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Inhibition effect of sodium concentrations on the anaerobic digestion performance of Sargassum sp. Yi Zhang, Lianhua Li, Xiaoying Kong, Feng Zhen, Zhongming Wang, Yongming Sun, pengyu Dong, and Pengmei Lv Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 24 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017
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Inhibition effect of sodium concentrations on the anaerobic digestion performance
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of Sargassum sp.
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Yi Zhanga,b, Lianhua Lia,b,d, Xiaoying Konga,c,d, Feng Zhen a,c,d, Zhongming Wanga,c,d*, Yongming Suna,c,d,
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Pengyu Dong a,b, Pengmei Lva,c,d
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a
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b
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c
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Guangzhou 510640, China.
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China. University of Chinese Academy of Sciences, Beijing 100049, China.
Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development,
d
CAS Key Laboratory of Renewable Energy, Guangzhou 510640, China;
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*
Corresponding authors
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Tel: +86-20-87067783; Fax: +86-20-87057737
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E-mail address:
[email protected] (ZM Wang);
[email protected](XY Kong)
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Abstract: The effect of variable sodium concentrations on the anaerobic digestion performance of
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Sargassum sp. was investigated and the variation in bacterial and archaeal communities was analyzed by
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high-throughput gene sequencing. Results showed that the maximum methane yield of 290.41 ± 34.21 mL
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CH4 g-1 VS was obtained at a sodium concentration of 4.42 g L-1, increased by 38.1% compared to the
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control group. Meanwhile, higher volatile fatty acids production was observed at the sodium concentration
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of 4.42 g L-1. The inhibitory concentration value of 10%, 50% and 90% were occurred at the sodium
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concentration of 6.3 g L-1, 11.3 g L-1 and 18.7 g L-1, respectively. The hydrolytic, acidogenic, acetogenic
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bacteria and the hydrogenotrophic methanogens could normally metabolize at sodium concentrations of
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2.46-24.08 g L-1, whereas the acetoclastic methanogens were severely inhibited at sodium concentrations
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greater than 20.15 g L-1. The microbial community structure showed that Paludibacter and Fibrobacter
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played a key role in the step of acidogenesis/acetogenesis at sodium concentration of 4.42 g L-1, while
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Defluviitalea and Levilinea were the major acidogenic/acetogenic bacteria at sodium concentration of 20.15
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g L-1. Methanosaeta and Methanosarcina were predominated methanogens at sodium concentration of 4.42
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g L-1.
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Table of Contents Graphic -1
Specific methane yields (mLCH4 gVS )
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Methane yields
300 250 200 150 100 50 0
0.49
2.46
4.42 8.35 12.28 16.22 20.15 -1 Sodium concentrations (g L )
24.08
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Keywords: Anaerobic digestion, sodium concentration, Sargassum sp., inhibition concentration, microbial
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community
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1. Introduction
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Seaweed is highly attractive energy crop as feedstock for anaerobic digestion with the advantages of
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non-requirement for land or fertilizers, rapid growth rates, high biomass yields, high content of easily
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hydrolysable sugars, and low lignin content. 1, 2 Sargassum sp., brown seaweed, is widely distributed in the
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South China Sea and the East China Sea with 130 taxonomically accepted species and has been commonly
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used as seafood, heath products for its antioxidant production, and adsorbent for heavy metal sorption in
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the environment; it can also be used for bioenergy production.
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for methane production, and the biochemical methane potential (BMP) of Sargassum sp. can reach to 380
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mL CH4 g-1 VS (volatile solids) by anaerobic digestion. 5
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Sargassum sp. is a suitable raw material
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However, the high mineral content of 179-554 g kg-1 TS (total solids) in seaweed has a detrimental
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effect on anaerobic digestion performance. 6 Among these mineral salts, sodium chloride is the primary part
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with the content of 51-384 g kg-1 TS and has been proved to be the major inhibitory factor for anaerobic
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digestion in high content.
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significant role in the enzymatic mechanism of adenosine triphosphate (ATP) synthesis or nicotinamide
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adenine dinucleotide (NADH) oxidation.
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obtained for microbial growth and anaerobic digestion performance. 10 However, different inhibitory levels
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of sodium concentration varying from 3.5 g L-1 to 53 g L-1 have been reported due to the difference in
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material and anaerobic digestion condition. Higher sodium concentration could induce microbial cells to
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dehydrate or even death due to increased osmotic pressure, and the toxicity of sodium concentration was
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related to inoculum resources and its associated environment. 8 11 Sodium concentration ranging from 3.5 g
7, 8
Sodium is an essential metal element for microbial growth due to its
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Beneficial sodium concentration of 0.046-0.115 g L-1 was
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L-1 to 5.5 g L-1 caused a moderate inhibition on the performance of anaerobic digestion.
