Spatial Configuration of Extracellular Organic Substances

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Spatial Configuration of Extracellular Organic Substances Responsible for the Biogas Conversion of Sewage Sludge Ying Xu, Yiqing Lu, Xiaohu Dai, Minghao Liu, Lingling Dai, and Bin Dong ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00313 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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ACS Sustainable Chemistry & Engineering

Spatial Configuration of Extracellular Organic Substances Responsible for the Biogas Conversion of Sewage Sludge Ying Xua, Yiqing Lua, Xiaohu Daia*, Minghao Liub, Lingling Daia, Bin Donga a

State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental

Science and Engineering, Tongji University, Shanghai, 200092, China b

Huai’ an Urban Construction Design and Research Institute Co. Ltd., Huai’ an, 223001, China

*Corresponding author. E-mail addresses: [email protected] (Xiaohu Dai) [email protected] (Ying Xu) Address: No. 1239, Siping Road, Shanghai, 200092, PR China No. 108, Beijing North Road, Huai’an, 223001, PR China (Minghao Liu)

ACS Paragon Plus Environment

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Abstract The influence of the key structural features of sludge that are responsible for the low anaerobic conversion efficiency of sludge is poorly understood. In this study, sludge organic substances are reclassified into extracellular organic substances (EOS) and cell biomass on the basis of sludge structure. The roles of EOS in the biogas conversion of both sewage sludge (SS) and model sludge (MS) were investigated. It is observed that with increasing EOS content the net cumulative methane production (NCMP) of the sludge decreased by 36.4%, implying the crucial roles of EOS in anaerobic sludge digestion. The experimental results showed that with increasing EOS content in sludge the extracted EOS content decreased, indicating that the structural stability of EOS in sludge was reinforced. Considering that the biodegradation of EOS typically depend on structural stability, spatial configuration of EOS has been hypothesized to account for the low anaerobic digestion efficiency. Further analyses of the spatial configuration of EOS from the MS and SS revealed that the random-coil shape with extended chains in MS is more readily biodegradable than the dense globule shape with cross-linked chains in SS. These findings shed light on the underlying mechanism responsible for the low biogas conversion of sludge. Keywords: Anaerobic digestion; Methane production; Sludge structure; Extracellular polymeric substances; Sludge age

Introduction As a byproduct of biological wastewater treatment, large amounts of sewage sludge with a high content of pathogenic bacteria, heavy metals, micropollutants, and other harmful substances are inevitably produced. If these substances are not treated and disposed of appropriately, they will cause severe secondary pollution 1. Anaerobic digestion is of great promise for treating large


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amounts of sewage sludge as it can remove odours and pathogens, stabilise the sludge, and, more importantly, generate renewable energy such as methane 2. Anaerobic digestion can maximise the value of sludge organic substances; however, poor efficiency still prevents it from being widely commercialised. For example, its efficiency is limited to 30-45% volatile solid (VS) reduction for sewage sludge 3, and methane productivity from sewage sludge is always limited to 180-290 mL CH4 /g VS 4-5 which is far below the theoretical methane potential (450-600 mL/g VS) of the sewage sludge

6-7

. To improve the

efficiency of anaerobic digestion, many studies have been conducted. In principle, there are three main study orientations: the operational conditions of the digester, the methods of pretreating the sludge, and the properties of the sludge. Most studies have focused on the first two: for example, several operational factors, such as sludge feed, retention times, temperature, solids content, co-digestion, mechanical agitation, have been reported to affect the efficiency 6, 8-10, in addition, pretreatment methods such as ultrasonic treatment, alkaline treatment, microwave-acid treatment, thermo-chemical treatment, and even high-pressure treatment also have been studied to improve the hydrolysis of sludge which has been acknowledged as the rate-limiting step 11-13. Essentially, both the operational conditions and the pretreatment methods are external measures for improving anaerobic sludge digestion, which usually depends on the properties of the sludge. For example, Appels et al.2 proposed that the optimum pretreatment parameters and magnitude of the improvement vary considerably for different types of sewage sludge. Gavala et al.12 concluded that the temperature and duration of the optimum pretreatment depend on the nature of the sludge. It has been clear that most methane originates from sludge organic substances during the anaerobic digestion process 14, and that the properties of sludge organic substances are directly related to the biogas conversion, for example, Wang et al. found that cationic polyacrylamide can


