Bioresource Technology 159 (2014) 297–304

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Enhanced high-solids anaerobic digestion of waste activated sludge by the addition of scrap iron Yaobin Zhang 1, Yinghong Feng, Qilin Yu, Zibin Xu, Xie Quan ⇑ Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A new method to accelerate the

anaerobic digestion of sludge without pretreatment.  Iron scrap has better mass transformation efficiency than iron powder.  Acetobacteria and iron-reducing bacteria can be enriched by rusty scrap.  Adding rusty scrap made the methane production raise 29.51%.

a r t i c l e

i n f o

Article history: Received 4 January 2014 Received in revised form 22 February 2014 Accepted 25 February 2014 Available online 6 March 2014 Keywords: Waste activated sludge Anaerobic digestion Iron Methane production Sludge reduction

a b s t r a c t Anaerobic digestion of waste activated sludge usually requires pretreatment procedure to improve the bioavailability of sludge, which involves considerable energy and high expenditures. This study proposes a cost-effective method for enhanced anaerobic digestion of sludge without a pretreatment by directly adding iron into the digester. The results showed that addition of Fe0 powder could enhance 14.46% methane yield, and Fe scrap (clean scrap) could further enhance methane yield (improving rate 21.28%) because the scrap has better mass transfer efficiency with sludge and liquid than Fe0 powder. The scrap of Fe with rust (rusty scrap) could induce microbial Fe(III) reduction, which resulted in achieving the highest methane yield (improving rate 29.51%), and the reduction rate of volatile suspended solids (VSS) was also highest (48.27%) among Fe powder, clean scrap and rusty scrap. PCR–DGGE proved that the addition of rusty scrap could enhance diversity of acetobacteria and enrich iron-reducing bacteria to enhance degradation of complex substrates. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Yield of waste activated sludge generated in biological wastewater treatment processes has increased continuously in the recent decade, due to increasing population in cities and towns and ⇑ Corresponding author. Tel.: +86 411 8470 6140; fax: +86 411 8470 6263. E-mail addresses: [email protected] (Y. Zhang), (X. Quan). 1 Tel.: +86 411 8470 6460; fax: +86 411 8470 6263. http://dx.doi.org/10.1016/j.biortech.2014.02.114 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

[email protected]

construction of new waste water treatment plants (WWTPs). The total dry sludge amount in European Union countries has reached more than 10 million tons per year (Duan et al., 2012). In China, over 11.2 million tons of dry sludge is generated annually and almost 80% of it has not obtained necessary stabilization (Zhang et al., 2012). To minimize the volume of waste sludge, most municipal sludge (such as more than 80% in China) before discharge is dewatered to get high-solid sludge with total solid content typically greater than 10% (w/w). However, there is still a large amount

