earthworm activities in the VF signifi-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. Vermifilter setup and operation
Two sets (each set has three parallel reactors) of cylindrical fil-ters were set up. One set was the vermifilters (Fig. 1) with an initialearthworm density of 32 g/L (fresh weight basis) as suggested byZhao et al. (2010), while the conventional biofilters (BF) withoutearthworms were used as the control. Each filter (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 (10–20 mm indiameter). A layer of plastic fiber was placed on the top of the filterbed to avoid the direct hydraulic influence on the earthworms andensure an even influent sludge distribution. The earthworms,Eisenia fetida, used in this study were purchased from a farm inYancheng City, China. The influent sludge was obtained from thesecondary sedimentation tank of a municipal WWTP in Shanghai,
China. The hydraulic load of the two sets of filters was kept at4 m/d, and the organic load of the influent sludge was maintainedwithin the range of 1.10–1.28 kg-VSS/(m3 d). After passing throughthe filter bed continuously, the sludge entered into a sedimenta-tion tank. These filters ran steadily for 8 months to investigatetheir treatment performances on liquid-state sludge stabilizationafter about 30-day acclimation.
2.2. Sampling and chemical analysis
Biofilm samples were collected from the filter 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 profiles 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 asV1–V4. The biofilms on the ceramsites were rinsed into centrifugetubes with sterile water, and then centrifuged (9000 rpm) for15 min at 4 �C. The dewatered biofilm 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 firstly filtered through the 0.45 lm mixed cellulose estermembrane.
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 biofilms during vermifiltration of liquid-state sludgewas evaluated according to the method proposed by Caravelli et al.(2004). One unit of dehydrogenase activity was defined 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 biofilmsamples 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; AgilentTechnologies Inc., UK). The neutral lipids, glycolipids andphospholipids were eluted with chloroform, acetone and metha-nol, respectively (Frostegard and Baath, 1996). The Phospholipid
Fig. 2. Performance data of the BF and the VF reactors: (a) the VSS reduction ofsludge; (b) the ratios of VSS/SS in the influent and effluent sludge.
342 C. Zhao et al. / Bioresource Technology 151 (2014) 340–346
fatty acid (PLFA) fraction was transesterified into fatty acid methylesters (FAMEs) through mild alkaline methanolysis reaction (Amiret al., 2010). Then FAMEs were analyzed on a Trace DSQ gas chro-matography–mass spectrometer (Thermo, USA). The detailed GC–MS experimental conditions were obtained according to the meth-od proposed by Dungait et al. (2008). The PLFAs were quantifiedthrough 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, Sigma–Aldrich, 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 configurations are indicated by c andt, respectively. The anteiso and iso branching are designated bythe prefix a or i. Cyclopropyl fatty acids is expressed as cy.
The sum of all the identified PLFAs was used to estimate the to-tal viable microbial biomass (Zelles, 1999). Certain PLFAs wereused as biomarkers to determine the presence and abundance ofspecific 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-fied PLFAs (Zornoza et al., 2009). The Shannon index for the iden-tified PLFAs (HPLFA) was calculated as follows:
HPLFA ¼ �XR
i¼1
pi ln pi ð1Þ
where, pi is the relative abundance of each PLFA in the total sum; Ris the number of the identified 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 significance of the assays. Ifp < 0.05, the results were considered to be statistically significant.A principal component analysis of PLFA data was used to assessthe influences of earthworm presence and the depth of filter beddepth on the microbial community structures of BF and VF bio-films. 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 significantly enhancedsludge 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. 2b
shows that the average VSS/SS ratio of the VF effluent sludge(VES) was decreased to 0.63 from 0.73 (the influent sludge) afterVF treatment. The decrease of VSS/SS ratio of the BF effluent sludge(BES) was also observed, whereas the observed decrease was not assignificant 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 effluent 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
The total microbial biomass of the biofilms could be indicatedby total PLFAs. As the above has shown that the depth of the filteris 100 cm and different microbial biomass quantities in thebiofilms might correspond to certain depth. Thus, their microbialbiomass as a function of the depth distribution of biofilms in thetwo filters was investigated and shown in Fig. 3.
As shown in Fig. 3, the total PLFA concentrations of these twofilters were decreased with the deepening of the depth. Withinthe investigated depth of the VF and BF biofilms, total PLFA con-centrations in VF and BF biofilms were correspondingly decreasedfrom 1750 to 1401 lg g�1-VSS and 2271 to 1609 lg g�1-VSS,respectively. In the filtration systems, when the influent sludgepassed through the filter 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 VFbiofilms were significantly lower than those in BF biofilms, indicat-ing that earthworm activities led to the decrease in microbial bio-mass of biofilms 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 of
Table 1Concentrations of TCOD and SCOD in the influent, BF and VF effluent sludge.
Samples Average SCOD (mg/L) Average TCOD (mg/L) SCOD/TCOD
Influent 65.4 ± 7.4 411 ± 11.1 0.16 ± 0.08BF effluent 36.3 ± 4.1 279 ± 9.4 0.13 ± 0.05VF effluent 68.6 ± 10.0 210 ± 8.9 0.32 ± 0.09
Fig. 3. Variation of microbial biomass in the BF and VF biofilms versus the depthdistribution. Different letters indicate statistical differences from each other(p < 0.05).
Fig. 4. Variations of dehydrogenase activity in the BF and VF biofilms versus depthdistribution. Different letters indicate statistical differences from each other(p < 0.05).
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that in the control without earthworms during the vermicompo-sting 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 biofilms was ob-served, the results of dehydrogenase activity variation (Fig. 4)showed that the presence of earthworms enhanced the totalmicrobial activity in the VF biofilms compared with the BFbiofilms.
