Bioresource Technology 123 (2012) 439–444

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Bioresource Technology

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Using fluorescence excitation–emission matrix spectroscopy to monitorthe conversion of organic matter during anaerobic co-digestion of cattledung and duck manure

Shuming Wan a,b, Beidou Xi a,⇑, Xunfeng Xia a, Mingxiao Li a, Dandan lv a, Lei Wang a, Caihong Song a,b

a Laboratory of Water Environmental System Engineering, Chinese Research Academy of Environmental Science, Beijing 100012, Chinab Agriculture College, Northeast Agricultural University, Harbin 150030, China

h i g h l i g h t s

" Spectroscopic characteristics of dissolved organic matter in anaerobic digestion." Co-digestion obtains remarkable organic matters removal rate." Tyrosine-like/fulvic-like fluorescence intensity indicates nitrogen form conversion." Tryptophan fluorescence intensity variations reflect microbial activity." Co-digestion corrects the limitation of single manure digestion.

a r t i c l e i n f o

Article history:Received 29 December 2011Received in revised form 30 March 2012Accepted 2 April 2012Available online 7 April 2012

Keywords:Excitation–emission matrix (EEM)spectroscopyAnaerobic co-digestion (AD)Cattle dung (CD)Duck manure (DM)Fluorescence intensity ratio

0960-8524/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.biortech.2012.04.001

⇑ Corresponding author. Address: No. 8, DayangChina. Fax: +86 10 84913133.

E-mail address: xibeidou@263.net (B. Xi).

a b s t r a c t

In this study, the removal of volatile solids (VSs) and soluble chemical oxygen demand (SCOD) by co-digesting cattle dung (CD) and duck manure (DM) was determined and compared with the reductionachieved with CD or DM digestion alone. Moreover, fluorescence excitation–emission matrix spectros-copy was utilised to characterise the conversion mechanisms of organic nitrogen. It was found that theco-digestion provided 71% VS reduction compared with 58% for CD and 61% for DM. The amounts ofCOD removed were 28%, 23% and 31% for CD, DM and the mixture, respectively. Tyrosine-like/fulvic-likefluorescence intensity (FI) ratios increased during the initial 15 days of co-digestion and were associatedwith an increase in total nitrogen in the supernatant. After 15 days, CD and DM exhibited a lower tryp-tophan-like/fulvic-like FI ratio (0.8–1.6), whereas the co-digestion remained stable at a high level (3.0–3.6), rendering an improved microbial population and biochemical activity.

� 2012 Published by Elsevier Ltd.

1. Introduction

Anaerobic digestion (AD) is a biochemical technology used totreat organic waste and produce biogas. As one of the most impor-tant substrates in the AD process, cattle dung (CD) has been exten-sively used (Ahn et al., 2006; Angelidaki et al., 2006). However, itsdigestion efficiency is not favourable due to the relatively low bio-degradability and biogas yield. One approach for improving its effi-ciency is to increase its biogas production rate using co-digestion,where CD is mixed with highly degradable wastes as long as theyare available in the vicinity of farms (El-Mashad et al., 2010).

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The mixture of different materials can stimulate digestion dueto a better carbon and nutrient balance (Mshandete et al., 2004;Parawira et al., 2004). According to Angelidaki et al. (2005), thereare two advantages to using cattle manure for co-digestion. First,cattle manure supplies a wide variety of sources of nutrients,trace metals, vitamins and other compounds necessary for micro-bial growth. Second, it can neutralise pH levels and improve buf-fering capacity. Braun et al. (2003) and Weiland (2000) reportedthat the co-digestion of animal manure with biodegradable wasteappears to be a robust technology that can increase biogas pro-duction by 80–400% in biogas plants. Moreover, many studieshave shown that the sensitivity of the AD process to environmen-tal changes can be improved by combining different sources oforganic wastes (Creamer et al., 2010; Zhang et al., 2011). Duckmanure (DM) is a desirable material to co-digest with CD becauseof its high biodegradability and nutrient content. Thus, the

440 S. Wan et al. / Bioresource Technology 123 (2012) 439–444

limitations of single CD digestion can be corrected and theefficiency thereby improved.

