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International Biodeterioration & Biodegradation 103 (2015) 38e50

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Characterisation of dissolved organic matter extracted from thebio-oxidative phase of co-composting of biogas residues and livestockmanure using spectroscopic techniques

Caihong Song a, b, Mingxiao Li a, *, Beidou Xi a, *, Zimin Wei b, Yue Zhao b, Xuan Jia a,Hui Qi c, Chaowei Zhu a

a Innovation Base of Groundwater and Environmental Systems Engineering, Chinese Research Academy of Environmental Science, Beijing 100012, Chinab Life Science College, Northeast Agricultural University, Harbin 150030, Chinac College of Agronomy, Liaocheng University, Liaocheng, Shandong 252059, China

a r t i c l e i n f o

Article history:Received 20 November 2014Received in revised form9 March 2015Accepted 21 March 2015Available online 29 April 2015

Keywords:Biogas residuesComposting efficiencyRaw materialsSpectroscopic techniquesParallel factor analysisPCR-DGGE

* Corresponding author. No. 8, Dayangfang, BeiyuBeijing 100012, China.

E-mail addresses: limingxiao8122@163.com (M. Li)(B. Xi).

http://dx.doi.org/10.1016/j.ibiod.2015.03.0320964-8305/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

The composition of composting substrate significantly influences the composting process. To evaluatethe effect of biogas residue content of initial composting mixture on the composting efficiency, co-composting processes of biogas residues and livestock manure (BRLM) were performed in terms ofweight fractions of biogas residues (T1: 30%, T2: 40%, T3: 50% and T4: 60%). The dissolved organic matter(DOM) transformation was characterised. Fractionation of DOM, FTIR, UVevis and fluorescence spectraindicated that the degradation efficiency of alcohols, ether and polysaccharides, and molecular weight,aromaticity and polycondensation degree of composts were in the order T3 > T2 > T1 > T4. Parallel factoranalysis also showed that the content of humic-like substances was in the same order. Hierarchicalcluster analysis showed that humified and stabilised degree of compost was optimal when the weightfraction of biogas residues was 40e50%. Bacterial profiles implied that biogas residue content of com-posting substrate significantly influenced bacterial dynamics. Bacteria were mainly active in the degra-dation of easily biodegradable organic matter and lignin. The abundance of bacteria involved in thedegradation of easily biodegradable organic matter and lignin in the course of composting was closelyrelated to composting efficiency and humification degree of compost.

© 2015 Elsevier Ltd. All rights reserved.

Introduction

At present, with the rapid development of biogas engineering inChina, there is an urgent need to dispose of a large amount of biogasresidues from anaerobic digestion. In addition, the amount of ani-mal manure is increasing because of the swift development oflivestock farming. For example, the annual yield of animal manurein China is over 3 billion tons (Duan et al., 2012). Therefore, biogasresidues and livestock manure have become two of the mostimportant sources of agricultural pollution. Traditionally, biogasresidues are often utilised directly as organic fertiliser (Yuan et al.,2011), which could result in the addition of hormones, chemical

an Road, Chaoyang District,

, m15910793842_1@163.com

pesticides and potential ammonia oxidation-inhibiting substances,which are not conducive to plant growth. Livestock manure beingused as the co-substrate for biogas residues composting can notonly balance the C/N ratio of initial composting materials but alsoprovide microbial biomass and a large amount of easily degradableorganic matter (Creamer et al., 2010). More importantly, thenegative influence of traditional biogas residue land utilisation onthe soil could be eliminated ormitigated via composting (Singh andKalamdhad, 2012, 2013; Ho et al., 2013).

Composting is a process involving continuous mineralisationand humification of organic matter (Gea et al., 2007). A typicalcomposting process includes two general stages: the bio-oxidativephase and the followingmaturation phase. The former also consistsof a mesophilic stage, a thermophilic stage and a falling-temperature stage (Yu et al., 2007). The rapid degradation oforganic matter and reduction of volume and weight of compostpiles are observed during the bio-oxidative phase of composting(BPC). This fact is convenient for the management of composting

Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:limingxiao8122@163.commailto:m15910793842_1@163.comhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.ibiod.2015.03.032&domain=pdfwww.sciencedirect.com/science/journal/09648305http://www.elsevier.com/locate/ibiodhttp://dx.doi.org/10.1016/j.ibiod.2015.03.032http://dx.doi.org/10.1016/j.ibiod.2015.03.032http://dx.doi.org/10.1016/j.ibiod.2015.03.032

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 39

plants when considering the cost and the environmental impacts ofcomposting. Therefore, research on organic matter dynamics dur-ing the BPC is important for shortening composting time andreducing the covering area of composting pile, thereby reducing thecomposting cost and environmental pollution.

As a highly active component, the characteristics of dissolvedorganic matter (DOM) and its transformations could reflect thecomposting process and the humification degree of organic matter.Extensive research has been conducted to explore the chemicalstructure and molecular weight changes of DOM during compost-ing (Said-Pullicino et al., 2007;Wang et al., 2013). The application ofspectroscopic methods, especially excitation-emission matrix(EEM), has become increasingly common (Tang et al., 2011; Wanet al., 2012). EEM spectra coupled with fluorescence regionalintegration (FRI) is often used to quantitatively analyse DOM (Tianet al., 2012; Lv et al., 2013). However, FRI cannot essentially solvethe problem of overlap among the fluorescence peaks. Parallelfactor analysis (PARAFAC) can decompose the three-way data intoindividual fluorescence components and quantitatively analyseDOMnotably well because of its scientific nature (Yu et al., 2010; Heet al., 2013a). However, the available information on DOM duringthe bio-oxidative phase of biogas residues and livestockmanure co-composting is still limited, and biogas residue composting effi-ciency is also rarely reported.

The objectives of this study were to explore the dynamics ofDOM during the bio-oxidative phase of biogas residues and live-stock manure co-composting by spectroscopic techniques coupledwith PARAFAC and to evaluate the effect of biogas residue contentof initial composting mixture on the composting efficiency.

Materials and methods

Preparation of composting materials

Pig manure (PM) and chicken manure (CM) were collected froma pig farm and a chicken farm, respectively, in Hebei Province,China. After scraping off the hog-hair and feathers, the sampleswere collected, transported immediately to the laboratory andstored in a refrigerator at 4 �C until being used (less than 15 days).Biogas residues were collected from a farm in Beijing. It was pre-treated by screening out the stones. Some characteristics ofbiogas residues and livestock manure (BRLM) are shown in Table 1.