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significantly change the solubilisation and hydrolysis properties of sludge organic substances and thus restricted methane production from sludge

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; Zhao et al utilized aged refuse to adjust the

properties of sludge organic substances and found that the methane production from sludge was improved

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. Thus, recognising the sludge organic substances and exploring the roles of key

sludge organic substances in the anaerobic biogas conversion of sewage sludge are very important for fundamentally improving anaerobic digestion efficiency. Commonly, the main components of sludge organic substances have been classified into proteins, polysaccharides, humic matter, lipids etc. 17. However, these components are not independent, and they typically interact with each other in the sludge. For example, it has been suggested that extracellular proteins can interact with polysaccharides and inorganic substances 18. Thus, it is difficult to recognise the key sludge organic substances for biogas conversion based on this kind of classification. In fact, sewage sludge typically consists of water, extracellular polymeric substances (EPS), adsorbed organic substances (AOS), inorganic compounds, and bacterial cells 19-20

. Sludge organic substances can thus be classified into EPS, AOS, and cell biomass (CB). In

this classification, both EPS and AOS are extracellular substances, and thus they can be collectively called extracellular organic substances (EOS). EPS are widely acknowledged to be the backbone of sludge as it allow the accumulation of organic matter from wastewater, mediate the mechanical stability of sludge in conjunction with multivalent cations, bridge bacterial cells into a three-dimensional matrix, and protect the cells from eradication 19, 21-22. AOS are usually entangled in EPS as nutrients for bacterial cell. It thus can be inferred that EOS containing the EPS and AOS are directly responsible for the structural stability of sludge organic substances. In addition, it has been reported that the CB constitutes 10 to 20% of the organic substances in plants treating domestic wastewater 19, 23, which suggests that most sludge organic substances are


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in the form of EOS. From this viewpoint, we hypothesize that EOS are the key sludge organic substances and are responsible for the biogas conversion of sewage sludge, essentially, the stability of sludge organic substances could be reinforced by enhancing the structural stability of EOS associated with changing the spatial configuration of EOS, and thus resulting in poor biogas conversion of sewage sludge. The objective of this study was to investigate the role of EOS in the biogas conversion of sewage sludge. In the first part, the EOS contents in sludge with different sludge ages were calculated by measuring the CB contents based on the sludge structure, and the net cumulative methane production (NCMP) from sludge with different EOS contents were measured via biochemical methane potential (BMP) tests. The contributions of EOS to the biogas conversion of sludge were explored using sewage sludge (SS) and model sludge (MS) in the second part of the study. According to the analyses of the biochemical compositions and spatial configuration of the EOS, the underlying mechanism responsible for the effect of EOS on NCMP from sludge was then identified. To our knowledge, this is the first study to reveal the roles of EOS in biogas conversion from sewage sludge. These findings may provide a new understanding of the characteristics of sludge and may guide scientists in the development of more effective methods to improve anaerobic sludge digestion.

Experimental Section Sludge samples and characteristics Sludge samples with different sludge ages (10-day, 15-day, 25-day, 35-day, 45-day, and 150-day) were collected from a lab-scale anoxic/oxic sequencing batch reactor treating real municipal wastewater. Detailed information about the experimental setup can be found in the Supporting Information (SI, Text S1, Table S1 and Figure S1). MS was collected from the 20-day


