Bacterial bioaugmentation for improving methane and hydrogen production from microalgae
© Lü et al.; licensee BioMed Central Ltd. 2013
Received: 5 April 2013
Accepted: 26 June 2013
Published: 1 July 2013
The recalcitrant cell walls of microalgae may limit their digestibility for bioenergy production. Considering that cellulose contributes to the cell wall recalcitrance of the microalgae Chlorella vulgaris, this study investigated bioaugmentation with a cellulolytic and hydrogenogenic bacterium, Clostridium thermocellum, at different inoculum ratios as a possible method to improve CH4 and H2 production of microalgae.
Methane production was found to increase by 17?~?24% with the addition of C. thermocellum, as a result of enhanced cell disruption and excess hydrogen production. Furthermore, addition of C. thermocellum enhanced the bacterial diversity and quantities, leading to higher fermentation efficiency. A two-step process of addition of C. thermocellum first and methanogenic sludge subsequently could recover both hydrogen and methane, with a 9.4% increase in bioenergy yield, when compared with the one-step process of simultaneous addition of C. thermocellum and methanogenic sludge. The fluorescence peaks of excitation-emission matrix spectra associated with chlorophyll can serve as biomarkers for algal cell degradation.
Bioaugmentation with C. thermocellum improved the degradation of C. vulgaris biomass, producing higher levels of methane and hydrogen. The two-step process, with methanogenic inoculum added after the hydrogen production reached saturation, was found to be an energy-efficiency method for hydrogen and methane production.
Microalgae have enormous potential as a source for biofuel and bioenergy production due to their high photosynthetic efficiencies, high growth rates, and characteristics of not requiring external organic carbon supply. Anaerobic digestion of algal biomass to biogas containing methane or hydrogen is one of the most energy-efficient and environmentally beneficial technologies. The process is highly dependent on both substrate degradability as well as environmental conditions which regulate the microbial activity.
Anaerobic digestion could be carried out on microalgal residues after lipid extraction[3–6] or directly on freshly collected algae. With regard to the latter, the resistance of the microalgal cell wall could be one of the limiting factors for cell digestibility[7, 8]. The cell wall of some microalgal species such as Chlorella sp. and Scenedesmus sp. is known to contain recalcitrant cellulose, which could protect the microalgae against enzyme attack, thus restricting algal biodegradability[3, 10]. Lakaniemi et al. found that only approximately 50% of Chlorella vulgaris biomass was degraded during methanogenic fermentation. Various mechanical (high-pressure homogenization, bead beating), physical (ultrasonication), thermal, and chemical (acids, bases, and oxidizing agents) pretreatment methods have been investigated to improve the digestion efficiency[3, 8, 12–14]. However, although these pretreatment technologies could enhance methane production from algae with thick cell wall, the energy cost of pretreatment is high. For example, the amount of energy consumed in heating and pretreatment was found to be higher than or equal to the corresponding energy gain from increased methane production[3, 15, 16]. Besides, the use of thermochemical pretreatment may also lead to a possible formation of inhibitory substances (e.g. furfurals). Enzymatic hydrolysis is a well-known biological pretreatment process. Sander and Murthy found that cell walls of mixed algae are susceptible to degradation by cellulase and lipase. Ehimen et al. reported a pretreatment process of addition of a combined enzyme mixture and individual enzymes to the Rhizoclonium biomass prior to anaerobic digestion. The researchers observed that the enzymatic pretreatment led to greater methane conversions than the mechanical methods, and that the action of cellulase resulted in maximum methane yield, when compared with that of other enzymes. However, enzymes are usually only effective at the initial stage after addition and become inactive soon afterwards. Comparatively, living bacteria can continuously hydrolyze the materials through growth and proliferation. Nevertheless, appropriate bacterial species should be carefully selected to be effective for microalgae hydrolysis and be compatible with subsequent or synchronous anaerobic digestion.
Considering that cellulose contributes to the cell wall recalcitrance in the microalgae C. vulgaris, this study investigated bioaugmentation with a thermophilic, anaerobic, cellulolytic, and hydrogenogenic bacterium, Clostridium thermocellum, which is also available from cellulose-fed anaerobic digester, as a possible method to improve the degradation of C. vulgaris biomass to enhance the efficiency of methane and hydrogen production. To our best knowledge, the present study is the first report on improving C. vulgaris degradation by bioaugmentation using C. thermocellum.