Composting set-up and sampling

Four composting experiments with different biogas residuecontent (shown in Supplementary material (Table S1)) were con-ducted using aerobic composting reactors with the volume of 34 l.The basic characteristics of BRLM were as follows: C/N ratio, 21 to26; water content, approximately 60% and pH value, 7.7 to 7.8. Theoxygen was supplemented by ventilation (0.5 l kg�1 h�1).

Compost samples were collected at different points from the topto the bottom of the piles after 0, 6, 14 and 30 days to follow the

Table 1Characteristics of the composting substrates.

Parameters Pig manure Chicken manure Biogas residues

pH (1:10) 8.05 ± 0.19 7.82 ± 0.15 7.58 ± 0.11Moisture content (%) 71.23 ± 7.36 73.42 ± 7.54 82.45 ± 8.13Organic matter (%, d.m.) 80.90 ± 3.11 45.30 ± 1.57 35.70 ± 1.34TN (g/kg, d.m.a) 33.69 ± 4.23 27.88 ± 3.27 13.12 ± 2.31TP (g/kg, d.m.) 35.56 ± 3.21 18.57 ± 1.55 15.03 ± 1.63TK (g/kg, d.m.) 16.16 ± 0.87 11.39 ± 0.72 4.18 ± 0.46C/N ratio 25.30 ± 1.29 9.10 ± 0.88 31.40 ± 1.43

a d.m. e dry matter.

evolution of the DOM fraction. The sample was dried at 65 �C,ground and sieved to

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e5040

of 1200 nm min�1 over a range of 250e600 nm with a constantoffset (Dl ¼ 30 nm). Protein-like materials (PLF) and humic-likesubstances (HLF) were calculated according to Hur et al. (2009).The EEM spectra were obtained at a scan speed of 2400 nm min�1

over 200e450 nm and 280e520 nm ranges of excitation andemission wave-lengths, and the excitation wavelength incrementwas set at 5 nm. The scattering was regulated before PARAFAC.

PARAFAC modellingPARAFAC statistically decomposes three-way data into individ-

ual fluorescence components. A detailed description of PARAFACcan be found in the protocol of Bro (1997).

Analysis of bacterial community

Bacterial succession rule during the four different compostingtreatments was determined by the PCReDGGE technique. Theextraction and purification of DNA, preparation of PCR reactionmixture and operation of PCR program, and DGGE analyses werecarried out according to previous work (Song et al., 2014b). DNAwas extracted directly from compost samples using the E.Z.N.A.™Soil DNA kit (Omega Bio-tek, Guangzhou, China) in accordancewiththe manufacturer's instructions. PCR amplification was conductedusing the primers R534 (50-ATT ACC GCG GCT GCT GG-30) and GC-F341 (50- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGGGGG CCT ACG GGA GGC AGC AG-30) (Sangon Biotech., China). DGGEanalyses were carried out using 20 mL of PCR product loaded intopolyacrylamide (8%) gels with gradients of 35e60 % of denaturants(urea/formamide). A Gene Mutation Detection System (Bio-Rad,USA) was run at 80 V for 16 h at 60 �C to separate the fragments.Dominant bands were excised and eluted in sterile water overnight.The re-amplification of bands for sequencing was conducted ac-cording to Song et al. (2014b). The PCR product was purified andcloned according to Maeda et al. (2010).

Data processing

The physico-chemical analysis, GI data and spectral character-istic parameters were analysed by analysis of variance (ANOVA)using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A value ofp < 0.05 was considered statistically significant.

The EEM spectra of 16 samples were analysed by PARAFAC. Theanalysis was performed in Matlab 7.8.0 (R2009a) (MathWorks Inc.,Massachusetts, USA). The fluorescence intensity of each componentwas represented by Fmax (R.U., i.e., Raman units), which gives es-timates of the relative concentrations of each component.

The DGGE band profiles were digitised and band numbers werecounted using Quantity one v4.62 (Bio-Rad, USA) software. TheShannon Weaver diversity index was calculated from the relativeintensity data of the DNA bands in each lane according to Shannonand Weaver (1963).

Pearson correlation analysis was conducted using SPSS softwareto analyse the correlation among all parameters except for tem-perature. Based on the aforementioned analysis, principal compo-nent analysis (PCA) was also performed. Hierarchical clusteranalysis (HCA) on all spectral parameters was performed using theFurthest neighbour cluster method, which uses the squaredEuclidean distance as a similarity measure.

Results and discussion

Temperature profile

The temperature changes in the four different compostingtreatments are shown in Supplementary material (Table S2). The

pile temperatures increased from 25 �C to maximumvalues of 63.5,61, 60.5 and 60 �C for T3, T2, T1 and T4, respectively, after 103, 107,111 and 124 h. The thermophilic stage (above 50 �C) persisted forapproximately 144, 120, 108 and 108 h in T3, T2, T1 and T4,respectively. Thereafter, the temperature started to decline andthen remained fairly stable at a final value of approximately 35 �C.These findings indicate that the BPC ended at this time. Accordingto the US EPA (USEPA, 1994), T3 and T2 could meet hygienic re-quirements for organic waste, whereas T1 and T4 could not. Theseresults indicate that in comparisonwith T1 and T4, the degradationof organic matter in T3 and T2 was more robust. More heat pro-duced by active microbial metabolism could maintain a highertemperature and longer thermophilic stage in T3 and T2. It has beenrecognised that lignocellulose is one of the aromatic compoundsresistant to biodegradation (Blanchette, 1995), and biogas residuesare rich in lignocelluloses (Liu et al., 2010). PM and CM are mainlycomposed of easily degradable organic matter, such as aliphatics,saccharides and proteins (Song et al., 2014a). Although comparedwith T3 and T2, the percentage of easily degradable organic matterwas higher in T1, the microbial metabolismwas not as robust. Poorventilation due to less bulking agent (biogas residues) might ac-count for this phenomenon, whereas T4 also exhibited poor tem-perature, which might be attributable to more refractory organiccompounds in the composting materials of T4.

pH values, ammoniumeN, and germination index

The pH exhibited a complex and similar change in the fourdifferent composting treatments (Table 2). An increase rise was firstobserved on day 6, which may be as a consequence of the accu-mulation of ammonium. The degradation of organic matter con-taining nitrogen leads to the accumulation of ammonium. Inaddition, low-molecular-weight organic acids produced from thedegradation of easily degradable organic matter, such as solublecarbohydrates, lipids and proteins, were consumed by microbialmetabolism. Thereafter, the pH value decreased due to the volati-lisation of ammonia and consumption of ammonium, which wasconfirmed by the change of ammoniumeN content at the samestage (Table 2). On day 30, pH increased, which might be attributedto the exhaustion of organic acids. During the entire BPC, the pHremained above 7.5, which demonstrated that the BRLM substrateswere alkaline in nature.