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of biodegradable compounds contained in the dewatering sludge which gives rise to secondary pollution including leaching of liquid and odor (Lee and Han, 2013). Anaerobic digestion of waste sludge is an efficient and sustainable technology to stabilize sludge by means of mass reduction and methane production simultaneously. Methane is an alternative energy source to limited fossil fuels which helps cut down the operation cost of treatment plants. However, the application of anaerobic digestion of sludge is often limited by low methane yield and sludge reduction rate (Duan et al., 2012). The limiting factors are generally associated with the slow hydrolysis of sludge. To improve the rate-limiting hydrolysis and digestion performance, many types of pretreatments such as chemical, mechanical and biological sludge disintegration have been developed (Lagerkvist and Morgan-Sagastume, 2012). Pretreatment may result in lysis or disintegration of sludge cells, thus releasing and solubilizing intracellular material into the water phase and transforming refractory organic materials into biodegradable species, and therefore making more materials readily available for microorganisms. However, mechanical and thermal methods require the input of considerable amount of energy (Weemaes and Verstraete, 1998). Chemical treatment, including acid/alkali hydrolysis, ozone and advanced oxidation, requires large amount of chemicals to maintain the reaction conditions (Navia et al., 2002). The operating cost of the present pretreatment is high and often not easy to be acceptable for application. Fe0, which is a cheap reductant, was found to be able to accelerate anaerobic hydrolysis of simple and soluble materials when it was added into the anaerobic system (Liu et al., 2012; Zhang et al., 2011a). The mechanism was that Fe0 could decrease oxidative-reductive potential (ORP) to create a more favorable environment for anaerobic digestion. The activity of the enzymes associated with hydrolysis–acidification was observed to increase 2–34 times with the addition of Fe0 powder (Meng et al., 2013). It was possible that the Fe0 could accelerate the anaerobic digestion of sludge because Fe0 might enhance the hydrolysis–acidification. Moreover, Fe0 was reported to enhance the growth of methanogens to increase methane production (Chastain and Kral, 2010). In previous work, adding Fe0 powder in an anaerobic digestion of diluted sludge (1% solid content) after pretreatment by alkaline was confirmed to enhance methane production (Feng et al., 2014). However, there has been no study that investigated the effects of Fe0 on sludge digestion without a pretreatment till date. It is unknown whether addition of Fe0, especially iron scrap, could replace the tedious pretreatment processes mentioned above to enhance anaerobic digestion of high-solid sludge. If scrap or waste scrap iron could directly accelerate the sludge digestion, it is expected to provide a practical and economical alternative to reduce the complicated and high-cost procedures prior to digestion. In this study, the performance of anaerobic digestion of high-solid sludge was investigated in comparison with adding different types of iron including Fe0 powder, clean scrap and rusty scrap. Also, the microorganism communities functioning in the anaerobic digestion were identified and explored. 2. Methods 2.1. Substrates and inoculum Dewatered sewage sludge from the Chunliu Wastewater Treatment Plant (Dalian, China) was used as substrates for this study. The inoculums were collected from an anaerobic digester at a Waste Sludge Treatment Plant of Dalian (China). Before the digestion, the substrates were mixed with the inoculum in a ratio of 9:1. Two batch tests were conducted, and characteristics of sludge mixture are listed in Table 1.

2.2. Preparation of iron Three kinds of iron were used: Fe0 powder, clean scrap and rusty scrap (see Fig. S1, Supporting Information). The Fe0 powder (diameter of 0.2 mm, BET surface area of 0.05 m2/g, purity > 98%) was purchased from Shenyang Chemical Reagent Plant. The iron scrap (about 8 mm  4 mm  0.5 mm, purity > 95%) was obtained from a machinery workshop. Before the use, the scrap was soaked in 0.1 mol/L of NaOH solution for 24 h to remove oil and then washed with dilute HCl solution and tap water to remove the rusty cover. The rusty scrap (about 8 mm  4 mm  0.5 mm) was the same as the clean scrap mentioned above but without rust removal. In other words, the rusty scrap had a corrosion layer covering the surface of the scrap. 2.3. Batch experiments The mixture (200 mL) of substrates and inoculum was incubated in 250 mL serum bottles placed in an air-bath shaker (120 rpm) at 35 ± 1 °C for 22 days. After adding a certain amount of iron (in the form of the powder, clean scrap or rusty scrap), the bottles were capped with silica gel stoppers and oxygen was removed from the headspace by exchanging it with nitrogen gas for 10 min. A silica tube across the silica gel stoppers was connected to the gasbag. During the digestion, the biogas produced from each bottle was collected into gasbag for analysis. Two batch experiments were conducted in this study. The first experiment was to investigate the effects of different dosage of Fe0 powder and the clean scrap on anaerobic sludge digestion under seven dosage levels (0, 1, 6, 8, 10, 14 and 20 g/L). The other experiment was conducted to compare the effect of the clean and rusty scrap under a specific dosage (10 g/L). All experiments conducted triplicate at the same time. 2.4. Analytical methods Sludge samples from the reactors were analyzed for pH, ORP, total suspended solid (SS), volatile suspended solids (VSS), total protein and total polysaccharide. Then the samples were centrifuged at 8000 rpm for 10 min and immediately filtered through a 0.45 lm pore size cellulose membrane filters for analyzing soluble chemical oxygen demand (SCOD), soluble protein, soluble polysaccharide and VFAs. SS, VSS and SCOD were determined according to Standard Methods (APHA, 1998). The ORP was immediately measured by an ORP combination class-body redox electrode (Sartorius PY-R01, Germany) after the batch experiment was finished. The pH was recorded using a pH analyzer (Sartorius PB-20, Germany). The settlement percentage of sludge was tested using 25 mL of sludge. To avoid the highly dense and no homogenous of sludge, the sludge was fully mixed before sampling and the settling time was extended from generally 30–60 min. The settlement experiment was conducted under the optimal iron dose (10 g/L). Proteins were measured with a Lowry’s method using bovine serum albumin as a standard solution (Fr et al., 1995). Polysaccharides were measured with phenol–sulfuric acid method using glucose as a standard solution (Masuko et al., 2005). After the batch experiment, the reminding iron scraps was recovered by a magnet, and then it was cleaned, dried and weighed to calculate the consumption of iron. Fe2+ and total iron were analyzed by an adaptation of the ferrozine technique (Cooper et al., 2000). The concentration of CH4 and CO2 in the biogas was analyzed with a gas chromatograph (Shimadzu, GC-14C) equipped with a thermal conductivity detector and a 1.5 m stainless-steel column (Molecular Sieve, 80/100 mesh). The temperatures of injector, detector and column were kept at 100, 105 and 60 °C according to the reference (Zhao and Yu, 2008). Nitrogen was used as the carrier gas at a flow rate