At different investigated filter depths, the dehydrogenase activ-ity in VF biofilms was always higher than that in the BF biofilms(the maximum increase level was 0.58 mg�h�1g�1-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 filter 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 vermifiltration of liquid-state sludge.
3.4. Microbial community structure
3.4.1. PLFA profilesPLFA profiles can provide the insights into the microbial com-
munity structure because that a relative abundance of characteris-tic PLFAs is considerably different among specific groups ofmicroorganisms (Li et al., 2010). PLFA profiles of the BF and VF bio-films at different depths were investigated in this study and shownin Fig. 5. It was found that there were 16 identified PLFAs in VF
biofilms at every investigated depth (V1–V4), and the correspond-ing numbers in B1–B4 were 14, 14, 13 and 12, respectively. The10Me18:0 (PLFA of actinomycetes) and cy17:0 (PLFA of bacteria)were only detected in the VF biofilms. 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 biofilms were lower thanthose in BF. These results were consistent with the above observa-tion that the microbial biomass of biofilm in VF was lower thanthat in BF biofilms.
The data of PLFA profiles (the 16 identified PLFAs) were furtheranalyzed with the principal component analysis (PCA) to assessoverall differences in the microbial community structures of BFand VF biofilms of the two filters at different depths as well asthe influence of the earthworm presence and the different depthsof filter bed on microbial community structures. The first 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), biofilmsamples could be clearly distinguished from each other based onsampling depth (12, 37, 62 and 87 cm) and the presence of earth-worms. Biofilm 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 reflected the effect of earthwormactivity on the variation of PLFA profiles. Both BF and VF biofilmssampled along the depth showed a positive relationship withPC2. Therefore, the PC2 mainly reflected the variation betweenPLFA profiles and depths of the filter beds. Additionally, the bio-films sampled along the depth showed a negative relationship withPC2 due to the type and concentration of PLFAs were decreasedwith the deepening of the filter bed. The factor loadings of the 16identified PLFAs (Fig. 6b) were divided into three domains (I–III).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) biofilm samples from VF reactor were mainly clusteredon the positive side of PC1, indicating a correlative relationshipwith Domain I of PLFA profiles. The results suggested that earth-worm activities could benefit the microorganisms containing thosetypes of PLFAs (Domain I) and thus modified the structure of themicrobial communities in VF reactor.
Fig. 5. PLFA profiles of BF and VF biofilms at different depths of 12 cm (B1, V1), 37 cm (B2, V2), 62 cm (B3, V3) and 87 cm (B4, V4).
Fig. 6. Principal component analysis of 16 identified PLFAs at different sampling depths of 12 cm (B1, V1), 37 cm (B2, V2), 62 cm (B3, V3) and 87 cm (B4, V4) from the BF andVF biofilms. (a) the score plot of PC1 versus PC2; (b) the factor loadings of the 16 identified PLFAs.
344 C. Zhao et al. / Bioresource Technology 151 (2014) 340–346
3.4.2. Microbial community diversityThe Shannon index for the identified PLFAs (HPLFA) was used to
calculate the degree of microbial diversity in the biofilms of thetwo filters and the high value of HPLFA corresponded to the highdiversity and complex system. As shown in Table 2, the totalHPLFA in VF biofilms ranging from 3.69 to 3.79 (with the averageof 3.77), was higher than those in BF biofilms (with the averageof 3.49). The higher Shannon index indicated that the populationof microbial community structure in VF reactor had been diversi-fied 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
and BF biofilms was not significant (p > 0.05). These results re-vealed that the enrichment of fungi was the main reason forthe intensification of microbial community diversity in the VFreactor. The effect of earthworms on fungi might be caused byeither physicochemical modification 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.
Table 2The Shannon diversity index for total PLFAs and the specific microbial groups (bacteria, fungi and actinomycetes) of both BF and VF biofilms at different depths.
Depth (cm) Total HPLFA Bacteria HPLFA Fungi HPLFA Actinomycetes HPLFA
BF VF BF VF BF VF BF VF
12 3.50 3.69 2.13 2.07 1.03 1.21 0.34 0.4137 3.54 3.75 2.11 2.14 1.08 1.26 0.35 0.3562 3.48 3.84 2.03 2.15 1.07 1.27 0.38 0.4287 3.44 3.79 2.00 2.21 1.06 1.20 0.38 0.38Average 3.49 3.77 2.07 2.14 1.06 1.23 0.36 0.39
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3.4.3. Metabolic property of the microbial community structureThe PLFA profiles 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 influent sludge passedthrough the filters. Moreover, the higher mono:sat PLFA ratioswas found in the VF biofilms than that in the BF biofilms, 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 influent and BF and VF effluentswere 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 biofilms 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 identified as the PLFAs of aerobic bacteria, andthe branched PLFAs such as a15:0, i15:0, i16:0 and i17:0 wereidentified 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 insufficient 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).
indicating that the aerobic conditions had been improved by earth-worms. Yang et al. (2013a) also reported that sufficient 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 filter bed to significantly promote the aeration ofthe filter bed. It is well known that the degradation of organichad a higher efficiency under aeration condition, thus the presenceand distribution of earthworms improved aeration condition in theVF and thereby enhanced organic degradation during liquid-statesludge stabilization (Liu et al., 2012).
4. Conclusion
Earthworms significantly enhanced the microbial activity andmicrobial community diversity in the VF biofilms. 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 filter bed by the burrowing action ofearthworms. Those results suggested that the improved microbialcommunity diversity and metabolic properties of biofilms causedby earthworms brought about the overall optimization of the ver-mifiltration 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).
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