AmmoniumeN content also displayed a similar change ten-dency in the four different composting treatments regardless of thesubstrate compositions (Table 2). The samples collected from day 6displayed the highest concentrations of ammoniumeN:5.00 mg g�1 for T1, 3.31 mg g�1 for T2, 3.17 mg g�1 for T3, and3.81 mg g�1 for T4. At this moment, the composting was in thethermophilic stage, and the rapid degradation of organic nitrogendue to active microbial activity might account for the peakammoniumeN values. However, L�opez-Gonz�alez et al. (2013)demonstrated a decline in ammoniumeN in the initial phase ofcomposting of lignocellulose-rich substrates, which was differentfrom the composting of nitrogen-rich substrates, such as livestockmanure in this study. Thereafter, ammoniumeN continuallydecreased until the BPC ended. Cofie et al. (2009) reported ananalogous finding in the co-composting of faecal sludge andorganic solid waste. Bernal et al. (2009) and Xue et al. (2010) re-ported nitrification started when the temperature fell below 40 �C.The robust nitrification as the temperature decreased may beresponsible for the decrease of ammoniumeN.

GI is a sensitive indicator reflecting the phytotoxicity ofcompost. As shown in Table 2, there was a marked decrease inphytotoxicity during the entire BPC, which indicated that phyto-toxic substances were being degraded and transformed. On day 0,

Table 2Changes in pH, ammoniumeN and GI during composting.

NH4þ � N pH GI

T1 0d 3.71 (0.12)b* 7.80 (0.16) cd 4.54 (0.12)m

6d 5.00 (0.15)a 8.61 (0.24)a 22.01 (0.58)k

14d 2.33 (0.06)g 8.19 (0.27) bc 35.37 (0.89)j

30d 0.34 (0.02)jk 8.40 (0.32) ab 96.72 (2.33)b

T2 0d 3.12 (0.13)d 7.79 (0.23) cd 50.33 (1.00)h

6d 3.31 (0.15)c 8.65 (0.27)a 55.57 (1.02)g

14d 1.09 (0.03)i 8.44 (0.31) ab 96.16 (2.06)b

30d 0.25 (0.01)k 8.49 (0.35) ab 103.35 (2.21)a

T3 0d 2.67 (0.08)f 7.77 (0.31)d 67.13 (1.13)f

6d 3.17 (0.15)cd 8.35 (0.28) ab 67.37 (1.20)ef

14d 2.83 (0.09)e 7.87 (0.28) cd 72.52 (1.19)d

30d 0.42 (0.02)j 7.90 (0.32) cd 98.42 (2.34)b

T4 0d 1.53 (0.04)h 7.71 (0.32)d 7.28 (0.22)l

6d 3.81 (0.10)b 8.51 (0.26) ab 38.91 (0.93)i

14d 1.54 (0.04)h 7.76 (0.21)d 69.70 (1.24)e

30d 0.35 (0.01)jk 7.91 (0.29) cd 85.13 (2.06)c

* Values followed by different letters (aem) are statistically significantly different (p < 0.05). Values in parenthesis are standard deviations.

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 41

the GI in T1, T2, T3 and T4 was 4.54%, 50.33%, 67.13% and 7.28%,respectively. Several factors affect the phytotoxicity of compost,such as a lack of oxygen during composting, the accumulation oftoxic compounds and the excessive presence of ammonia, heavymetals and mineral salts (El Fels et al., 2014). At the end of the BPC,the GI in T1, T2, T3 and T4 was 96.72%, 103.35%, 98.42% and 85.13%,respectively. When the GI is more than 80%, the compost isconsidered mature (Wei et al., 2000). Therefore, all four composttreatments were considered phytotoxin-free andmature on day 30.

The distribution of MW fractions of DOM

Fig. 1 illustrates the MW distribution of DOM in different com-posting treatments. The MW distribution of DOM was similaramong all the 0-d-old samples. The proportion of fractionMW < 65 Da decreased with the increase of biogas residue contentof initial composting mixture, whereas those of fractions of65 Da 5 kDa) substances showed a marked increase and became themajor component of DOM, accounting for 70.46%, 74.24%, 84.52%and 63.88% in T1, T2, T3 and T4, respectively. Wei et al. (2014)conducted an analogous study and found that fractionMW > 5 kDa was the major component of DOM in all mature

Fig. 1. Distribution of MW fractions in DOM derived from different compostingtreatments.

composts. In addition, they reported that the lower MW andsimpler structural components in DOM were the biodegradableorganic compounds, and the higher MW components were lessdegradable bymicroorganism.MW¼ 5 kDamaybe a line of lowandhigh MW in DOM and the MW > 5 kDa fraction was likely humicsubstances. These results indicated that simultaneous degradationof simpler structural components, such as free amino acids, sugarsand simple protein-liked materials, and formation of humic acid-like materials in DOM in the course of BPC. As shown in Fig. 1, theproportion of fraction MW > 5 kDa was in the orderT4 T1 > T2 > T3. The MWdistribution of DOM was related to compost stability and humifi-cation degree. In this study, the MW distribution of DOM indicatedthat compost stability and humification degree were in the orderT4 < T1 < T2 < T3.

FTIR spectra

The FTIR spectra of the DOM samples at the start and end of theBPC are illustrated in Fig. 2a. The spectra were characterised by (a)an intense broad band around 3437 cm�1 and at 3263 cm�1, pri-marily due to the OeH stretching vibrations of the hydroxyl groupsof alcohols, phenols, and organic acids; (b) a weak band around2964 cm�1, attributed to aliphatic CeH stretching; (c) a strongabsorbance band at about 1642 cm�1, assigned to the C]Cstretching vibrations of aromatic rings; (d) a weak band atapproximately 1571 cm�1, ascribed to NeH deformation and C]Nstretching of amides (e) a weak band at approximately 1408 cm�1,assigned to the CeO asymmetric stretching of carboxyl groups; (f) asharp band around 1385 cm�1, attributed to OeH deformation ofphenolic structures and/or antisymmetric stretching of COOegroups; (g) an intense band at about 1111 cm�1, mainly caused bythe CeO stretching of secondary alcohols and/or ethers and (h) aweak band around 1003 cm�1, mainly due to CeO stretching ofpolysaccharides (Xi et al., 2012a; He et al., 2013b).