Methane and hydrogen production
Calculated result using the modified gompertz equation for the cumulative methane production
Ultimate methane yieldP(ml/g-VS)
Maximum methane production rateR max (ml/g VS?·?d)
Hydrogen is a key intermediate during anaerobic digestion as well as a product synthesized by C. thermocellum. Hence, in the enrichment cultures of Series 1 with mixed inoculum and in the one-step treatment of Series 2, hydrogen was accumulated in the first few days and then was rapidly consumed by methanogens (Figure 1c). Hydrogen production increased with the increase in the inoculum ratio of C. thermocellum. Comparatively, in the two-step treatment of Series 2, the hydrogen produced in the first step with only C. thermocellum was further consumed and reached a maximum plateau value of 53.4 ml H2/g VS after 5 days (Figure 1d), equivalent to 0.167 mol/mol of the corresponding methane production in the second step.
Production of ethanol and volatile fatty acids (VFAs)
Microalgal degradation and bacterial growth monitored by fluorescent method
Electron microscopic observation of microalgal cell degradation
Automated ribosomal intergenic spacer analysis (ARISA)
Similarly, for methanogens in liquid phase, the H values of treatments with C. thermocellum were significantly higher than those of treatment without C. thermocellum. The H value of treatment with 1% C. thermocellum inoculum ratio was also the highest for methanogens in solid phase as in liquid phase, and showed a significant increase from Day 5 to Day 20. The H values of treatment with 0% bacterial inoculum ratio were almost stable for the duration. However, the H values of treatment with 5% and 10% inoculum ratios were close to those of treatment with 0% inoculum ratio during the first 12 days and then exhibited an obvious decrease.
The cells of Chlorella are surrounded by a recalcitrant cellulosic cell wall, which encloses a parietal and cup-shaped chloroplast with a pyrenoid[9, 22]. While stained with multiple fluorochromes, the C. vulgaris cell wall consisted of β-polysaccharides could be clearly observed (Additional file1: Figure S5 and Figure S6) [Note: cellulose belongs to β-polysaccharides]. If the microalgal biomass is not subjected to any cell disruption process, then the cell walls could be very resistant to hydrolysis, protecting the cells against the enzymes produced by the anaerobic consortium, and thus restricting cell biodegradability. C. thermocellum is an acetogenic, thermophilic, and anaerobic bacterium with a high rate of cellulose degradation and propensity to synthesize hydrogen. The present study found that C. thermocellum could utilize C. vulgaris as a substrate for growth (Figure 4). Furthermore, addition of C. thermocellum resulted in the production of higher levels of hydrogen (Figure 1), along with higher concentrations of ethanol, acetate, and butyrate (Figure 2). These results imply the contribution of C. thermocellum to algal cell degradation (Figure 5). The improved cell wall breakage resulted in the release of more organic matter, thus enhancing the diversity of bacteria in both suspension and granular phases, and the diversity of methanogens in the suspension phase (Figure 6). In addition, their quantities (as suggested by more SMP in Figure 4) were also ameliorated, favoring improvement in fermentation efficiency and process stability. Meanwhile, the hydrogen generated from C. thermocellum activity could promote the development of hydrogenotrophic methanogenesis, resulting in higher methane yield (Figure 1, Table 1) and an increase in the abundance of hydrogenotrophic methanogens, thus reducing the diversity of methanogens in granular phase (Figure 6). Therefore, anaerobic digestion of C. vulgaris biomass could be improved by the addition of C. thermocellum through enhanced cell disruption and excess hydrogen production.
The methane yield achieved from C. vulgaris degradation without bioaugmentation was 322 ml CH4/g VS, which is equivalent to 50% of the theoretical methane yield estimated by the carbohydrates, proteins, and lipid content of C. vulgaris and calculated according to Becker. CH4 yield from microalgae was bound up with chemical composition of microalgal biomass and process parameters such as the bioreactor type and the digestion temperature. Lakaniemi et al. reported 286 ml CH4/g VS from C. vulgaris at 37°C. Bruhn et al. reported 271 ml CH4/g VS from macerated Ulva lactuca at 52°C and found that a decrease of the digestion temperature from 52°C to 37°C lowered the final methane yield by 7%. Thus, the CH4 yield from C. vulgaris without bioaugmentation was comparable with previous results. With the addition of C. thermocellum, the methane production could be further increased by 17?~?24%.