Although several studies have investigated the impact of anaer-obic co-digestion using different animal manures (Fang et al.,2011; Wu et al., 2010), there are few studies that have been under-taken regarding the fluorescence properties of these manures.Three-dimensional excitation–emission matrix (EEM) fluorescencespectroscopy can provide an overall view of the fluorescent prop-erties of dissolved organic matter (DOM) over a selected spectralrange by characterising the locations and intensities of fluores-cence peaks. Baker (2002) employed EEM spectra to analyse thefluorescence properties of some farm wastes. EEM spectra havebeen widely employed in the structural identification and stabilityassessment of solid wastes (Marhuenda-Egea et al., 2007; Yu et al.,2010). He et al. (2011a) investigated the EEM spectral characteris-tics of DOM extracted from municipal solid waste and its influenceon the biological stability of a landfill. Wang et al. (2011) used EEMspectra to investigate the destruction mechanisms of sludge diges-tion. However, there is limited research on the evolution of thefluorescence properties of different animal manures during anaer-obic co-digestion.

Therefore, this study applied EEM to characterise the evolutionof the fluorescence properties of DOM during the digestion of dif-ferent wastes. The objective of this study was to investigate theEEM fluorescence properties of the co-digestion process using CDand DM and to compare the removal of organic matter during thisprocess with that achieved during the digestion of CD and DM sep-arately. In addition, the transformation of nitrogen was specificallyexplored in characterising the fluorescence spectrum by monitor-ing the variation in the intensity ratio (Ri) during the digestionprocess.

2. Methods

2.1. Preparation of substrates

DM was collected from a farm near Baoding, He Bei Provinceduring March 2011. After scraping off the feathers, the sampleswere collected, transported immediately to the laboratory andstored in a refrigerator at approximately 4 �C until use as sub-strates for co-digestion experiments (less than 15 days). CD wascollected during March 2011 from a farm in Shunyi District, Bei-jing. It was pre-treated by screening out the straw and thenground. Some characteristics of the substrate are shown in Table 1.

Table 1Characteristics of CD and DM.

Parameter Unit CD DM

pH 7.53 8.64TS g/kg 252.4 ± 0.4 386.2 ± 0.2VS g/kg 196.2 ± 0.1 292.3 ± 0.2TOC g/kg TS 448.1 ± 0.3 263.8 ± 0.3TN g/kg TS 16.6 ± 0.2 25.2 ± 0.1TP g/kg TS 7.8 ± 0.1 8.4 ± 0.1

Table 2Compositions of the substrates fed into the reactors.

Experiment Composition of feedstock(%w/w) HRT (day)

Duck manure Cattle Dung Water

CD 0 32 68 40DM 18 0 82 40CD + DM 11 14 75 40

2.2. Experimental

Batch digestion tests were performed on CD, DM, and mixturesof CD and DM. The mixtures contained 14% CD and 11% DM. Theseloadings were determined according to previous experimental re-sults and were considered appropriate levels for giving a relativelyshort lag phase of less than 6 days at the beginning of the digestion.All of the tests were carried out in duplicate using six 2.5-L anaer-obic reactors at a mesophilic temperature of 35 ± 1 �C for 40 daystable 2.

In each digestion test, 200 mL of inoculum was used. The inoc-ulum was obtained from a full-scale biogas plant co-digestingmanure with agricultural organic wastes. The TS and VS/TS of theinoculum used were 16.8 g/kg and 0.52, respectively. After theinoculum was mixed with the substrates (CD, DM or mixture) inthe reactors, tap water was added to bring the liquid volumes upto an effective volume of 2 L. All reactors were tightly closed withrubber septa and screw caps, and each one had a sampling portnear the bottom. The head space of each reactor was purged withhelium gas for approximately 5 min to assure anaerobic conditionsprior to starting the digestion tests. The biogas produced from thereactor was collected in the gas-collection tank using a water-dis-placement system, which was designed to impart a small and con-stant back pressure to the reactor vessel. Water displaced by thegas overflowed from the base of the vessel to drain. Prior to themeasurement of biogas volume, all reactors were shaken manuallyfor approximately 1 min once a day to completely mix the materi-als. The pH, SCOD, VS and other parameters were measured every5 days.