As shown in Fig. 2a, the FTIR spectra of DOM at different com-posting treatments presented similar peak locations and variedonly in the relative intensity. Peaks at 2964 and 1571 cm�1 for theraw composting materials almost disappeared after the BPC, indi-cating a relatively rapid biodegradation of aliphatics and amidesduring composting. In addition, for the raw composting materials,peaks at 1408 cm�1 (carboxyl C) also almost disappeared after theBPC. He et al. (2013b) showed that carboxyl C originated from thedegradation of organic carbon during aerobic composting. The

Fig. 2. The FTIR spectra (a) and the parameters from the FTIR spectra (b) of DOM in the four different composting treatments. T1: 30%; T2: 40%; T3: 50%; T4: 60%.

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 43

carboxyl C content fluctuated in the course of composting, whichwas inconsistent with the result in the present study. Compared tothe samples on day 0, the peaks at about 1385 cm�1 for the sampleson day 30 had higher relative intensity (transmittance), indicatingthe degradation of OeH and/or COOegroups.

In order to compare the different composting treatments andfurther analyse the changes those took place in the course ofcomposting, the ratios between the peak intensities at about 2964,1642, 1111 and 1003 cm�1were calculated according to He et al.(2013b). As shown in Fig. 2b, except for T4, the ratios 1642/1111(aromatic C/alcohol C) and 1642/1003 (aromatic C/polysaccharideC) both increased after the BPC. The increased levels were in theorder T1 < T2 < T3. These results implied that the biodegradation ofalcohols, ether and polysaccharides occurred in the course ofcomposting. T3 exhibited the highest degradation efficiency. Exceptfor T4, on day 30, the 1642/2964 peak ratio (aromatic C/aliphatic C)also increased. The increased levels were in the order T2 < T1 < T3.These findings implied that aliphatic compounds were degraded bythe composting treatment. T3 showed the best performance.Analogous results had been previously observed by several in-vestigators. He et al. (2013b) showed that the ratios 1645/1103(aromatic C/alcohol C) and 1645/2923 (aromatic C/aliphatic C) bothsteadily increased during the composting of cattle manure. Xi et al.(2012a) reported that the 1662/1000 peak ratio (aromatic C/poly-saccharide C) increased during composting of municipal solidwaste. These can be attributed to the preferential degradation ofaliphatics, alcohols, ethers, and polysaccharides used for the energyrequirements of the microorganisms and an increase in aromaticityduring composting.

UVevis spectroscopy

UVevis spectra are extensively used to characterise the molec-ular structure of organic matter (Xi et al., 2012a). As shown in Fig. 3,

Fig. 3. UVevis spectra of DOM in the four different composting t

the UVevis spectra of DOM extracted from T1, T2, T3 and T4 werecharacterised by a decrease in absorbance with wavelength and anincrease in absorbance with composting time. The degree ofaromaticity and molecular weight of DOMwere strongly correlatedwith their UV (250e280 nm) molar absorptivities, and they couldbe reliably determined using molar absorptivities measured atapproximately 250e280 nm (Chin et al., 1997). In this study, the UV(250e280 nm) molar absorptivities of DOM at the end of the BPCwere in the order of T3 > T2 > T1 > T4, which indicated that thedegree of aromaticity andmolecular weight of DOM extracted fromthe four composting treatments on day 30 were also in the sameorder.

To obtain more information on the chemical composition andtransformation of DOM, four parameters (SUVA254, SUVA280, E250/E365, E253/E203) were employed to investigate these characteristics.As shown in Table 3, the SUVA254 of the raw samples for T3, T2, T1and T4 was 0.227, 0.240, 0.235 and 0.138, respectively, whereas thatof the samples on day 30 for T3, T2, T1 and T4 was 0.440, 0.418,0.380 and 0.245, respectively. Nishijima and Jr. Speitle (2004) re-ported that an increase of the SUVA254 implied a higher degree ofaromaticity and higher molecular weight. Therefore, the afore-mentioned results indicated that the aromatic polycondensationand molecular weight of DOM increased after the BPC, which wasconsistent with the results reported by He et al. (2011). When theBPC ended, the SUVA254 values increased by 61.70% for T1, 74.17%for T2, 93.83% for T3 and 77.54% for T4. These results suggest thatthe substrate compositions of T3 and T2weremore beneficial to thehumification of DOM.

Westerhoff and Anning (2000) demonstrated that the SUVA280could be used as an index for the amount of aromatic compounds.As shown in Table 3, the SUVA280 value increased after compostingand was in the order T3 > T2 > T1 > T4, indicating that the amountof water-extractable aromatic compounds after composting wasalso in the order T3 > T2 > T1 > T4.

reatments. (a) T1: 30%; (b) T2: 40%; (c) T3: 50%; (d) T4: 60%.

Table

3Chan

gesof

characteristic

param

etersof

DOM

duringthefourdifferentco

mpostingtrea

tmen

ts.