Unlike the one-step process, hydrogen accumulated in the cultures inoculated only with C. thermocellum in the first stage of the two-step treatment was not consumed by methanogens. As a result, both the bioenergy gases, hydrogen and methane, could be recovered. In Series 2, the hydrogen and methane yield from two-step treatment was 53 and 321 ml/g VS, respectively, equivalent to 13.4 kJ/g VS of energy, which is 9.4% higher than the corresponding yield obtained from one-step treatment. When compared with the hydrogen yield reported in the literature, the yield obtained in the present study is of average magnitude. For example, Park et al. reported a hydrogen yield of 28 ml H2/g dry weight from the microalgae Laminaria japonica pretreated by ball milling and heat treatment at 120°C for 30 min, using anaerobic sewage sludge as an inoculum. Yang et al. achieved a hydrogen yield of 27.27 ml H2/g VS from lipid-extracted Scenedesmus biomass subjected to heat pretreatment at 95°C for 30 min. Carver et al. reported hydrogen yields of 82 and 114 ml H2/g VS from C. vulgaris using microalgae-associated bacteria and a thermophilic consortium at 60°C, respectively, while Lakaniemi et al. obtained a much lower hydrogen yield of 10.8 ml H2/g VS from the same algal biomass at 37°C.
It should be noted that bioaugmentation with C. thermocellum made the anaerobic digestion system complex. The activity of this acidogenic phase bacteria might be coupled with a probable inhibition of the methanogens and/or a slower rate of acid intermediate consumption by the methanogenic process. The longer lag time of C. thermocellum at a high inoculum ratio could probably be due to the need for the methanogens to alter their physiological state according to the new environment. In addition to higher production of hydrogen, the two-step treatment presented shortest lag time and a comparable level of methane production, providing an energy efficiency method worthy of consideration. The proportions of algal biomass and methanogenic inoculum may also be an important parameter for methane production. The low proportion of granular sludge resulted in a much lower methane yield, longer lag time, and lower maximum methane production rate. This might be due to low methanogenic activity or the number of methanogens, which could result in the accumulation of VFAs. Therefore, proper methanogenic inoculum ratio relative to the amount of algal biomass and C. thermocellum should be considered.
Bioaugmentation with C. thermocellum improved the degradation of C. vulgaris biomass, producing higher levels of methane and hydrogen. However, the increases in methane yield were in the same order of magnitude with different inoculum ratios of C. thermocellum. The two-step process, with methanogenic inoculum added after the hydrogen production reached saturation, was found to be an energy-efficiency method for hydrogen and methane production. The fluorescence peaks of EEM spectra associated with chlorophyll can serve as biomarkers for algal cell degradation.
Substrate and inoculum
The microalgae C. vulgaris (strain ESP-6, Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan) were grown photoautotrophically in Liquid Bold’s Basal Medium (BBM) with 0.1 vvm CO2 (5% CO2 and 95% 0.45-μm filtered air) sparging. Light was provided by 8000–10000 lux LED lights (WD-TM-D35W, Widen Photodiode Technology Co., China). After 7 days of incubation, the microalgal biomass was harvested and concentrated by centrifugation at 3600?×?g for 15 min. The solid concentrate was subjected to anaerobic digestion. The concentrated algal biomass contained 12.9% (on wet weight basis) of total solid (TS), 93.5% (on dry weight basis) of volatile solid (VS), and 58, 11, and 14% (on dry weight basis) of proteins, lipids, and sugars, respectively.
C. thermocellum (strain DSM2360) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. Fresh cultures were maintained by routinely transferring 5% (v/v) inoculum into fresh medium containing 5 g/L of absorbent cotton. Other compounds contained in the fresh medium included (per liter of distilled water): KH2PO4, 0.50 g; K2HPO4?·?3H2O, 1.00 g; urea, 2.00 g; MgCl2?·?6H2O, 0.50 g; CaCl2?·?2H2O, 0.05 g; FeSO4?·?7H2O, 1.25 mg; morpholinopropane sulfonic acid, 10.00 g; resazurin, 1.00 mg; yeast extract, 6.00 g; glucose, 5.00 g; cysteine-HCl?·?H2O, 1.00 g. C. thermocellum was freshly harvested after 4 days of incubation when no more hydrogen production was detected.
Granular sludge, cultivated in a laboratory-scale (3.5 L) anaerobic sequenced batch reactor (ASBR), was added as the methanogenic inoculum. The ASBR was operated at 55°C, and glucose and acetate (80%:20%, calculated as COD) were utilized as the feedstock at an organic loading rate of 2 g COD/(L-day). The methanogenic sludge was taken after being acclimated for more than 50 days and rinsed with anaerobic preheated (55°C) buffer solution to remove the residual carbon. The buffer solution was the same as that used in the subsequent batch experiments. The TS of the granular sludge was 11.1% (w/w) and the VS was 77.8% (w/w) of the TS.