2.3. Analytical methods

2.3.1. Chemical analysisTotal solids and VS were measured according to the Standard

Methods (APHA, 2005).The soluble chemical oxygen demand(SCOD) in the suspended fraction was measured using COD am-poules and a spectrophotometer (Shimadzu UV-1700, Japan). Thetotal nitrogen (TN) and ammonium–nitrogen (NHþ4 -N) in the sam-ple supernatant were measured according to the Standard Meth-ods (APHA, 2005) after being centrifuged and filtered. The TOC ofall samples were measured using an Analytik Jena Multi N/C2100 TOC analyser (Analytik Jena, Germany).

2.3.2. Fluorescence spectroscopyThe samples were centrifuged at 8000 rpm for 10 min and fil-

tered through a 0.45-lm membrane to extract DOM. Before fluo-rescence analysis, the dissolved organic carbon (DOC) of allsamples was measured using an Analytik Jena Multi N/C 2100TOC analyser (Analytik Jena, Jena, Germany). All samples were di-luted with 0.1 mol/L phosphate buffer (pH 7). The final DOC con-tent was set to approximately 30 mg/L. EEM fluorescencespectroscopy was performed on each sample using a Hitachi Fluo-rescence Spectrophotometer (F-7000) at room temperature (25 �C).Excitation and emission were simultaneously scanned at wave-lengths ranging from 200 to 450 nm and from 250 to 550 nm,respectively, at 5-nm intervals. The slit widths were set to 10 nmfor both the excitation and emission monochromators, and thescan speed was 2400 nm/min.

2.4. Data processing

The fluorescence intensity (FI) and ratio (Ri) were adopted forEEM spectrum analysis. The concentration of different fractionsand EEM data analysis were calculated using the SPSS 16.0 (SPSSInternational, Chicago, USA) software package. Finally, Origin 8.0(Origin Lab, Los Angeles, USA) was used for figure processing.

Fig. 1. VS and SCOD concentration in cattle dung, duck manure and the mixtures of cattle dung (CD) and duck manure (DM).

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3. Results and discussion

3.1. Performance of the digesters

Laboratory-scale anaerobic digestion systems were used todetermine the performance of CD, DM and the mixture. The anal-ysis was performed throughout the entire process and was evalu-ated by monitoring pH, biogas production and solids removal(data not shown). Different performance parameters such as VSdestruction, SCOD removal, nitrogen concentration were mea-sured. All of the digesters performed well during the digestionprocess.

3.1.1. VS and SCOD removalFig. 1a shows the VS concentrations during the digestion of CD,

DM and the mixture. A marked variation in the VS was observedover the experimental period. After 5 days of digestion, the VS ofCD rapidly decreased to 11.2 g/kg, which was lower than that ofDM (14.6 g/kg) and the mixture (16.3 g/kg). However, the VS oftwo single manure digestions fluctuated in the following days,whereas the mixture showed a more constant decreasing trend.After 40 days of digestion, average VS removal rates of 58%, 61%and 71% were obtained for CD, DM and the mixtures, respectively.The removal from the mixtures was much higher than that fromthe CD and DM. Considering the poor digestibility characteristicsof the fed manure, the additional removal efficiency gained inthe mixtures can be considered a significant improvement in thedigestion.

Compared with the single digestion, co-digestions might reducethe accumulation of intermediates during the initial stage of

Fig. 2. TN and NHþ4 -N concentration in cattle dung (CD), duck manure

digestion (El-Mashad et al., 2010). These results suggest that somefactors were limited during single CD and single DM digestion andwere corrected by providing essential factors from the mixture viaco-digestion (Zhang et al., 2011).

The same form of representation was chosen for SCOD; the con-centration profiles are shown in Fig. 1b. The average SCOD removalduring anaerobic digestion was 28%, 23% and 31% for CD, DM andthe mixture, respectively. The parallel data analysis of VS and SCODwas useful in characterising the evolution and the biodegradationmechanisms of the soluble and particulate organic matter duringthe digestion process. Demirer and Chen (2005) also reported thatthe VS and SCOD data obtained using dairy manure were highlycorrelated.

When the SCOD removal was considered, it could also be seenthat the values showed great variation during anaerobic digestion.The concentration of SCOD in the mixture reached a peak valueafter 15 days, whereas DM achieved its peak value 6 days later.This finding indicates the decomposition of organic matter andthe formation of soluble fractions. During the initial stage of AD,the increase in SCOD could also be due to the solubilisation of vol-atile fatty acids (VFAs). Compared with DM and the mixture, theCD contained higher cellulose and coarse fractions, which weremuch more difficult to hydrolyse. Therefore, CD digestion showedless VFA accumulation and lower SCOD levels throughout thewhole process.