SUVA254

SUVA280

E 250/E

365

E 253/E

203

PLH

HLF

F max

ofco

mpon

ent

12

34

T10d

0.23

5(0.026

)fgh

*0.19

3(0.018

)g5.38

2(0.412

)b0.22

0(0.028

)de

0.63

9(0.054

)cd

0.30

3(0.020

)d1.92

17(0.183

)cd

0.30

17(0.038

)h0.17

85(0.011

)j1.78

06(0.100

)a

6d0.30

6(0.031

)cdef

0.25

6(0.016

)de

3.84

3(0.384

)defg

0.26

6(0.026

)abcd

0.52

2(0.058

)ef

0.39

2(0.023

)b1.62

93(0.179

)def

0.36

15(0.043

)gh

0.23

44(0.023

)i1.23

52(0.091

)c

14d

0.33

0(0.034

)bcd

e0.27

2(0.019

)d3.65

8(0.391

)efg

0.28

9(0.032

)abc

0.44

6(0.050

)f0.45

0(0.025

)a1.03

13(0.130

)hi

0.41

45(0.039

)fg

0.27

3(0.019

)h0.78

75(0.051

)f

30d

0.38

0(0.027

)abc

0.31

6(0.020

)bc

3.64

3(0.388

)efg

0.30

8(0.029

)a0.43

6(0.062

)f0.45

7(0.024

)a0.97

19(0.111

)i0.60

4(0.055

)c0.33

02(0.024

)fg

0.62

57(0.048

)g

T20d

0.24

0(0.021

)efgh

0.20

1(0.017

)gf

4.94

3(0.435

)bc

0.23

6(0.033

)cde

0.79

0(0.069

)ab

0.17

6(0.019

)fg

2.05

68(0.194

)bc

0.41

15(0.036

)fg

0.30

34(0.026

)gh

1.61

86(0.112

)b

6d0.27

7(0.020

)defg

0.23

0(0.018

)ef

4.28

4(0.437

)cde

0.24

6(0.034

)bcd

e0.70

0(0.067

)bc

0.24

8(0.027

)e1.51

22(0.163

)efg

0.52

78(0.042

)de

0.37

9(0.025

)cde

0.52

35(0.046

)hi

14d

0.36

3(0.029

)abcd

0.28

5(0.017

)cd

4.10

7(0.366

)def

0.27

8(0.030

)abc

0.52

1(0.055

)ef

0.38

6(0.026

)bc

1.28

42(0.124

)ghi

0.59

29(0.045

)cd

0.43

26(0.037

)b0.60

7(0.053

)gh

30d

0.41

8(0.030

)ab

0.35

1(0.023

)ab

3.53

9(0.383

)efg

0.31

4(0.025

)a0.46

6(0.061

)ef

0.41

8(0.030

)ab

1.72

82(0.168

)de

0.57

61(0.046

)cd

0.50

68(0.039

)a0.38

01(0.045

)j

T30d

0.22

7(0.018

)fgh

i0.20

9(0.019

)fg

3.18

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C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e5044

The absorbance ratio at 250 and 365 nm (E250/E365) is often usedto characterise the molecular weight, aromaticity and poly-condensation degree of organic molecules (Santos et al., 2009). Asshown in Table 3, the E250/E365 ratio was decreased by the com-posting treatment and in the order T3 < T2 < T1 < T4 on day 30.E250/E365 was inversely proportional to the degree of humificationand molecular weight of organic matter. Therefore, the aforemen-tioned results also indicate that the degree of humification andmolecular weight of DOM were in the order T4 < T1 < T2 < T3,which is in agreement with the results obtained by Li et al. (2010).

Vieyra et al. (2009) reported that a low E253/E203 ratio is asso-ciated with scarce substitution in the aromatic rings or with thesubstitution with aliphatic functional groups, whereas higher E253/E203 ratios indicate the presence of polar functional groups on thearomatic ring. As shown in Table 3, the E253/E203 ratio in the currentstudy increased after composting and was in the orderT4 < T1 < T2 < T3, indicating that the quantity of polar functionalgroups on the aromatic ring was also in the order T4 < T1 < T2 < T3on day 30. During the biodegradation of composting materials,hydroxyl, carbonyl and carboxyl groups on the aromatic rings wereproduced, which resulted in an increase in the E253/E203 ratio (Heet al., 2013b).

Synchronous fluorescence spectra

Generally, in comparison with EEM spectra, the synchronous-scan excitation spectra are representative of the spectral summa-tion of different fluorophores in DOM, and they exhibit betterresolution (He et al., 2011). As shown in Fig. 4, the synchronousspectra of DOM exhibited a major peak at 278e288 nm, which wasassociated with protein-like substances. Two small peaks at334e344 nm and 366e374 nmwere also detected. Guo et al. (2012)demonstrated that peaks at 324e335 nm and 365 nmwere relatedto fulvic-like and fluorescent humic-like substances. In this study,these two fluorescent peaks underwent a significant red shift. Thisphenomenon indicated an increase in condensation degree of DOMmolecular structure. A fourth peak centred at 426e434 nm, whichwas associated with humic-like substances, was also observed.

A main peak (260e300 nm) was identified in all compostingtreatments and associated with the presence of proteinaceousmaterials and monoaromatic compounds (He et al., 2012). Thepercentage of the fluorescence area of the peak was assigned toPLH. Santín et al. (2008) reported that a fluorescent area at300e420 nm is related to humic-like substances and indicates thepresence of polycyclic aromatic compounds. The percentage of thefluorescence area was assigned to HLF. As shown in Table 3, PLHdecreased as the composting process proceeded by 31.77% in T1,41.01% in T2, 42.11% in T3 and 41.52% in T4, whereas HLF increasedby 50.83% in T1, 137.50% in T2, 142.62% in T3 and 169.28% in T4.Comparative analysis of these data indicated a decrease in pro-teinaceous materials and monoaromatic compounds and an in-crease in humic-like materials in the four composting treatmentswith increasing composting time. The composting process wascharacterised by the biodegradation of fresh organic materials andthe biosynthesis of humic-like substances.

Excitation-emission matrix spectra

The three-dimensional EEM fluorescence spectra of the DOMsamples at the start and end of the BPC are illustrated in Fig. 5. TheEEM contours of the DOM from the 0-d-old samples were obviouslydifferent from those of the 30-d-old samples. The former mainlyexhibited two peaks: A and B, whereas in addition to peaks A and B,the latter also contained three peaks: C, D and E. The presence ofthese fluorescence EEM peaks in compost had been previously

Fig. 4. Synchronous-scan excitation spectra of the four different composting treatments. (a) T1: 30%; (b) T2: 40%; (c) T3: 50%; (d) T4: 60%.

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 45

observed by several investigators (Xi et al., 2012b; Lv et al., 2013).Peaks A, B and C represented the tyrosine-like, tryptophan-like andfulvic acid-like materials, respectively. Peaks D and E were repre-sentative of humic acid-like materials.

The fluorescence intensity (FI) of peaks A and B displayed adecrease after composting in T1, T2, T3 and T4, which indicated thatprotein-like substances were degraded into non-fluorescentstructures or converted to other forms during the BPC (Wan et al.,2012). The FI of peaks A and B varied for different raw compost-ing substrates, which might be ascribed to the different composi-tions of composting substrates because organic matter compositioninfluences the fluorescence characteristics of composting sub-strates (Wan et al., 2012). The FI of peaks C, D and Ewas in the orderT3 > T2 > T1 > T4, which could reflect the amount of humic sub-stances in T1, T2, T3 and T4.

Fig. 5. Excitationeemission matrix spectra of the four different c

Fluorescent components

Ohno et al. (2008) reported that each PARAFAC componentcould represent one fluorophore, and the scores of componentsrepresent the relative contents of different fluorophores. Fig. 6presents the EEM contours of the four fluorescent components, asidentified by the DOM FluorePARAFAC model.

Component 1 was composed of two excitation maxima at 224and 276 nm, with one emission peak centred at 338 nm, which wascomparable to component 1 described by Guo et al. (2012).Component 1 represented tryptophan-like substances and wassimilar to the peak B traditionally identified in Fig. 5 and occupied adominant Fmax value. Component 2 had two primary (and sec-ondary) fluorescence peaks at excitation/emission wavelengths of239 (216)/404 nm and 318 (286)/404 nm, respectively. It was

omposting treatments. T1: 30%; T2: 40%; T3: 50%; T4: 60%.