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Y. Zhang et al. / Bioresource Technology 159 (2014) 297–304 Table 1 Characteristics of sludge mixture.a

c d

pH

SSb (g/L)

VSSc (g/L)

TCODd (g/L)

Total protein (g/L)

Total polysaccharide (g/L)

Sludge mixture 1 Sludge mixture 2

7.46 ± 0.04 7.49 ± 0.05

98.2 ± 5.9 103.4 ± 5.4

56.7 ± 3.8 57.3 ± 2.5

92.5 ± 8.1 96.0 ± 9.6

23.69 ± 1.56 25.13 ± 2.14

8.99 ± 0.79 8.72 ± 0.87

Average data and standard deviation obtained from three tests. SS: total suspended solids. VSS: volatile suspended solids. TCOD: total chemical oxygen demand.

of 30 mL/min. VFAs (acetic acid, propionic acid, butyric acid and valeric acid) were measured in another gas chromatograph (Shimadzu, GC2010) with GC-flame ionization detector, FID (Shimadzu, Model 14B) and a 30 m  0.25 mm  0.25 lm fused-silica capillary column (DB-FFAP). The operating temperatures for the injection port and the FID were 170 °C. The temperature in the oven was gradually increased from 100 to 130 °C at a rate of 5 °C/min according to the reference Fan et al. (2006)). Nitrogen was the carrier gas at a flow rate of 30 mL/min. Analysis of Variance (ANOVA) were used to analyze the statistical significance of the results, which was conducted at 95% confidence level, using the software SPSS. 2.5. DNA extraction, amplification by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and sequencing The genomic DNA of the sample was extracted using an extraction kit (Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions. A primer combination of 341f/907r was used to selectively amplify the 16S ribosomal RNA sequences. A 40 base pair GC clamp was added to the forward primer at the 50 -end to improve detection of the sequence variation in DNA fragments by subsequent DGGE (Buchholz-Cleven et al., 1997). The 16S rDNA fragment was amplified using a PCR thermal cycler Dice (BioRad Co. Ltd., USA) with a touchdown PCR method (Zhang et al., 2011b). The PCR products obtained were applied in the DGGE analysis using a BioRad Dcode system (BioRad Co. Ltd., USA). A DGGE gel of 6% polyacrylamide with a linear denaturing gradient ranging from 30% to 60% (100% denaturing gradient contains 7 M urea and 40% formamide) was applied. Electrophoresis was conducted at a constant voltage of 180 V in 1 TAE buffer and 60 °C for 6 h. The gels were then stained with SYBR Gold (Dalian TaKaRa, China) in 1 TAE buffer for 40 min, after which the UV transillumination image of the gel was photographed using the Gel Doc 2000 System (BioRad Co. Ltd., USA). Selected DGGE bands were excised and re-amplified by PCR with the aforementioned primers without the GC clamp. The PCR products were sequenced at TaKaRa Biotechnology Co. Ltd., (Dalian, China). The obtained sequences were then compared to the reference microorganisms in the GenBank database using the BLAST program. 3. Results and discussion 3.1. Comparison between clean scrap and Fe0 powder for enhanced anaerobic digestion of sludge To investigate the effects of clean scrap and Fe0 powder, the anaerobic digestion of sludge was conducted with addition of clean scrap or powder at seven different levels (0, 1, 6, 8, 10, 14, 20 g/L). The cumulative production of methane from the digesters during the digestion of 22 d is shown in Fig. 1. Biogas, as a by-product from anaerobic digestion of sludge, is mainly composed of methane and carbon dioxide. Because