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reported that a sodium concentration of 10 g L-1 caused 50% inhibition on the acetoclastic methanogens
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activity. Some microorganisms could tolerate and survive in condtions of high sodium concentration by
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acclimation. Chen et al
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22.8 g L-1 after the inoculum acclimated. The reason might be osmotic adaptation, acclimated
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microorganisms could maintain the normal structure of cells and ensure the proper function of intracellular
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enzymes.
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cultivation were the family Fusobacteriaceae and the genus Methanosaeta respectively, which can tolerate
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the concentration of sodium up 19.66 g L-1. Although anaerobic digestion of seaweeds has been
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investigated on a number of occasions, many studies have focused on the tolerance of methanogens to
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highly saline conditions, and paid little attention to the underlying microbial ecology in anaerobic digestion
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of Sargassum sp. at different sodium concentrations.
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Miura et al
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Rinzema et al 12
reported that the tolerance of methanogens clearly increased from 12.7 g L-1 to
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reported that the predominant bacteria and archaea in marine sediments after
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Anaerobic digestion comprises three steps, hydrolysis/acidogenesis, acetogenesis, and methanogenesis,
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which are mediated by various microbial populations of different physiological and biochemical
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characteristics. It is important to understand the microbial behavior involved in methane production under
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diverse saline conditions. Therefore, this study aimed to evaluate the amplitude of the inhibition effect of
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sodium ion on anaerobic digestion over a wide range of concentrations. The influence of different sodium
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concentrations on the anaerobic digestion performances and the shifts of the bacterial and archaeal
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community structure were investigated during the anaerobic digestion of Sargassum sp.
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2. Materials and methods
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2.1. Raw material
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Sargassum sp. was obtained from the Institute of Oceanology, Chinese Academy of Sciences, Qingdao,
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Shandong province, China. The dried material was washed to remove debris and salt, air dried, and then
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milled into 1-mm particles using a blender. The content of TS, VS, ash, element sulfur (S), carbon (C),
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nitrogen (N) and the ratio of carbon to nitrogen (C/N) were 86.95%, 67.55%, 19.40%, 0.54%, 19.40%, 1.38%
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and 21.47, respectively.
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The inocula, sieved through a 1-mm mesh to remove large particles, were collected from mesophilic
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(35 °C) continuous stirred tank reactor (CSTR) by treating seaweed as feedstocks with a working volume
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of 60 L for 90 days, and the organic loading rate (OLR) and hydraulic retention time (HRT) were
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3 g VS L-1 and 15 days. The TS, VS, salinity, pH value, sulfide, ammonia nitrogen (NH3-N) content and
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the sodium concentration in the inocula were 1.45%, 0.85%, 4.54 g L-1, 8.10, 1.53 mg L-1, 641 mg L-1 and
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0.49 g L-1, respectively.
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2.2. Experimental setup
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Anaerobic digestion tests were performed in glass bottles with a working volume of 1200 mL at 35 °C.
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Inocula (1000 g) were added to each bottle followed by the addition of 20 g dried samples. The initial total
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sodium concentration was the sum of the background and exogenous sodium concentration in
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digesters. Initial total sodium concentration was adjusted with sodium chloride, and the corresponding
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concentrations were 2.46 g L-1 (N1), 4.42 g L-1 (N2), 8.35 g L-1 (N3), 12.28 g L-1 (N4), 16.22 g L-1 (N5),
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20.15 g L-1 (N6) and 24.08 g L-1 (N7). The substrate and inoculum without sodium chloride addition was
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used as control (N0). The blanks containing the same amount of inoculum and water were used as
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corrections. The digestion reactors were filled with N2 and then sealed with butyl rubber plugs. The period
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of anaerobic digestion tests was carried out for 60 days.
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2.3. Analytical methods The TS, VS, C, N, and S content were analyzed by previously described methods.
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The salinity was
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determined by a portable conductivity meter (CON200G1, Clean, Shanghai, China). The values of pH were
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measured by pH meter (pHS-3C, Rex, Shanghai, China). The concentration of NH3-N was determined by a
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commercially available kit according to the manufacturer’s protocol (DR2800, Hach, Loveland, USA). The
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contents of formate and volatile fatty acids (VFAs), mainly including acetate, propionate and butyrate, were
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analyzed by high-performance liquid chromatography (e2695, Waters, Boston, USA) using a refractive
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index detector and Shodex sugar SH-1011 column. The mobile phase was 0.005 M H2SO4 with a flow rate
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of 0.5 mL min-1 and the temperature of column was 50 °C. Biogas produced from the digesters was
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excluded when calculating the biogas yield of the substrates. The content of methane and carbon dioxide
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were measured by gas chromatography (GC2014, Shimadzu, Kyoto, Japan). And the temperature of
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thermal conductivity detector and column were respectively 120 °C and 50 °C. The sodium concentration
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of inocula was determined by inductively coupled plasma-optical emission spectroscopy (Optima 8000,
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PerkinElmer, Waltham, USA). The modified Gompertz model could statistically describe the growth of
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microorganisms by fitting the cumulative methane production in the following equation (Eq.1) where B is
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the cumulative methane production (mL) at digestion time t (d), P is ultimate methane production (mL),
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Rmax is maximum methane production rate (mL d-1), λ is lag time (day) and e is exponential (2.718). 16 ோೌೣ ×
B = P × exp ቄ−exp ቂ
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ሺߣ − ݐሻ + 1ቃቅ
(1)
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The effect of sodium concentration on the performance of anaerobic digestion was evaluated by calculating
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inhibitory concentration (IC) value. The Rmax value of each sodium concentration was fitted to the Hill
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model.