Fig. 6. Excitationeemission matrix spectra of fluorescent components identified by the PARAFAC model.

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e5046

similar to the component 1 described by Yu et al. (2010) andattributable to short-wavelength humic-like materials, which pri-marily originated from fulvic-like substances. Stedmon and Bro(2008) also reported terrestrial-like and marine-like humic fluo-rophores, which were similar to the component 2 in this study.Component 3, fluorescing at a primary (secondary) excitation/emission wavelength pair of 265 (207)/459 nm and an excitation/emission wavelength pair of 360/459 nm, was also observed. Itrepresented long-wavelength humic-like substances (He et al.,2013a). Component 4 contained two excitation maxima at 222and 274 nm, with one emission peak centred at 305 nm, which issimilar to the peak A traditionally identified in Fig. 5. According toHe et al. (2014), component 4 was related to tyrosine-likesubstances.

In the entire BPC, the Fmax values (shown in Table 3) ofcomponent 1 displayed different changes in the four differentcomposting treatments. However, the Fmax values of component 1from all four composting treatments markedly decreased after 30days of composting, which suggested a marked decrease in theoccurrence of tryptophan-like substances. The Fmax values ofcomponent 4 in the four different composting treatments changedin an irregular manner. However, when the BPC ended, the Fmaxvalues of component 4 from all composting treatments exhibited adecrease, which indicated that tyrosine-like substances weredegraded and transformed by microbial metabolism (Xi et al.,2012a). On the contrary, the Fmax values of components 2 and 3in the four composting displayed an upward trend during the entireBPC, which revealed that the humification degree of DOM washigher with the composting time. This is in agreement withaforementioned results of UVevis and fluorescence spectra andcomparable to the finding described by Yu et al. (2010). On day 30,the sum of Fmax values of components 2 and 3 in T1, T2, T3 and T4was 0.9342, 1.0829, 1.072 and 0.8887, respectively, which revealedthat the content of humic-like substances in T2 and T3 was higherthan in T1 and T4, whereas that of components 1 and 4 in T1, T2, T3

and T4 was 1.5976, 2.1083, 2.1248 and 2.0442, respectively.Compared with T1 and T4, the content of protein-likematerials wasalso higher in T2 and T3, which might result from soluble microbialbyproduct-like materials due to vigorous microbial metabolism inT2 and T3.

In comparisonwith the peaks traditionally identified in Fig. 5, inraw composting materials, PARAFAC also exhibited components 2and 3, which represented fulvic-like and long-wavelength humic-like substances, respectively. This finding suggests that fulvic-likeand humic-like substances were easily covered in the peaks tradi-tionally identified. However, PARAFAC could effectively separateoverlapped fluorescence peaks.

Based on all spectral parameters, the composting efficiency ofBRLM was evaluated by HCA (shown in Fig. 8a). In the currentstudy, two main clusters were observed. The samples from days0 and 30 formed the first and second large clusters, respectively.This demonstrates that DOM underwent a significant change after30 days of composting. The first large cluster of the dendrogramalso displayed two groups. The squared Euclidean distance be-tween the sample from T2 on day 30 and the second large clusterwas the highest. These results suggest that the highest degree oftransformation of organic matter occurred in T2, and the com-posting efficiency of T2 was the highest among the four differentcomposting treatments. In the first large cluster, the squaredEuclidean distance between the sample from T3 and that from T2was the lowest, and these two samples formed a group, whichsuggests that T3 also had higher composting efficiency. The sam-ples from T1 and T4 also formed a group, and they were the nearestto the second large cluster and had the lowest compostingefficiency.

Bacterial community composition

The study of the functional diversity of microbial communitiesin compost is instrumental for understanding the composting

Fig. 7. DGGE profile of 16S rRNA gene fragments amplified with the primers R534 andGC-F341. Band patterns of four composting treatments (T1: 30%, T2: 40%, T3: 40% andT4: 50%) are presented. The letters (white font) above each lane indicate the samplingtime. Methe mesophilic stage, Tethe thermophilic stage, Fethe falling-temperaturestage.

Fig. 8. Hierarchical cluster analysis (a) of the spectral parameters and principalcomponent analysis (b) of all parameters from the four different compostingtreatments.

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 47

process. DGGE profiles of amplified 16S rDNA fragments forcompost samples derived from the four different composting pro-cesses are presented in Fig. 7. Closest relatives of bands excisedfrom DGGE profile are shown in Table 4. PCR-DGGE profiles ofbacterial communities implied that bacterial dynamics for differentcomposting treatments visibly differed. The differences suggestedthe effect of biogas residue content of composting substrate onbacterial community composition. As shown in Fig. 7, bacterial di-versity was markedly higher in T3 than in other trials during theentire composting process (data shown in Supplementary material(Table S3)). This finding revealed that the composition of com-posting substrate could be optimal for bacterial growth in T3. 26different bands (Fig. 7) were successfully sequenced in this study.As shown in Table 4, from the functional standpoint, 26 bacterialspecies fell into two primary groups being responsible for thedegradation of easily degradable substances, such as aliphatics,proteins and polysaccharides, and humification and stabilisation ofthe composting substrates. The degradation of lignin in the courseof composting is closely associatedwith the humification process ofcomposting materials (Song et al., 2014b). Some bacterial species,such as Flavobacterium, Pseudoxanthomonas, Sphingobacteriumcomposti (Karadag et al., 2013), Bacillus (Tian et al., 2013), Ure-ibacillus (Ting et al., 2013) and Streptomyces (Lu et al., 2013) wererelated to the degradation of lignin.

In the present study, more abundant bacteria involved in thedegradation of lignin were observed in T3 than in other threetreatments throughout the entire composting process. More spe-cifically, at the mesophilic stage, the quantity of bacterial speciesrelated to lignin degradation was markedly higher in T3 (6) than inT1 (1), T2 (2) and T4 (4). At the thermophilic stage, it was alsohigher in T3 (4) and T2 (4) than in T1 (3) and T4 (2). At the falling-temperature stage, it was the highest in T3 (5), markedly higherthan in T1 (1), T2 (2) and T4 (3). These results indicated that morebacterial species involved in lignin degradation may be responsiblefor markedly higher humification degree of organic matter in T3

than in other composing treatments. It can be deduced that thenutrient composition and physicochemical environment of T3 maybe more suitable for the growth of lignin decompositionmicroorganisms.