Methane production (mL/g-VSS)

a b

Parameters

200

Iron scrap Fe0 powder

180 160 140 120 100

0

1

8 6 10 Dosage (g/L)

14

20

Fig. 1. Methane production during the digestion with various dosages of Fe0. The bars designate the standard deviations of triplicate tests.

methane is a recyclable energy and methanogenesis is susceptible to the environment, methane yield is an important index to assess performance of anaerobic sludge digestion. From Fig. 1, methane production was significantly increased with adding iron to a certain extent. With an increase of iron from 0 to 10 g/L, the methane production raised from 145.8 to 179.9 mL/g-VSS for the scrap and to 165.1 mL/g-VSS for the powder. It appeared that the scrap had a higher performance in enhanced methanogenesis from the sludge digestion than the powder. The maximum methane yield observed was 179.91 mL/g-VSS at a dose of 10 g/L scrap, which was 23.42% higher than those of the control group and 8.96% higher than the Fe0 powder group. Further increase in iron dosage did not get more increment in the methane production, and even the methane production at 20 g/L of both the powder and scrap was found to decrease compared with that at 14 g/L. This experiment clearly indicated that the scrap iron had better effects on the enhanced anaerobic digestion of sludge. The release of electron from Fe0 was associated with the surface reaction. Although the powder Fe0 had greater surface area available for its function in the digestion than the scrap, the powder was more easily immersed into sludge to hinder its effects due to its small size. The test on settleability of sludge showed that the settlement ratios of the control sludge, the sludge added with the powder and the sludge added with the scrap were 76%, 56.8% and 73.6%, respectively (Fig. S2, Supporting Information). It indicated that the powder increased the sludge aggregation but decreased the mobility. The powder might play a role like a ‘‘settling seed’’ to drive the sludge enclosed itself tightly. Comparatively, the scrap iron had little effect on the settleability, predicting that the scrap was ‘‘free’’ to sludge, which ensured that it had a good mass transfer with sludge and bacteria under stirring or shaking conditions. The function of Fe0 could be made only through the reductive reaction occurring on its surface, i.e., Fe0 = Fe2+ + 2e. Thus, Fe2+ release is an indicator to reflect the extent of its participation in the process. From Table 2, the Fe2+ concentration in the scrap groups was always higher than that in the powder ones. This result suggested that the scrap was more effective to enhance anaerobic digestion of sludge than the powder. Also, the Fe2+ release in the

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Table 2 Release of Fe2+ during the digestion.a Dosage (g/L)

Fe2+ from the scrap (mg/L) Fe2+ from the powder (mg/L)

1

6

8

10

14

20

14.12 ± 0.67 /

15.88 ± 0.58 16.44 ± 0.38

25.91 ± 0.72 20.67 ± 0.59

33.81 ± 0.64 26.97 ± 0.44

33.26 ± 0.78 22.11 ± 0.28

32.09 ± 0.69 19.69 ± 0.37

32.72 ± 0.83 18.64 ± 0.32

Mean values with standard deviation obtained from three tests.

powder and scrap groups at the dosage of 8–10 g/L reached the highest levels (Table 2). Higher dosage might get the sludge easily settled making the sludge unable to equally distribute in the bulk. It might be a reason for the decreased methane production at the dosage higher than 10 g/L. Considering that the scrap is cheaper and more easily accessible, the scrap is preferred with regard to practical application. Therefore, following investigation in this work was focused on the scrap iron. 3.2. Sludge reduction with addition of the iron scrap Protein and polysaccharide are the two main substrates accounting for a majority of COD in the sludge. During the anaerobic digestion, those substrates would be finally mineralized into methane and carbon dioxide, accompanied with the sludge reduction. The changes in VSS, total protein and total polysaccharide in the sludge before and after the digestion with the addition of different amount of scrap are shown in Fig. 2. The raw sludge contained 23.69 g/L of protein and 8.99 g/L of polysaccharide, representing 41.78% and 15.56% of volatile solid content, respectively. The VSS in the raw sludge was 56.74 g/L. At 10 g/L of the dosage, the removals of VSS, total protein and total polysaccharide reached 47.07%, 57.85% and 61.07%, respectively, while their removals in the control group were only 34.67%, 48.06% and 49.33%, respectively. The results were in agreement with that of the methane production. The addition of scrap iron could significantly enhance the reduction of VSS. It was observed that the VSS reduction increased with the dosage up from 1 to 10 g/L. However the dosage of more than 10 g/L seemed not to further improve the sludge reduction. The enhancement of organic matters reduction may be attributed mainly to the activity of the enzymes associated with hydrolysisacidification which was observed to significantly increase with the addition of Fe in previous study (Meng et al., 2013). 3.3. Comparison between the clean scrap and the rusty scrap