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IC10, IC50 and IC90 were the sodium concentration of 10%, 50% and 90% inhibition on the
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anaerobic digestion performance.
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2.4. Microbial diversity analysis
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2.4.1. Microbial DNA isolation
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For analyzing the bacterial and archaeal communities, the inoculum and samples (N0, N2, N4 and N6)
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were collected on days 1, 5, 15 and 45, which were stored at the temperature of -20 °C for further analysis.
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The sample DNA was extracted according to the instructions of the E.Z.N.ATM Mag-Bind Soil DNA Kit
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(OMEGA, D5625-01, USA). After purification, the concentration of the extracted genomic DNA was
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quantified by Qubit2.0 DNA Kit (Life, Q10212, USA).
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2.4.2. PCR amplification
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2.4.2.1. Bacteria PCR amplification
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The forward primer 341F (5′-CCTACGGGNGGCWGCAG-3′) and the reverse primer 805R
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(5′-GACTACHVGGGTATCTAATCC-3′) was used to amplify the V3-V4 regions of the bacterial 16S
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ribosomal RNA gene. Polymerase chain reaction (PCR) was performed on PCR equipment (BIO-RAD,
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T100TM Thermal Cycler, U.S.). In the first round of amplification, the reactions were performed in a 50-µL
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mixture containing 5 µL of 10× PCR Buffer, 0.5 µL of 10 mM dNTPs, 0.5 µL of forward and reverse
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primer (50 µM), 0.5 µL of 5 U µL-1 Platinum Taq, and 10 ng of template DNA. The steps of the PCR
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included a hot start at 94 °C for 3 min, followed by 5 cycles of denaturation at 94 °C for 30 s, annealing at
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45 °C for 20 s, elongation at 30 °C for 45 s, 20 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s,
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and extension at 72 °C for 5 min. In the second round of amplification, bridge PCR was conducted in the
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same PCR system, except that the template DNA concentration was 20 ng. The reaction was then amplified
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in the mixture at 95 °C for 30 s, by 5 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s, and extension
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at 72 °C for 5 min.
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2.4.2.2. Archaea PCR amplification
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Nested PCR, including three rounds, was performed in the archaea PCR amplification. In the first
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round amplification, the forward primer 340F (5′-CCCT AYGGGGYGCASCAG-3′) and reverse primer
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1000R (5′-GGCCATGCACYWCYT CTC-3′) was used to amplify the V3-V4 regions of the archaeal 16S
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ribosomal RNA gene.
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the bacterial DNA PCR. In the second round of amplification, the primers used were 349F
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(5′-GYGCASCAGKCGMGAAW-3′) and 806R (5′-GGACTACVSGGGT ATCTAAT-3′) in the V3-V4
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regions of the archaeal 16S ribosomal DNA gene, and there was no change in the reaction conditions. In the
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third round of amplification, the reaction conditions were the same as those in the second round
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amplification of the bacterial DNA PCR.
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2.4.3. Processing of pyrosequencing data
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The reaction conditions were the same as those in the first round amplification of
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The DNA samples were sequenced by paired-end sequencing on an Illumina MiSeq PE300 platform in
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the Shanghai Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). In order to ensure quality, any
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ambiguous and unmatched reads in the primer were removed. After filtering the unqualified sequences, the
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effective bacterial and archaeal sequences of all 17 samples summed up to 762276 and 844741, and the
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average length of each sequence was 417 bp and 379 bp, respectively. The non-amplified and
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non-continuous sequences were eliminated using UCHIME.
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database.
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taxonomic units (OTUs) by Usearch.
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concentrations, mainly including Chao, Shannon and coverage, were generated by using Mothur. 22 In order
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The sequences were matched by the Silva
The analysis of biological information was performed at 97% similarity level of operational 21
The diversities of microbial community at different sodium
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to obtain the information of species classification for each operational taxonomic unit (OTU), taxonomic
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classification at the phylum and genus level was performed using the Ribosome Database project (RDP). 23
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2.5. Statistical analysis
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One-way analysis of variance (ANOVA) was used to tested the significance of sodium concentration
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(p