Bacterial species associated with the degradation of easilybiodegradable organic matter contained Lactobacillus composti,Bacteroidetes and Acinetobacter (Karadag et al., 2013). In the presentstudy, L. compostiwas detected only at the thermophilic stage of T3.Bands 6 and 10 were closely related to Bacteroidetes. The band 6appeared during the entire composting process of T3, while it wasdetected only at one or two composting stages for other threetreatment groups. The band 10 was observed at the thermophilicstages of T3 and T4 and the falling-temperature stage of T4. Aci-netobacter strains can grow in aerobic or anaerobic conditions andare able to degrade a wide range of hydrocarbons. In this study,three bacterial species (bands 14, 16 and 17) belonged to Acineto-bacterwere observed. Band 14 appeared only at the mesophilic andthermophilic stages of T3. Band 16 was also detected only at thethermophilic stage of T3. Band 17 was observed at the mesophilicstages of T1, T3 and T4, the falling-temperature stage of T3 and thethermophilic stage of T2, respectively. The above-mentioned re-sults indicated that the abundance of bacteria involved in thedegradation of easily biodegradable organic matter was signifi-cantly higher in T3 than in other composting trials throughout the

Table 4Sequence analysis of bands excised from DGGE profile shown in Fig. 7.

Band name Accession No. Closest relatives Similarity (%)

1 AB268118 Lactobacillus composti strain NRIC 0689 992 FJ675663.1 Uncultured bacterium clone LL141-8P6 1003 FJ675661.1 Uncultured bacterium clone LL141-8P3 1004 HG934362.1 Flavobacterium sp. M1-I3 1005 HQ326817.1 Serpens flexibilis strain PM-37 1006 CU922272.1 Uncultured Bacteroidetes bacterium 997 AJ244699 Flavobacterium sp. V12 968 FJ598325.1 Pseudoxanthomonas sp. CTN-8 1009 JQ246806.2 Pseudomonas sp. XC1 10010 HQ727602.1 Uncultured Bacteroidetes bacterium clone BC_COM486 9711 KF032912.1 Psychrobacter sp. Bl39 10012 KF562753.1 Uncultured Ureibacillus sp. clone ABF-23 9513 KM242468.1 Streptomyces sp. INBio_4517H 10014 EU705749.1 Uncultured Acinetobacter sp. 3P-3-2-D21 10015 FR774815.1 Uncultured Desulfitobacterium sp. 9516 HQ860316.1 Acinetobacter seohaensis strain TN1 9717 DQ083508.1 Acinetobacter sp. HPC1331 9918 KF911272.1 Uncultured bacterium clone Comp5-1 10019 NR_044843.2 Caryophanon tenue strain NCDO 2324 9920 KM277365.1 Solibacillus silvestris strain CM3HG10 9921 KM101054.1 Bacillus sp. HGG-18 9922 KJ191871.1 Uncultured Paenibacillaceae bacterium clone MF36 9523 KF630644.1 Uncultured Bacillus sp. isolate DGGE gel band XJC-47 9524 EF122436 Sphingobacterium composti 9525 NR_116957.1 Pseudofulvimonas gallinarii strain Sa15 9726 HE804910.1 Uncultured bacterium clone ATB-AR-23770 98

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e5048

entire composting process. This might account for an advancedstage of organic matter transformation and high composting effi-ciency of T3.

In addition to aforementioned bacterial species, the dominantsequences from the BRLM composting included 12 bacterial spe-cies. Pseudomonas was associated with nitrogen metabolism, suchas N2 fixation and denitrification. Psychrotolerant Psychrobacterwas found in animal manure or soil. Solibacillus silvestris and Pae-nibacillaceae were thermophilic strains and ascribed to Bacillus-related bacteria. Their presence has been reported in the com-posting processes (Tian et al., 2013). Desulfitobacterium wasanaerobic bacteria and might originate from biogas residues.Serpens flexibilis, Caryophanon tenue and Pseudofulvimonas gallinariiwere seldom reported in previous studies, it was necessary toexplore their role during composting in future studies. At last, 4uncultured bacterium were also detected.

It should be noted that bacterial diversity presented a significantrise at the thermophilic stage of T2, which was inconsistent withprevious studies. Moreover, at the thermophilic stage, the Shan-noneWeaver diversity index was also markedly higher in T2 (2.55)than in T1 (1.93) and T4 (2.07) (data shown in Supplementarymaterial (Table S3)). These phenomena might be related to anadvanced stage of organic matter transformation, high compostingefficiency and humification degree of T2 (as indicated in afore-mentioned spectral analysis).

In conclusion, fractionation of DOM, FTIR, UVevis, synchronousand EEM fluorescence spectra as well as PARAFAC showed that theorganic matter transformation during composting was charac-terised by the increase of molecular weight, aromaticity and poly-condensation degree of organic molecules. The composting processwas characterised by the biodegradation of fresh organic materials,such as proteinaceous materials and the biosynthesis of humic-likesubstances. T3 compost exhibited the highest humification degree.It should be highlighted that more abundant bacteria involved inthe degradation of ligninwere also observed in T3. The degradationof lignin was closely related to the formation of humic-like sub-stances (Song et al., 2014b). Therefore, it was concluded that

abundant lignin decomposition bacteria could be responsible formarkedly higher humification degree in T3 than in other treat-ments. In addition, the degradation and transformation of easilybiodegradable organic matter, such as proteinaceous materialswere associated with composting efficiency (Hosseini and Aziz,2013). Aforementioned spectral analysis indicated that proteina-ceous materials, such as tyrosine-like and tryptophan-like sub-stances exhibited markedly higher biodegradation rate in T3 thanin other treatments. In combination with the results of spectralanalysis and bacterial community composition, markedly highercomposting efficiency of T3 maybe as a consequence of moreabundant bacteria involved in the degradation of easily biode-gradable organic matter in T3 than in other treatments.

Multivariate statistical analysis

Correlation analysisA correlation analysis among different parameters, including

chemical, biological and spectral indexes, was conducted. As shownin Table 5, a significant correlation with each other among the Fmaxvalues of four components was observed except between Fmax1 andFmax2 and between Fmax1 and Fmax3. The reason may be thatcomponent 1 represents protein-like materials, whereas compo-nents 2 and 3 are involved with fulvic-like and humic-like sub-stances, respectively.

The correlations between the Fmax values of four componentsand other parameters were also analysed (shown in Table 5). Fmax1exhibits a positive correlation with PLH (r ¼ 0.677, P ¼ 0.004) and anegative correlation with HLF (r ¼ 0.688, P ¼ 0.003). An obviouscorrelation between Fmax2 and the parameters originated fromUVevis spectra is observed. Fmax4 correlates well with E253/E203.