scrap from machinery industries is rusty iron. It was reported that Fe(III) could directly oxidize organics in anaerobic digestion depended upon microbial Fe(III) reduction, in which Fe3+ was reduced into soluble Fe2+ and organics were oxidized into simple materials and even were mineralized (Lovley and Phillips, 1988). When the rusty layer was used up, the inner metal Fe0 could continuously operate in the anaerobic sludge digestion. Thus it was assumed that the rusty scrap iron could also induce an enhanced anaerobic digestion of sludge. To clarify it, the experiment was conducted to investigate the effects of rusty scrap on the sludge digestion with clean scrap as comparison. According to the methane production and sludge reduction, the optimal dosage of iron scrap was 10 g/L. Therefore, the dosage in this experiment was fixed at this level. The cumulative methane production after the digestion for 22 d was 166.37, 201.77 and 215.47 mL/g-VSS for the control group, the clean scrap and rusty scrap groups, respectively (Fig. 3a). A statistical analysis for methane production among control group, the clean scrap group and rusty scrap group was conducted by ANOVA methods (see Table S1, Supporting Information) to obtain a low P-values (0.01 < 0.05), which confirmed that the improving effect caused by the clean scrap and rusty scrap was significant. And the results

Methane production (mL/g-VSS)

a

0

240

(a) 200 160 120 80

Control group Clean scrap Rusty scrap

40 0 0

4

8

The iron scrap is easily oxidized in environment to form a layer of iron oxides covering the surface of scrap. In most cases, waste

Total protein R-Total protein

Total polysaccharide R-Total polysaccharide

70 60

50 40

50

30

40

20 30

10 0

20 InitialControl 1

6

8

10

14

20

ZVI scrap dosage (g/L) Fig. 2. Effect of different dosage of iron scrap on VSS, total protein and total polysaccharide after 22 days digestion. The bars designate the standard deviations of triplicate tests.

Carbon dioxide production (mL/g-VSS)

60

VSS R-VSS

Removal efficiency (%)

Concentration (g/L)

70

12

16

20

24

Time (days)

140

(b)

120 100 80 60

Control group Clean scrap Rusty scrap

40 20 0

0

4

8

12

16

20

24

Time (days)

Fig. 3. Effect of clean scrap and rusty scrap on methane production (a) and carbon dioxide production (b) during sludge anaerobic digestion. The bars designate the standard deviations of triplicate tests.