Series 1: One-step methane production with different inoculum ratios of C. thermocellum
Series 2: Two-step co-production of hydrogen and methane
In the two-step experiment, 3 g VS/L of algal biomass was first incubated with 5% (v/v) of C. thermocellum containing culture medium at 55°C for 7 days in 500-ml buffer solution, as mentioned earlier, for hydrogen production. Subsequently, 2 g VS/L of methanogenic sludge was added to produce methane. As a reference, a one-step experiment with the same amount of algal biomass, granular sludge, and 5% (v/v) of C. thermocellum containing culture medium was set up. Furthermore, a control with granular sludge and 5% (v/v) of C. thermocellum containing culture medium, similar to that used in Series 1, was also included in this series. Two different inoculum to substrate ratios (methanogenic sludge to microalgal biomass: 3:2 in series 1 and 2:3 in series 2) were introduced to investigate the influence of inoculum to substrate ratio. All the experiments were carried out in duplicate and the results were expressed as means.
Analysis of gaseous and liquid samples
M(t) is the cumulative methane production (ml/g VS added) at time t (days), P is the highest methane yield (ml/g VS), Rmax is the maximum methane production rate (ml/g VS/day), and λ is the lag phase (days). Lag phase refers to the initial adaptive phase, during which methane production remains relatively constant prior to rapid growth.
The liquid samples were centrifuged at 16,000?×?g for 10 min. Subsequently, the supernatants were collected and analyzed for pH, volatile fatty acids (VFAs), alcohols, dissolved organic carbon (DOC), total inorganic carbon (TIC), dissolved nitrogen (DN), and three-dimensional fluorescent intensity. The pH was tested with a pHS-2 F Digital Meter. The DOC, TIC, and DN were analyzed on a TOC-VCPH Analyzer (Shimadzu, Japan). The concentrations of VFAs (including acetic, propionic, isobutyric, butyric, and isovaleric acids) and alcohols in the supernatant were determined using an Agilent 6890 N gas chromatography (GC) system equipped with a flame ionization detector (FID). The fluorescence excitation-emission matrixes (EEM) were recorded for the supernatant in a 10-mm quartz cuvette in a Varian Cary Eclipse fluorometer (Agilent, Santa Clara, CA, USA). The emission was scanned from 220 to 750 nm at 2-nm intervals and 10-nm bandwidth, while the excitation was produced with a Xenon flash lamp in 10-nm bandwidth at 10-nm intervals from 200 to 700 nm. The EEM signals were processed and subjected to parallel factor analysis (PARAFAC), as described in the study by Lu et al..
Transmission electron microscopy observation of the microalgal cell
The cells of C. vulgaris were observed using transmission electron microscopy (TEM). Samples were prepared according to the procedure developed by Yamamoto et al., and examined with a transmission electron microscope (JEM-1230, JEOL, Japan).
Multiple fluorochrome staining of the microalgal cell and spectral microscopy observation
The cells of C. vulgaris were stained successively by FITC for proteins, Con A for α-polysaccharides and calcofour white for β-polysaccharides according to Chen et al.. The samples were then examined with a Leica DMI 4000B spectral microscope imaging system.
Both the liquid samples and granules corresponding to different sampling dates were used for DNA extraction. The total DNA was extracted from the pellets using PowerSoil DNA isolation kit (MoBio Laboratories Inc., CA), according to the manufacturer’s protocol. The fingerprint technique of Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to monitor the microbial dynamics. The extracted DNA was amplified using primers 1389 F and 71R for archaea, and primers ITSF and ITSReub for bacteria, respectively. Polymerase chain reaction (PCR) and ARISA of the PCR product were carried out according to the method described by Qu et al.. Shannon diversity index was used to analyze the ARISA profiles and H value was calculated using the software PAlaeontological STatistics (PAST) version 2.17b, according to the procedure proposed by Hammer et al..