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anoxic/oxic sequencing batch reactor using synthetic wastewater. The basic constituents of the synthetic wastewater are summarised in Table S2 (SI). SS was collected from secondary sludge in the Quyang municipal wastewater treatment plant (Shanghai, China) with a 20-day sludge age and stored at 4°C until use. The inoculum was obtained from a 6.0-L lab-scale anaerobic sludge digester, which was operated semi-continuously with a 30-day solid retention time at 37 ± 1 °C. The organic load rate was 5.6 kg VS/m3·d. The basic characteristics of the sludge samples (10-day, 15-day, 25-day, 35-day, 45-day, and 150-day), SS, MS, and inoculum are listed in Table S3 (SI). The average values and standard deviations were obtained from two tests. Extraction of the EOS from the sludge Because EOS are mainly composed of EPS and AOS, the extraction of EOS are divided into two parts: the extraction of EPS and the extraction of AOS. 200 mL of the sludge sample was first centrifuged (6000 g, 15 min) to separate the solids from solution. The supernatant was corrected and denoted as AOS in this study. The remaining solids were resuspended to the original volume (200 mL) using deionized water and then were subjected to EPS extraction. For the extraction of EPS from SS and MS, the formaldehyde plus NaOH method 24 was used to avoid damaging the biopolymer-metal complex. For the extraction of EPS from sludge with different ages, the cation-exchange resin (CER) method was used according to Frølund et al.

25

. The detailed

procedures for the two kinds of EPS extraction methods are described in Text S2 (SI). Lastly, the AOS and the corresponding EPS were blended together to obtain the EOS. All the obtained EOS samples were divided into two parts: one part was stored at 4°C until use and the other part was further processed by freeze-drying, and the dried samples were ground into a powder and stored in a desiccator before use. Biogas production tests


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Thirty-day BMP assays were carried out at 37°C to measure the methane yields of the sludge samples (sludge with different sludge ages [10-day, 15-day, 25-day, 35-day, 45-day, and 150-day], SS, and MS), EOS from SS (SS-EOS), and EOS from MS (MS-EOS) using an automatic methane potential test system (AMPTS-II, Bioprocess Control Sweden AB). In the BMP tests, the inoculum-to-substrate ratio was set to 1:2 (calculated with chemical demand oxygen (COD)). Before the tests were conducted, the pH of all samples was carefully checked and neutralized to around 7.0 by adding 4 M NaOH or 4 M HCl. Bottles containing only inoculum were used as blank tests (1.0 g COD of inoculum for the sludge sample and 0.3 g COD of inoculum for the EOS sample). After flushing the bottles with nitrogen gas to remove oxygen, all bottles were sealed and put into the AMPTS-II system. Methane production from only the inoculum was subtracted from the total cumulative methane production and the NCMP was calculated and expressed in CH4 mL/g COD under standard conditions (0°C; 1.013 × 105 Pa). The basic parameters of the different sludge samples in the BMP tests are summarized in Table S4 (SI). All the experiments were conducted in duplicate, and the means and standard deviations are reported. Cell number measurement The total cell numbers (both the viable and dead cells) in the sludge samples of different sludge ages were measured by flow cytometry (BD FACSCalibur, USA) using SYBR-Green I plus propidium iodide 26. The detailed procedures for the measurement are described in Text S3 (SI). Laser light scattering measurements for SS-EOS and MS-EOS Laser light scattering (LLS) measurements were conducted on a DLS/SLS spectrometer (BI-200SM, USA) equipped with a solid-state laser (λ0 = 532 nm). The lyophilized EOS samples (SS-EOS and MS-EOS) were dissolved in the buffers (glycine-NaOH with pH 9.0 ± 0.1) with


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NaCl added to adjust the ionic strength (0.1 M) to make sure that the effect of ionic strength was of the same level. The concentrations of both SS-EOS and MS-EOS solutions were prepared at 0.10 g/L for the LLS tests. Each EOS solution was clarified by using a 0.45-um hydrophilic filter into a dust-free vial carried out at 25°C. The detailed methods for calculating the LLS parameters are described in Text S4 (SI). Analytical Methods The total solids (TS), VS/TS, total Kjeldahl nitrogen, ammonia, and COD were determined according to standard methods

27

. Ca, Mg, Fe, and Al were quantitatively measured using

inductively coupled plasma-mass spectroscopy (ICP/MS-7700, Agilent) in accordance with the standard method 27. The soluble protein content was determined by the Lowry-Folin method with bovine serum albumin (BSA) as standard