Y. Zhang et al. / Bioresource Technology 159 (2014) 297–304

showed that the methane production of the rusty group was 29.5% greater than that of the control group. Interestingly, the rusty scrap had a higher performance than the clean scrap to enhance the sludge digestion. Production of carbon dioxide was also in the following order, rusty scrap (136.9 mL/g-VSS) > clean scrap (129.9 mL/g-VSS) > control (113.4 mL/g-VSS) (Fig. 3b). The production of carbon dioxide was well in agreement with the methane results. Acetate, propionate, butyrate and valerate are several major VFA forms in anaerobic digestion, most of which would be converted to methane and carbon dioxide based on acetogens and methanogens operating harmoniously. As shown in Fig. 4, the VFAs concentration of the rusty scrap group was lowest. Specially, propionate was the dominating type of VFA in each digestion system. It was because conversion of propionate into acetate was unfavorable in thermodynamics (DG = +76.1 kJ/mol) (Schmidt and Ahring, 1993). The concentrations of propionate were 2715, 2113 and 1891 mg/L for the control, clean scrap and rusty crap groups, respectively. This result suggested that the scrap could enhance decomposition of propionate and the rusty scrap created further improvements on the propionate conversion. The results were in agreement with Liu et al. (2012), who found that Fe0 could decrease propionate content in anaerobic digestion of sucrose. Fe0 could decrease the ORP value of the anaerobic system to create beneficial environment for obligate anaerobes such as methanogens, hemoacetogens, etc. (Ren et al., 2007). It made these susceptible anaerobes ready for utilization of the relevant substrates to forward the anaerobic digestion. Moreover, the activities of several key enzymes associated with hydrolysis and acidification might be increased in presence of iron. It was reported that the activity of enzymes associated with acetogenesis increased 2–34 folds by Fe0 powder (Meng et al., 2013). In addition, the propionic type fermentation could be limited under a low-ORP environment because the propionic type fermentation was a facultative anaerobic process. Reducing propionate production was an important reason for the low propionate concentration (Wang et al., 2006). On the other hand, microbial Fe(III) reduction was the reason for the higher performance of the anaerobic sludge digestion with the addition of rusty scrap. Fe(III) oxides were insoluble at the neutral pH values, at which the microbial iron reduction could use insoluble Fe(III) oxides as electron acceptor. It is well recognized that most iron-reducing-bacteria can directly transfer the electron from organic oxidation to solid medium, such as iron oxide or electrode. The electron transfer from the bacteria to solid Fe(III) oxides and to the surface of electrode shared the same mechanisms, including direct contact, nanowire or pili, and electron shuttle. Fe(III) reduction is an energetically favorable process to oxidize organics compounds. In addition to oxidizing VFA, many complicated contaminants, such as aromatic hydrocarbons, halogenated solvents and chlorinated benzenes, could be decomposed by microbial

Concentration (mg/L)

3000

A n-B

P iso-V

iso-B n-V

2000

1000

0

Control group Clean scrap

Rusty scrap

Fig. 4. Composition of volatile fatty acid after anaerobic digestion. A (acetate), P (propionate), iso-B (isobutyrate), n-B (n-butyrate), iso-V (isovalerate) and n-V (n-valerate).

301

Fe(III) reduction (Häggblom et al., 2000). Several pure-culture isolates of iron reducing bacteria are known to oxidize short-chain and long-chain fatty acids. Therefore, Fe(III) oxides that covered the surface of rusty scrap could stimulate the Fe(III) reduction to enhance degradation of complex substrates. Accordingly, the degradation of VFAs with the rusty scrap was fastest (Fig. 4). Microbial Fe(III) reduction is more favorable in thermodynamics than methanogenesis (Zhang et al., 2009). Therefore methanogenesis may be inhibited by the Fe(III) reduction when the electron donor is limited such as low concentration of organics. However, the sufficient electron donors in the digested sludge supported different microbial respiratory processes to happen simultaneously. Coates et al. (2005) reported total VFAs decreased from 96 to 28 mM and methane production increased by 200% when adding Fe(III) compound in anaerobic treatment of hog manure. 3.4. Release of iron ion As shown in Fig. 5a, there was a little amount of iron in the control group after the digestion, likely a result from Fe-related coagulants used during the sludge dewatering in the WWTP. Almost all iron ions were ferrous due to the anaerobic environment. The iron concentration of the rusty scrap groups was higher than that of the clean scrap groups. It was an evidence for microbial Fe(III) reduction. After removing the remaining scrap in the end of digestion, the total iron content of the sludge in the rusty group was 1116.6 mg/L, also higher than that of the clean one (949.9 mg/L) (Fig. 5b). Comparatively, the iron in the liquid phase was very low than that in the sludge. The reason may attribute to the formation of iron precipitates. During the anaerobic digestion of sludge, phosphate was produced from decomposition of protein, which would react with iron ion to precipitate as phosphate iron. It could be verified from the changes in phosphate concentration (Fig. S3, Supporting Information). The phosphate for the control, clean and rusty scrap groups was 1.13, 0.90 and 0.47 g/L, respectively. The reduction of phosphate was 19.96% and 58.3% for the clean scrap and rusty scrap, respectively. It indicated that adding the rusty scrap is more effective to collect phosphate in the solid sludge. The capture of phosphate decreased its content in the effluent beneficial for reducing the cost in the following processes for treating the digested effluent. Such iron content (1000 mg/L) in the sludge is not a problem even if the sludge after the digestion is used as agricultural purposes because this content falls within the background value of iron in soil ranging from 5 to 50 g/L (Brady and Weil, 1996). From the consumption of clean scrap (0.584 g/L/Batch) and rusty scrap (0.766 g/L/Batch) (shown in Table S1, Supporting Information), the dosage of 10 g/L for clean and rusty scrap were expected to be reused for about 17 and 13 batches of the sludge digestion, or to be used for 228 and 376 d, respectively, with a sludge retention time of 22 d. Together with the methane production (Fig. 3), the increment of methane production depend on iron consumption calculated as follows: For clean scrap: V(mL CH4/g Fe) = DV(mL CH4/g VSS)  VSS(g/L)/Iron consumption(g/L/Batch) = (201.8–166.4)  56.74/0.584 = 3439 mL and for rusty scrap: V(mL CH4/g Fe) = (215.5–166.4)  56.74/0.766 = 3637 mL. It meant that one kg of clean scrap or rusty scrap could increase 3.4 or 3.6 m3 of methane, respectively. Assuming that the price of scrap iron (with rust) is $0.35/kg and the price of methane is $0.60/L, one kg of rusty iron scrap dosage could bring a profit of about $1.85, more than five folds of its cost. 3.5. Other parameters Other key parameters related to anaerobic digestion were detected after digestion (Table S1, Supporting Information). In an anaerobic system, a lower ORP was beneficial for acetate