3.1.2. Conversion of nitrogen formAccording to Zupancic and Ros (2008), the supernatant from

anaerobic sludge digestion is characterised by a high nitrogen con-tent and can constitute a significant fraction (up to 50%) of the

(DM) and the mixture of cattle dung and duck manure (CD + DM).

Fig. 3. Fluorescence EEM of DOM from each digestion at different stages of the process.

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nitrogen load. The concentrations of TN and NHþ4 -N in differentsample supernatants are reported in Fig. 2.

During the start-up, a rapid increase in TN in DM was observed.This increase indicates that insoluble nitrogen in the feedstock wasconverted to soluble forms during the hydrolysis stage. The TN in-creased to 198.5 mg/L (43.8% higher than the initial concentration)after 15 days of digestion and decreased slowly thereafter. The co-digestion showed a similar trend during the first 15 days: the TNincreased from 81.7 mg/L to 146.2 mg/L; however, it dropped sig-nificantly after day 15. Despite the variation in TN, the NHþ4 -N con-centration of all of the digestions remained stable. TN includes thefree NHþ4 -N as well as the NHþ4 -N released from the organic nitro-gen, which might be attributed to the degradation of organic mat-ter as a result of microbial activity (Uludag-Demirer et al., 2008).DM and the co-digestions exhibited a significant increase in TNfrom day 0 to day 15, but the ammonia concentration variedslightly throughout the whole process (Fig. 2b). The NHþ4 -N con-centrations remained relatively constant at approximately11.2 mg/L, 53.4 mg/L and 31.8 mg/L for CD, DM and the mixture,respectively. Hence, there are likely to be other mechanisms forthe conversion of different forms of nitrogen.

3.2. EEM fluorescence spectra of DOM

The three-dimensional EEM fluorescence spectra of DOM sam-ples at different stages of the AD process are illustrated in Fig. 3.Peak A was located at the excitation/emission wavelengths (Ex/Em) of 235–240/345 nm, whereas Peak B was detected at the Ex/Em of 280/320 nm. The two peaks have been reported as protein-like peaks, in which the fluorescence is associated with aromaticprotein-like substances (Peak A) and tryptophan protein-like sub-stances (Peak B). Two secondary peaks centred at approximately270/425 (Peak C) nm and 330/420 nm (Peak D) were also observedin all DOM samples throughout the entire digestion process. Chenet al. (2003) attributed Peak D to humic-like organic substances.Peak C is not frequently cited in the literature. He et al. (2011b) re-ported peaks characterised at the Ex/Em wavelength pair 288/455 nm, a region which is associated with humic acid-like sub-stances, and regarded these peaks as an indicator of maturity. Thus,the presence of Peak C could indicate the stability of organic sub-stances during the AD process.

It is apparent that each of the digestions had different fluores-cence properties during the AD process. The CD showed lower

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fluorescence intensity (FI: 80–800 intensity units) throughout theentire experiment and a significant decrease after digestion. DMand the mixture showed similar behaviour; the tryptophan fluo-rescence intensity was much higher than that of tyrosine. CDwas distinguished by the presence of tyrosine fluorescence at anintensity similar to that of tryptophan. The FI of Peak D, which isrelated to humic-like substances with low biodegradability, wasrelatively stable during the AD process.

In addition to peak location and fluorescence intensity (FI), thepeak intensity ratio is another parameter that can be used to deter-mine the characteristics of DOM. Each fluorescence peak may varyin fluorescence intensity relative to one another, depending on thesource of organic matter, whereas the variations in the overall fluo-rescence intensity reflect changes in concentration. Hence, bydetermining both the ratio of fluorescence intensity of each fluo-rescence peak and the absolute intensity, it may be possible to dif-ferentiate between different farm wastes (Baker, 2002).