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, and total protein in each sludge sample was

calculated based on the measurements of total Kjeldahl nitrogen and ammonia

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. Total

polysaccharide content was determined by the Anthrone method by glucose as standard 30, and the deoxyribonucleic acid (DNA) was quantified by a diphenylamine colorimetric assay 31. Humic matter was purified and measured according to the standard isolation protocol from the International Humic Substance Society (IHSS) 32. Total organic carbon (TOC) was determined with a TOC analyser (Shimadzu, TOC-L CPH/CPN). All statistical analyses were performed using the SPSS 19.0 package (SPSS International, Chicago), and fitting of the experimental data with a mathematical model was performed with SigmaPlot 10.0 package.

Results and Discussion Recognition on the sludge organic substances


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It is well known that the sludge organic substance is directly related to the biogas conversion of SS in the anaerobic digestion process. Therefore, recognizing the sludge organic substance is very important for improving the biogas conversion. According to the characteristics of sludge structure, sludge organic substances can be reclassified into CB and EOS, as depicted in Figure 1. In this paper, the CB is defined as the sum of cell membranes and intracellular organic substances, and the EOS consist of EPS and AOS from wastewater. It is therefore logical to surmise that the disintegration of EOS could be the prerequisite for the degradation of sludge floc and could be responsible for the biogas conversion in the anaerobic digestion process. To quantify the CB and EOS content, the cell densities in the different sludge samples were measured and the results are summarized in Table S5 (SI). The table shows that the cell numbers in different sludge samples varied between 1.3 and 3.0 × 1011 cells/g VS, which is consistent with the results of previous studies 19, 25-26, 33. In addition, the tendency of total cell numbers to decrease with the increase of sludge age was also found in the present study (Table S5, SI), and this result is in line with the report of Sanders et al., 34 who found that the number of viable cells decreased with increasing sludge age. Furthermore, it has been reported that one bacterial cell has a mass of 10-12 g 34, and thus the proportion of CB to the total amount of sludge organic substances (expressed in %VS) can be calculated. The EOS content can then be obtained by subtracting the CB content from the total sludge organic substances. The distributions of CB and EOS contents in the sludge organic substances are shown in Figure 2. It shows that the EOS contents accounted for more than 69.9 ± 1.0 %VS in the sludge samples and that the average value of EOS content was about 79.7 ± 6.7 %VS, indicating that most of the sludge organic substances are extracellular, which is in conformity with the previous studies of Frølund et al. 25 and Flemming et al.

35

. Frølund et al. found that 85-90 % of total organic carbon was


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extracellular in activated sludge, and Flemming et al. reported that the microorganisms accounted for less than 10 % of the dry mass in most biofilms. In addition, Jahn and Nielsen 36 found that the CB was only a minor fraction of the organic matter in biofilms, which also supports the results of the present study. It has been clear that biogas mainly originates from the sludge organic substances in anaerobic sludge digestion, and thus it can be inferred that EOS, which account for a substantial proportion in sludge organic substances, could be the crucial bioconversion organic substances in sludge. As depicted in Figure 2, an interesting phenomenon was found that the proportion of the EOS in sludge organic substances increased from 69.9 ± 1.0 %VS to 86.0 ± 1.3 %VS with the increase of sludge age, which is in accordance with the results of Brown and Lester

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, who found that the extracellular polymer concentrations in

activated sludge increased considerably with sludge age. One reasonable explanation for this is that sludge with a higher sludge age has a more mature microbial community structure with a low growth rate and more extracellular biopolymer storage, resulting in a high proportion of EOS in the sludge. In addition, the sludge with a higher sludge age adsorbs more organic matter from the wastewater, whereas the utilization rate of the AOS decreases, which also can lead to a high EOS content in the sludge. According to these results, it can be concluded that sludge age can influence the EOS content of sludge. Furthermore, it has been reported that the sludge age can influence the physicochemical properties of sludge

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, and that the physicochemical

properties typically have a significant effect on the biogas conversion of the sludge

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.

Therefore, it is proposed that the biogas conversion of sludge can be influenced by the EOS content.