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Y. Zhang et al. / Bioresource Technology 159 (2014) 297–304

50

1200

(b)

Concentration (mg/L)

Concentration (mg/L)

(a) 40

30

20

10

0

900

600

300

0

Control Clean group scrap

Rusty scrap

Control Clean group scrap

Rusty scrap

Fig. 5. Concentration of iron, after 22 days digestion. (a) Fe2+ in liquid phase, (b) Total iron in sludge. The bars designate the standard deviations of triplicate tests.

production and reducing the propionate accumulation. The ORP was decreased from 330 to 369 and 361 with the clean scrap and the rusty scrap, respectively. This result was corresponding to the VFA composition in which the lower OPR decrease the accumulation of propionate (Fig. 4). The pH value increased slightly with the addition of both the clean and rusty scraps, as a result of the enhanced VFAs decomposition and methanogenesis. After the anaerobic digestion, the concentration of SCOD, soluble protein and soluble polysaccharide were in the following order: control > clean scrap > rusty scrap. The results indicated that the rusty scrap was more efficient to decompose organics than the clean scrap. The reduction percentage of VSS, total protein and total polysaccharide with the dosage of rusty scrap was also highest, which was in agreement with the above results. Especially, the percentage of sludge reduction (VSS reduction) with adding the rusty scrap was 7.1% higher than that with the clean scrap. Iron also significantly affected the component of biogas. Generally, the improvement of hydrolysis–acidification may accelerate the anaerobic digestion to increase biogas yield as well as net methane production rather than to change the content of biogas. However, the methane content increased from 57.73% to 59.06% and to 62.96% by the clean scrap and the rusty scrap, respectively, which was probably attributed to improving the activity of methanogenesis. It was in agreement with the report that the abundant of methanogens increased from 64.9% to 70.3% after adding iron in an anaerobic reactor (Liu et al., 2011). In the anaerobic fermentation ammonia was produced by biological degradation of nitrogenous substrates, mostly in the form of sludge protein in this study, which was hydrolyzed to amino acids and further degraded to ammonia. After anaerobic digestion, the concentration of ammonia was 3.64 g/L for the control group, while 3.93 and 4.07 g/L for the clean scrap and the rusty scrap, respectively in line with the findings of protein decomposition (Fig. S3, Supporting Information). Many researchers found that the methanogenic activity was inhibited under high ammonia concentration (Westerholm et al., 2009). In this study, ammonia was increased in the group with adding iron, but no more inhibition of methanogenesis was observed which meant that the resistance of methanogens to ammonia was raised.

fragments (Fig. 6). Representative bands were selected from the DGGE gel for sequencing in order to identify the species presented in the anaerobic digester and the results are shown in Table 3. By

3.6. Microorganism analysis Bacterial community structure after the anaerobic digestion was studied by the DGGE analysis of PCR amplified 16S rRNA gene

Fig. 6. DGGE profiles of bacterial 16S rRNA genes from the sludge after 22 d of anaerobic digestion. The gels with band were collected from the DGGE gel and labeled as bands 1–14. The sequencing results of each band are shown in Table 3.