3.3. Fluorescence intensity ratio variations

The fluorescence EEM data of digestions conducted for over40 days are presented in Fig. 4a and are simplified to show thefluorescence intensity ratio. The results show that the tyrosine-like/fulvic-like fluorescence intensity ratio (RA/C) of CD declinedfrom approximately 4.0 to a ratio of approximately 2.0 duringthe digestion, although it was still higher than that of the DMand mixture. This decrease in RA/C is associated with a decreasein tyrosine fluorescence intensity and a relatively stable fulvic-likeand humic-like fluorescence intensity (Fig. 4d), suggesting thatprotein groups were broken down into non-fluorescent structuresor converted to other forms. Interestingly, the RA/C of the mixture

Fig. 4. Fluorescence intensity ratio of different peaks in cattle dung (CD), duck manure (Dfulvic-like fluorescence intensity; (b) tryptophan/fulvic-like fluorescence intensity; (c) prfluorescence intensity.

increased rapidly from day 0 to day 15, much like the TN concen-tration. This result indicates that the accumulation of tyrosine-likeorganic compounds is linked to an increase in the TN concentrationand that tyrosine might take part in the conversion of the mainform of nitrogen.

Fig. 4b shows that the initial tryptophan/fulvic-like fluorescenceintensity ratio (RB/C) ranged from 2.7 to 3.5. All samples exhibiteddifferent evolutions of this ratio; the RB/C of CD and DM decreasedsignificantly after digestion, whereas the co-digestion showed anincrease at the end of the process. The RB/C of CD varied from 3.5to 1.6; the high initial RB/C reflects the biochemical structure offeedstock, and the decrease in the protein-like fluorescence overtime most likely reflects the hydrolysis of proteins, which is themost important chain in the anaerobic digestion. DM behaved ina similar manner, although the overall tryptophan fluorescenceintensity was higher.

Samples from the co-digestion showed greater detail with re-spect to the evolution of RB/C (Fig. 4b). As a type of microbialbyproduct material (Chen et al., 2003; Reynolds and Ahmad,1997), the tryptophan intensity slightly increased during the firststage and remained high after 20 days. This result was correlatedwith the number of microorganisms and their activity. Anaerobicbacteria and Archaea are the dominant microorganisms presentduring digestion (Trzcinski et al., 2010); these bacteria decomposecelluloses and proteins into fermentable sugars and amino acidssuch as tryptophan; then, these utilisable materials are brokendown into finer structures or are used to form more-stable macro-molecules. The mixture of the CD and DM might modify the char-acteristics of the feedstock and overcome the limitations of single-manure digestion such that the associated microbial populationand biochemical activity are improved. This phenomenon could

M), and the mixtures of cattle dung and duck manure (CD + DM). (a) Tyrosine-like/otein-like/humic and fulvic-like fluorescence intensity and (d) fulvic-like/humic-like

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also explain the remarkable VS removal efficiency obtained by co-digestion.

Additionally, the co-digestion (Fig. 4c) showed a more complexevolution in the protein-like/humic and fulvic-like fluorescenceintensity ratios (RA+B/C+D). These differences can be attributed tothe more complex composition of the feedstock. Animal speciesand physiology affect the composition of excreted material, whichin turn affects the material’s fluorescence characteristics. Researchhas demonstrated that livestock faeces is typically composed of15–25% protein in wet manure (poultry, cattle and pigs); withinthis composition, a large fraction of protein N is attributed to tryp-tophan and tyrosine (Demirer and Chen, 2005). Hence, the protein-like fluorescence intensity was very strong, although humifiedmaterials had accumulated. The RC/D was observed to be stable,which suggested that the humic and fulvic acid were in a dynamicbalance; thus, the formation of humified materials is a long andcomplex process (Fig. 4b).

4. Conclusions

Compared with digesting CD and DM separately, co-digestionachieved a remarkable VS removal of 71%, and the SCOD removalefficiency was also notable. The RA/C of the co-digestion showed atrend similar to that of the TN concentration, which implies thattyrosine-like substrates take part in the conversion of organicforms of the main nitrogen fraction. Moreover, the high level ofRB/C indicates that mixing CD with DM can improve the microbialpopulation and biochemical activity. Thus, the EEM fluorescencespectra of DOM could be conveniently used to characterise theconversion of organic matter during anaerobic digestion.

Acknowledgements

This work was financially supported by the National NaturalScience Foundation of China (Nos. 51078340 and 50878201) andthe research project of organic wastes treatment in rural area usingnovel technology and equipment (No. 2012BAJ21B02).

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