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Figure 1. Schematic illustration of the distribution of sludge organic substances 100.0

Distribution of Sludge Organic Substances (%VS)

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CB EOS

CB EOS

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Figure 2. The distributions of EOS and CB contents in sludge of different sludge ages (calculated by the VS) Effect of EOS content on methane conversion of the corresponding sludge To explore the effect of EOS content on the biogas conversion of SS, BMP assays for sludge samples of different sludge ages were conducted. The cumulative methane yields in terms of


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millilitres of CH4 from the sludge samples with different EOS contents are summarized in Figure S2 (SI), and the relationships between the NCMP and the EOS contents (sludge ages) are shown in Figure 3. As shown in Figure 3, the NCMP from the sludge with a higher sludge age (150-day) was only 126 ± 11 mL CH4/g COD, which was lower than that from the sludge with a 10-day sludge age (198 ± 9 mL CH4/g COD ). These results are in accordance with observations by Muller et al. 41, who also found that sludge with a higher sludge age exhibited a lower degree of degradation. One possible reason for this phenomenon is that the structure of sludge floc may be reinforced as the sludge age increases, thus restricting the hydrolysis of the sludge, resulting in a low NCMP. This supposition is supported by the results of previous studies 33, 42, in which the sludge with long sludge age resisted deflocculation. However, the sludge age is just one parameter for biological wastewater treatment, and the low NCMP attributed to the sludge age is just one apparent reason. As depicted in Figure 2, it has been concluded that a high sludge age can lead to a great EOS content in the present study, suggesting that the EOS could be the substantial reason for the low NCMP. To further explore the substantial reason, a correlation analysis between the NCMP and the EOS content was conducted. Notably, an obvious negative correlation between the NCMP and the EOS contents was found in the present study (R2>0.83, P0.83

(10-day)

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(45-day)

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(150-day) 120

100 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0 84.0 86.0 88.0 90.0

EOS content in sludge organic matter (%VS)

Figure 3. The NCMP and the corresponding EOS content from the different sludge ages: The relationship between the NCMP and EOS was analyzed by the mathematical model and is marked by the red line; R2 is the goodness of fit; error bars represent the standard deviations of duplicate measurements. 100

Main organic component content of the extracted EOS

70.0 Protein (mg BSA/g TS) Ploysaccharide (mg Glc/g TS) TOC Content (mg/g TS)

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TOC content of the extracted EOS

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Net cumulative methane production from sludge (mL CH4/g COD)


0 10-day

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Figure 4. The main biopolymers (protein and polysaccharide) and TOC contents of the EOS extractions from different sludge ages. Error bars represent the standard deviations of duplicate measurements.


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Contributions of the EOS to the methane conversion of MS and SS In the present study, the content and biodegradability of EOS from different sludge samples are different, which is primarily attributed to the different sludge ages. According to the previous studies of Sheng et al.

43

and Durmaz et al. 44, there is reason to believe that the different

characteristics of wastewater can also lead to different EOS at the same sludge age, and thus the MS and SS obtained from the different characteristics of wastewater at the same sludge age (20 days) were used. To further explore the contributions of the different EOS to biogas production from the sludge, BMP assays for the MS, the SS, the MS-EOS, and the SS-EOS were conducted. The net cumulative methane yields in terms of millilitres of CH4 from the MS, SS, MS-EOS, and SS-EOS are summarized in Figure S3 (SI). It has been reported that the theoretical methane production of sludge is 350 mL CH4/g COD at the standard conditions (0°C; 1.013 × 105 Pa) 45, and thus the ratio of the NCMP to the theoretical methane production was calculated in the present study. The NCMP and the ratio from the MS, SS, MS-EOS, and SS-EOS are present in Figure 5. Notably, the NCMP from the MS-EOS was about 300 ± 17 mL CH4/g COD, which was higher than that from the MS (240 ± 10 mL CH4/g COD). The same tendency also can be found for the ratio values (85.8 ± 3.0% vs. 68.6 ± 3.5%). The NCMP and the ratio values from the SS were also respectively lower than those from the SS-EOS (230 ± 12 mL CH4/g COD vs. 159 ± 15 mL CH4/g COD; 65.6 ± 3.2% vs. 45.6 ± 2.7%). These results indicate that the biodegradability of the extracted EOS was higher than that of the corresponding sludge. One apparent reason for these results may have been that biopolymers in the extracted EOS were mainly in a soluble state, whereas the EOS in the sludge were mainly in a solid state, and thus the hydrolysis rate of the extracted EOS was higher than that of the corresponding sludge, resulting in higher NCMP and ratio values. Essentially, the mode of occurrence of the extracted