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Y. Zhang et al. / Bioresource Technology 159 (2014) 297–304 Table 3 Sequence analysis of DGGE bands. Band

Closest match

Identity (%)

Class

Origin

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Methylocella silvestris (NR074237) Uncultured bacterium (JQ726674) Clostridium sp. (JQ670700) Sphingopyxis witflariensis (NR028010) Novosphingobium indicum (NR044277) Clostridium orbiscindens DSM 6740 (NR029356) Acetobacter tropicalis (NR036881) Tsukamurella spumae (NR042800) Clostridium populeti (NR 026103) Roseomonas lacus (NR_042318) Caloramator proteoclasticus (NR026265) Syntrophobacter fumaroxidans (NR027598) Sporomusa silvacetica (NR 026378) Geobacter bemidjiensis Bem (NR042769)

97 98 94 99 99 99 95 99 93 98 95 94 98 97

Alphaproteobacteria Alphaproteobacteria Clostridia Alphaproteobacteria Alphaproteobacteria Clostridia Alphaproteobacteria Actinobacteridae Clostridia Alphaproteobacteria Clostridia Deltaproteobacteria Alphaproteobacteria Deltaproteobacteria

Acidic forest soil Denitrifying sludge Marine hot springs Activated sludge Deep-sea environment No recorded Anaerobic digester Activated sludge Anaerobic digester Freshwater lake sediment Mesophilic granular methanogenic sludge Mesophilic anaerobic sludge bed reactor Forest soil Subsurface sediments

comparing the bands number, it seemed that relatively high bacterial diversity was found in the digester with the clean scrap (containing 10 bands) and rusty scrap (containing 12 bands), as compared to the control group (containing 8 bands). Among all bands, the main bacteria found in the digester belonged to Alphaproteobacteria and Clostridia (comprising 50% and 29% of the total band number, respectively). Band 1 had the highest similarity with Methylocella silvestris, a facultative methanotroph that could attenuate methane emissions. This microorganism was only found in the control group, which indicated that addition of iron could remove this kind of bacteria to avoid the consumption of methane biologically (Dunfield et al., 2003). Some detected microorganisms are capable of degrading complex organic matters to form organic acid, i.e., bands 2 (Uncultured bacterium), 4 (Sphingopyxis witflariensis), 5 (Novosphingobium indicum), 6 (Clostridium orbiscindens), 9 (Clostridium populeti) and 11 (Caloramator proteoclasticus). C. populeti (band 9) was an anaerobic cellulolytic microorganism and C. proteoclasticus (band 11) had strong ability to degrade proteins and amino acids (Tarlera and Stams, 1999). These two microorganisms have been found in the reactor with clean scrap or rusty scrap indicating that iron can enhance the ability of cellulose and protein hydrolysis. Hydrogen production bacteria and acetobacteria may play important roles in anaerobic fermentation since hydrogen and acetate can be directly utilized by methanogenesis (Zeikus et al., 1975). Band 3 was closest to Clostridium sp. (with identity of 94%) belonging to H2 producing bacteria, which had been found in the three reactors. Syntrophobacter fumaroxidans (band 12) was enriched in the digester with the rusty scrap which was capable of producing both hydrogen and formate by propionate oxidation. This was consistent with the results of VFA component after digestion that the residual propionate in the reactor added rusty scrap was lowest. Bands 7, 10 and 13 showed high sequence similarity to acetobacteria, belonging to Acetobacter tropicalis (band 7), Roseomonas lacus and Sporomusa silvacetica, respectively. A. tropicalis had been found in the three reactors but the other two microorganisms only generated in the reactors with the clean scrap or rusty scrap, indicating that the iron enhanced the diversity of acetobacteria in the digesters so as to increase acetate production. Two deltaproteobacteria (bands 12 and 14) were only observed in the digester with rusty scrap and did not appear in other two digesters. Bands 14 showed high sequence similarity to Geobacter bemidjiensis Bem which had the ability to reduce Fe(III) and oxidize acetate and other multi-carbon organic substrates to carbon dioxide (Nevin et al., 2005). Iron-reducing bacteria was only found in the digester with the rusty scrap, which further proved that rusty scrap could enrich iron-reducing bacteria to enhance degradation of complex substrates, which contributed higher sludge reduction.

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Enhanced high-solids anaerobic digestion of waste activated sludge by the addition of scrap iron.

Anaerobic digestion of waste activated sludge usually requires pretreatment procedure to improve the bioavailability of sludge, which involves conside...
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