A highly significant correlation with each other six spectralcharacteristic parameters (SUVA254, SUVA280, E250/E365, E253/E203,PLH and HLF) is observed, which agrees well with previous reports(He et al., Li et al., 2010). There is an obvious correlation betweenammoniumeN and spectral characteristic parameters (apart fromE250/E365). Interestingly, the aforementioned five parameters all

Table 5Pearson correlation between spectral parameters and maturity indices from the four different composting (n ¼ 16).

Fmax1 Fmax2 Fmax3 Fmax4 SUVA254 SUVA280 E250/E365 E253/E203 PLH HLF pH NH4þ � N GI

Fmax1 1 �0.297 �0.217 0.506a �0.413 �0.365 0.4 �0.401 0.677b �0.688b �0.482 0.295 �0.342Fmax2 1 0.700b �0.710b 0.572a 0.583a �0.570a 0.594a �0.361 0.348 0.274 �0.308 0.800bFmax3 1 �0.802b 0.35 0.33 �0.304 0.462 �0.288 0.252 0.273 �0.267 0.729bFmax4 1 �0.408 �0.395 0.43 �0.573a 0.477 �0.45 �0.505a 0.359 �0.656bSUVA254 1 0.995b �0.629b 0.798b �0.777b 0.779b 0.321 �0.508a 0.654bSUVA280 1 �0.665b 0.813b �0.754b 0.756b 0.312 �0.503a 0.658bE250/E365 1 �0.810b 0.627b �0.622a �0.326 0.311 �0.678bE253/E203 1 �0.805b 0.791b 0.179 �0.659b 0.813bPLH 1 �0.999b �0.255 0.652b �0.552aHLF 1 0.252 �0.641b 0.528apH 1 0.256 0.236NH4

þ � N 1 �0.477GI 1

a Correlation is significant at the 0.05 level (2-tailed).b Correlation is significant at the 0.01 level (2-tailed).

C. Song et al. / International Biodeterioration & Biodegradation 103 (2015) 38e50 49

comprise the spectral information on protein-like substances. Thisalso further illustrates that ammoniumeN is closely related to theprotein-like materials during the BRLM composting. As a repre-sentative indicator for evaluating compost maturity, GI correlateswell with all parameters (not including Fmax1) in this study.Component 1 is related to tryptophan-like substances, whichmightaccount for the lack of obvious correlation between Fmax1 and GI. Heet al. (2013a) reported that no obvious correlation is observed be-tween Fmax1 and the humification parameters, which is similar tothe results in the current study. It should be noted that as animportant index for reflecting composting progress, pH exhibits nosignificant correlation with all parameters, except for Fmax4 in thisstudy, which implies that pH change is not directly associated withDOM evolution in the entire composting process.

Principal component analysisBased on aforementioned correlation analysis, PCA was con-

ducted (shown in Fig. 8b). All parameters were divided into twogroups. Group I mainly comprises the parameters related toprotein-like substances, such as PLH, Fmax1, Fmax4, and mightrepresent the biodegradation phase of easily biodegradable organicmatter. Group II might represent the humification and polymeri-sation stage of composting materials because it contained severalparameters related to the aromatic polycondensation and molec-ular weight of DOM. He et al. (2013b) reported analogous findingsin the evolution of DOM from cattle manure composting.

Humified and stabilised DOM is indicative of a quality compost.In the present study, according to spectroscopic information andthe results of HCA, it could be concluded that T2 and T3 composttreatments had a markedly higher degree of humification andstabilisation. Certain chemical characteristics of livestock manureare not adequate for composting and might hinder compostingprogress, such as an excess of moisture, low porosity, high N con-centration for the organic-C (which gives a low C/N ratio) and, incertain cases, high pH values. Biogas residues being used as bulkingagent for manure composting could improve the properties ofcomposting substrate, such as C/N ratio, particle density and me-chanical structure, positively affecting the decomposition rate oforganic matter. In addition, owing to fewer biosynthetic pathwaysandmore metabolic pathways in the anaerobic composting process(Shao et al., 2013), the structure of lignin in biogas residues ispartially damaged andmore easily attacked bymicrobes. In the BPCof BRLM, as the “core”, the oxidation products of lignin interactwith amino acids and peptides to form the humic-like substances(He et al., 2014). Livestock manure is rich in amino acids, suchtyrosine and tryptophan, and could supply the rawmaterials for theformation of humic-like substances. However, excess biogas

residues could introduce a mass of refractory lignocellulose, whichmight result in a low heating rate and temperature during the BPCand an excessively long maturing stage and slow humificationprocess. In this study, the optimal biogas residues content appearedin T2 and T3.

Conclusions

Fractionation of DOM, spectroscopic techniques, PARAFAC andHCA revealed that the degradation efficiency of alcohols, ether andpolysaccharides, and molecular weight, aromaticity, and poly-condensation degree of compost were optimal when the weightfraction of biogas residues in BRLM substrate was 40e50%. PCR-DGGE profiles of bacterial communities indicated that the abun-dance of bacteria involved in the degradation of easily biodegrad-able organic matter and lignin during composting was closelyassociated with composting efficiency and humification degree ofcompost. Generally, thermophilic stage was more rich in bacterialdiversity than mesophilic and falling-temperature stages positivelyaffected the organic matter transformation during composting.

In the bio-oxidative phase of biogas residues and livestockmanure co-composting, the parameters obtained from the DOManalysis fell into two main groups according to correlation analysisand principal component analysis. Group I represented thebiodegradation phase of easily biodegradable organic matter, andGroup II represented the humification and stabilisation stage of thecomposting substrates.

Acknowledgements

The authors wish to thank the National Key Technology R&DProgram of China (2012BAJ21B02), the National Natural ScienceFoundation of China (Nos. 51325804, 51178090 and 51378097) andthe National Public Benefit (Environmental) Research Foundationof China (No. 2011467010) for their financial support.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2015.03.032.

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Characterisation of dissolved organic matter extracted from the bio-oxidative phase of co-composting of biogas residues and ...IntroductionMaterials and methodsPreparation of composting materialsComposting set-up and samplingPhysico-chemical analysis and seed germination testExtraction of DOMFractionation of DOMSpectral analysis of DOMFTIR spectraUV–vis spectraFluorescence spectraPARAFAC modelling

Analysis of bacterial communityData processing

Results and discussionTemperature profilepH values, ammonium–N, and germination indexThe distribution of MW fractions of DOMFTIR spectraUV–vis spectroscopySynchronous fluorescence spectraExcitation-emission matrix spectraFluorescent componentsBacterial community compositionMultivariate statistical analysisCorrelation analysisPrincipal component analysis

ConclusionsAcknowledgementsAppendix A. Supplementary dataReferences