Volatile fatty acid
Parallel factor analysis
Soluble microbial products
Transmission electron microscopy
Automated ribosomal intergenic spacer analysis
Polymerase chain reaction
Dissolved organic carbon
Total inorganic carbon
This research was partially sponsored by the National Basic Research Program of China (973 Program, No. 2012CB719801), the National Natural Science Foundation of China (No. 51178327; 21177096), the Innovation Program of Shanghai Municipal Education Commission (13ZZ030), the Shanghai Pujiang Program (No. 11PJ1409200), the Fundamental Research Funds for the Central Universities (No. 0400219195), and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRY11008).
- Hughes AD, Kelly MS, Black KD, Stanley MS: Biogas from Macroalgae: is it time to revisit the idea? Biotechnol Biofuels 2012, 5: 86. 10.1186/1754-6834-5-86View ArticleGoogle Scholar
- Vandenbroucke M, Largeau C: Kerogen origin, evolution and structure. Org Geochem 2007, 38: 719-833. 10.1016/j.orggeochem.2007.01.001View ArticleGoogle Scholar
- Sialve B, Bernet N, Bernard O: Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol Adv 2009, 27: 409-416.View ArticleGoogle Scholar
- Ehimen EA, Sun ZF, Carrington CG, Birch EJ, Eaton-Rye JJ: Anaerobic digestion of microalgae residues resulting from the biodiesel production process. Appl Energ 2011, 88: 3454-3463. 10.1016/j.apenergy.2010.10.020View ArticleGoogle Scholar
- Ehimen EA, Connaughton S, Sun Z, Carrington GC: Energy recovery from lipid extracted, transesterified and glycerol codigested microalgae biomass. GCB Bioenergy 2009, 1: 371-381. 10.1111/j.1757-1707.2009.01029.xView ArticleGoogle Scholar
- Yang ZM, Guo RB, Xu XH, Fan XL, Luo SJ: Hydrogen and methane production from lipid-extracted microalgal biomass residues. Int J Hydrogen Energ 2011, 36: 3465-3470. 10.1016/j.ijhydene.2010.12.018View ArticleGoogle Scholar
- Afi L, Metzger P, Largeau C, Connan J, Berkaloff C, Rousseau B: Bacterial degradation of green microalgae: incubation of Chlorella emersonii and Chlorella vulgaris with Pseudomonas oleovorans and Flavobacterium aquatile . Org Geochem 1996, 25: 117-130. 10.1016/S0146-6380(96)00113-1View ArticleGoogle Scholar
- Chen PH, Oswald WJ: Thermochemical treatment for algal fermentation. Environ Int 1998, 24: 889-897. 10.1016/S0160-4120(98)00080-4View ArticleGoogle Scholar
- Okuda K: Structure and phylogeny of cell coverings. J Plant Res 2002, 115: 283-288. 10.1007/s10265-002-0034-xView ArticleGoogle Scholar
- Sánchez Hernández EP, Travieso Córdoba L: Anaerobic digestion of Chlorella vulgaris for energy production. Resour, Conserv Recy 1993, 9: 127-132. 10.1016/0921-3449(93)90037-GView ArticleGoogle Scholar
- Lakaniemi AM, Hulatt CJ, Thomas DN, Tuovinen OH, Puhakka JA: Biogenic hydrogen and methane production from Chlorella vulgaris and Dunaliella tertiolecta biomass. Biotechnol Biofuels 2011., 4:Google Scholar
- Jeon B-H, Choi J-A, Kim H-C, Hwang J-H, Abou-Shanab R, Dempsey B, Regan J, Kim J: Ultrasonic disintegration of microalgal biomass and consequent improvement of bioaccessibility/bioavailability in microbial fermentation. Biotechnol Biofuels 2013, 6: 37. 10.1186/1754-6834-6-37View ArticleGoogle Scholar
- Ehimen EA, Holm-Nielsen JB, Poulsen M, Boelsmand JE: Influence of different pre-treatment routes on the anaerobic digestion of a filamentous algae. Renew Energ 2013, 50: 476-480.View ArticleGoogle Scholar
- McMillan JR, Watson IA, Ali M, Jaafar W: Evaluation and comparison of algal cell disruption methods: microwave, waterbath, blender, ultrasonic and laser treatment. Appl Energ 2013, 103: 128-134.View ArticleGoogle Scholar
- Yen HW, Brune DE: Anaerobic co-digestion of algal sludge and waste paper to produce methane. Bioresour Technol 2007, 98: 130-134. 10.1016/j.biortech.2005.11.010View ArticleGoogle Scholar