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EOS is different from that of the EOS in the sludge, and the biodegradability of the extracted EOS can be improved by changing the mode of occurrence of the EOS. These results also imply that the EOS in the sludge may have great methane bioconversion potential and the EOS configuration in sludge could be an important parameter for the biogas conversion of sludge. In addition, as shown in Figure 5, the NCMP and the ratio values from the MS-EOS were respectively higher than those from the SS-EOS, which indicates that the biodegradability of the EOS extracted from the MS was higher than that of the EOS extracted from the SS. One logical explanation for this observation is that the biopolymer configuration in the MS-EOS may have been better for biodegradation than that in the SS-EOS, and this is in line with the study of Witt et al.

46

, who concluded that the structure of the biopolymer is a decisive factor in

biodegradability. Therefore, it can be proposed that the biodegradability of the EOS is higher in

Net cumulative methane production from sludge (NCMP, mL CH 4/g COD)

the MS than in the SS, which is mainly attributed to the different EOS configuration.

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Ratios of actual and theoretical methane production (%)

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SS-EOS

Figure 5. The NCMP from the MS, SS, and their corresponding EOS (MS-EOS and SS-EOS): the ratio of NCMP to theoretical methane production was labeled on the right y-axis. Error bars represent the standard deviations of duplicate measurements.


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Spatial configuration of the MS-EOS and SS-EOS To further reveal the underlying differences in the biodegradability between the MS-EOS and SS-EOS, the main components and the zeta potentials of the MS-EOS and SS-EOS are summarized in Table 1. The spatial configuration of the EOS was determined using the LLS measurement data shown in Table 2. Schematic illustrations of the spatial configuration of the MS-EOS and SS-EOS are shown in Figure 6. As shown in Table 1, the humic matter content was about 8.4 ± 2.7 mg/L in the MS-EOS, which was far less than that in the SS-EOS (116.1 ± 5.4 mg/L), which suggests that the hydrolysis of the biopolymers in the SS-EOS was lower than that in the MS-EOS due to the interactions between the hydrolytic enzymes and the humic matter according to the study of Hao et al. 47. It also shows that the main metal content (Ca, Mg, Al, and Fe) in the MS-EOS was only 7.981 ± 1.415 mg/L, which was about one-fifth of that in the SS-EOS. Moreover, it has been reported that the structural stability of biopolymers can be enhanced through bridging and electrostatic interactions by the metal ions 20-21, thus, it can be inferred that the spatial configuration of the biopolymers in the SS-EOS was more stable than that in the MS-EOS. In addition, the zeta potential of the MS-EOS was -36.7 ± 1.2 mV, which was lower than that of the SS-EOS, suggesting that there were more negatively charged free functional groups in the MS-EOS than in the SS-EOS. One of the logical explanations for this observation is that the metal ions can react with the biopolymers to form organic-metal complex by the negatively charged groups in the SS-EOS, and the biopolymers in the SS-EOS are prone to cross-linked via the metal-bridging, resulting in a high zeta potential. This explanation is also supported by previous studies 48-49, which concluded that polyvalent cations can hold sludge flocs together by intermolecular cross-linking.


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Furthermore, it has been reported that the anaerobic biodegradability of a biopolymer is typically associated with its spatial configuration 50, and the Mw, Rg, Rh, and VR values of the biopolymer are usually used to characterize the biopolymer structure 51-52. In general, biopolymers of low Mw are biodegraded more rapidly than those with high Mw 50. As shown in Table 2, the biopolymers of low Mw were found in the MS-EOS, whereas the SS-EOS contained high Mw biopolymers, which indicates that the MS-EOS could be biodegraded more rapidly than the SS-EOS. This result also can be corroborated by the NCMP from the MS-EOS and the SS-EOS (Figure 5). The Rg and Rh values of biopolymers are typically used to ascertain the shape properties of the biopolymers 51, which can directly influence the enzymatic reaction by changing the spatial location of the active sites of the biopolymer. Notably, although a higher Mw was found in the SS-EOS, the of the SS-EOS was only 52.6 nm, which was less than half that of the MS-EOS (Table 2), implying that there could be a significant difference in the spatial configuration between the SS-EOS and the MS-EOS. This is also supported by the Rh values of the MS-EOS and the SS-EOS in Table 2. It is worth noting that the ratio ρ = Rg/Rh is an important parameter directly related to the chain architecture and conformation. From the ρ value, the shape of the EOS, e.g., spherical, random-coiled, or rod-like, can be known. Specifically, the ρ value is typically around 0.78 for a homogeneous non-draining sphere, less than 0.5 for microgels, 1.0-2.0 for random coil, above 2.0 for a rigid rod 53-54. As depicted in Table 2, the ρ value of the MS-EOS was about 1.45, which is within 1.0-2.0, indicating that the shape of the MS-EOS was that of a random coil. Moreover, the ρ value for completely extended chains is 1.5. Here, the ρ is about 1.45, suggesting that the MS-EOS in the present study had nearly completely extended chains (Figure 6). However, the ρ value of the SS-EOS was about 0.81, which is close to 0.78, implying that the shape of the SS-EOS was that of a dense globule (Figure 6). It has been


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found that the main differences in the components between the MS-EOS and SS-EOS were the humic matter and metals (Table 1), which can interact with biopolymers. One logical explanation for this observation is that large amounts of the humic matter and metal ions can react with the biopolymers to form the cross-linked and dense structure of the SS-EOS, whereas the structure of the MS-EOS was loose because of the lack of humic matter and metal ions. This explanation is also supported by the results of zeta potential (Table 1). In addition, the VR values of the MS-EOS and the SS-EOS were about 0.91 and 0.04 (Table 2), respectively, which were less than 1.00, indicating that both the MS-EOS and the SS-EOS were colloids, which is in accordance with the study of Wang et al. 52, who found that the EPS were usually in the form of colloids. Interestingly, the GSSA of the MS-EOS was about ten times greater than that of the SS-EOS, indicating that the shape of the MS-EOS was more dispersed than that of the SS-EOS, which suggests that the active sites of the MS-EOS biopolymers more readily interact with outward substances such as the enzymes. This result also implies that the enzymes have more chances to form enzyme-substrate binary complexes with the biopolymers of the MS-EOS than with those of the SS-EOS, which is the first step in enzymatic reaction processes. It is thus reasonable that the random-coil shape of the MS-EOS with the completely extended chains is more easily biodegraded in the anaerobic digestion process than the dense globule shape of the SS-EOS with its highly cross-linked chains.


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Table 1. The main components and basic parameters of the MS-EOS and SS-EOS MS-EOS SS-EOS Protein (mg BSA/L) 487.4 ± 8.1 401.3 ± 7.6 Polysaccharide (mg Glc/L) 381.5 ± 6.1 146.8 ± 4.2 Humic matter (mg/L) 8.4 ± 2.7 116.1 ± 5.4 DNA content (mg/L) 15.5 ± 1.6 10.4 ± 2.5 COD (mg/L) 4786 ± 116 3929 ± 124 Ca (mg/L) 3.518 ± 1.158 15.12 ± 3.701 Mg (mg/L) 0.623 ± 0.145 1.218 ± 0.453 Al (mg/L) 1.814 ± 0.011 11.89 ± 1.566 Fe (mg/L) 2.026 ± 0.016 9.418 ± 0.569 Zeta potential (mV) -36.7 ± 1.2 -17.4 ± 2.6 BSA, bovine serum albumin; Glc, glucose; DNA, deoxyribonucleic acid; COD, chemical oxygen demand

Table 2. LLS data summary for the MS-EOS and SS-EOS solutions at pH = 9.0 ± 0.1 C C* GSSA ρ Mw (g/mol) VR (g/L) (nm) (nm) (g/L) (m2/g) MS-EOS 0.10 76.0 110.0 1.45 3.54 × 105 0.11 0.91 2.59 × 105 5 SS-EOS 0.10 65.0 52.6 0.81 9.83 × 10 2.68 0.04 2.13 × 104 C: EOS concentration; C*: overlap concentration; : mean hydrodynamic radius; : z-average root-mean-square radius of gyration; ρ: ratio of Rg to Rh; Mw: apparent weight-averaged molecular weight; VR: volume ratio of EOS; GSSA: geometrical specific surface area

Humic Matter Ca Ca Fe Fe Mg

Ca

Ca

Fe Fe

Ca Ca

Mg Al

Fe Fe

Mg

Model for the SS-EOS

Model for the MS-EOS


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Figure 6. Schematic illustrations of the shapes of MS-EOS and SS-EOS: The highly cross-linked biopolymer in the presence of many metal ions and humic matter is shown in the SS-EOS model; the nearly completely extended biopolymer chains in the absence of many metal ions and humic matter is shown in the MS-EOS model. Implications and future considerations for the anaerobic sludge digestion Although the anaerobic digestion of SS has been studied for over 10 years, many questions remain in terms of understanding its efficiency. The poorly understood mechanism of the influence of the key sludge organic substances could be responsible for the low anaerobic conversion efficiency of sludge. EOS are regard as the key sludge organic substances, and two main reasons can be expounded. One reason for the stabilization of SS is that a stable EOS structure can be regarded as a shield for protecting bacterial cells from disruption, resulting in poor disinfection of the sludge, which also can be supported by the study of Nguyen et al. who found that removing extracellular polymeric substances (EPS) can increase sludge disintegration and enhance methane production 55. Another reason for the resource utilization of SS is that EOS account for a substantial proportion of the sludge organic matter, and possess significant biogas conversion potential. In this paper, a framework for identifying how EOS are responsible for the biogas conversion of SS is provided. The sludge organic substances were reclassified based on the sludge structure (Figure 1), and the EOS content in the sludge organic substances were confirmed (Figure 2). An interesting relationship between the EOS contents and NCMP from the corresponding sludge was explored (Figure 3), and the hypothesis that the structural stability of EOS could be responsible for the relationship was proposed according to the Figure 4. To authenticate this proposition, two kinds of EOS with different structures (MS-EOS and SS-EOS) were used to explore the correlation between the NCMP and the structural stability (Figure 5). Further study revealed that the spatial configuration of the EOS is responsible for the


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biodegradability (Tables 1-2 and Figure 6). These findings imply that changing the spatial configuration of EOS in sludge could be an effective method for improving anaerobic sludge digestion. However, although the main influence mechanism of the EOS on biogas conversion was elucidated in this work, much more mechanisms need to be studied. The key factors and main driving forces for retaining the spatial configuration of the EOS still need to be expounded. The mechanisms behind the influence of the spatial configuration on the biodegradability of EOS and how to change the dense globular structure into a readily biodegradable structure also need to be further explored. Quantitative identification of the readily biodegradable structure of the EOS in situ is another challenge. Moreover, establishment of a mathematical model that can calculate the contribution of EOS to the NCMP from the corresponding sludge also requires further exploration.

Conclusion Sludge organic substances were reclassified based on the sludge structure in this paper, and the hypothesis that the EOS are responsible for the biogas conversion of SS was proposed. EOS, whose content increased from 69.9 ± 1.0 to 86.0 ± 1.3 %VS in SS with increasing sludge age, were the dominant sludge organic substances, and there is an obvious negative correlation between the NCMP and the EOS contents was found in the present study (R2>0.83, P

Spatial Configuration of Extracellular Organic Substances

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