- Lakaniemi AM, Tuovinen OH, Puhakka JA: Anaerobic conversion of microalgal biomass to sustainable energy carriers – a review. Bioresour Technol 2013, 135: 222-231.View ArticleGoogle Scholar
- Taherzadeh MJ, Karimi K: Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci 2008, 9: 1621-1651. 10.3390/ijms9091621View ArticleGoogle Scholar
- Sander K, Murthy GS: Enzymatic degradation of microalgal cell walls. Reno, Nevada, USA: ASABE annual international meeting; 2009.Google Scholar
- Lü F, Bize A, Guillot A, Monnet V, Madigou C, Chapleur O, Mazéas L, He PJ, Bouchez T: Metaproteomics of cellulose methanization under thermophilic conditions reveals a surprisingly high proteolytic activity. ISME J 2013. 10.1038/ismej.2013.120Google Scholar
- Simis SGH, Huot Y, Babin M, Seppala J, Metsamaa L: Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria. Photosynth Res 2012, 112: 13-30. 10.1007/s11120-012-9729-6View ArticleGoogle Scholar
- Erokhina LG, Shatilovich AV, Kaminskaya OP, Gilichinskii DA: The absorption and fluorescence spectra of the cyanobacterial phycobionts of cryptoendolithic lichens in the high-polar regions of Antarctica. Microbiology 2002, 71: 601-607. 10.1023/A:1020523206526View ArticleGoogle Scholar
- Phukan MM, Chutia RS, Konwar BK, Kataki R: Microalgae Chlorella as a potential bio-energy feedstock. Appl Energ 2011, 88: 3307-3312. 10.1016/j.apenergy.2010.11.026View ArticleGoogle Scholar
- Becker EW: Micro-algae as a source of protein. Biotechnol Adv 2007, 25: 207-210.View ArticleGoogle Scholar
- Bruhn A, Dahl J, Nielsen HB, Nikolaisen L, Rasmussen MB, Markager S, Olesen B, Arias C, Jensen PD: Bioenergy potential of Ulva lactuca : Biomass yield, methane production and combustion. Bioresour Technol 2011, 102: 2595-2604. 10.1016/j.biortech.2010.10.010View ArticleGoogle Scholar
- Park JI, Lee J, Sim SJ, Lee JH: Production of hydrogen from marine macro-algae biomass using anaerobic sewage sludge microflora. Biotechnol Bioprocess Eng 2009, 14: 307-315. 10.1007/s12257-008-0241-yView ArticleGoogle Scholar
- Yang ZM, Guo RB, Xu XH, Fan XL, Luo SJ: Fermentative hydrogen production from lipid-extracted microalgal biomass residues. Appl Energ 2011, 88: 3468-3472. 10.1016/j.apenergy.2010.09.009View ArticleGoogle Scholar
- Carver SM, Hulatt CJ, Thomas DN, Tuovinen OH: Thermophilic, anaerobic co-digestion of microalgal biomass and cellulose for H 2 production. Biodegradation 2011, 22: 805-814. 10.1007/s10532-010-9419-zView ArticleGoogle Scholar
- Cheong DY, Hansen CL: Feasibility of hydrogen production in thermophilic mixed fermentation by natural anaerobes. Bioresour Technol 2007, 98: 2229-2239. 10.1016/j.biortech.2006.09.039View ArticleGoogle Scholar
- He PJ, Mao B, Shen CM, Shao LM, Lee DJ, Chang JS: Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia for biodiesel production. Bioresour Technol 2013, 129: 177-181.View ArticleGoogle Scholar
- Chen CL, Macarie H, Ramirez I, Olmos A, Ong SL, Monroy O, Liu WT: Microbial community structure in a thermophilic anaerobic hybrid reactor degrading terephthalate. Microbiology 2004, 150: 3429-3440. 10.1099/mic.0.27193-0View ArticleGoogle Scholar
- Lü F, Hao L, Zhu M, Shao L, He P: Initiating methanogenesis of vegetable waste at low inoculum-to-substrate ratio: Importance of spatial separation. Bioresour Technol 2012, 105: 169-173.View ArticleGoogle Scholar
- Lu F, Chang CH, Lee DJ, He PJ, Shao LM, Su A: Dissolved organic matter with multi-peak fluorophores in landfill leachate. Chemosphere 2009, 74: 575-582. 10.1016/j.chemosphere.2008.09.060View ArticleGoogle Scholar
- Yamamoto M, Kurihara I, Kawano S: Late type of daughter cell wall synthesis in one of the Chlorellaceae , P arachlorella kessleri (Chlorophyta, Trebouxiophyceae). Planta 2005, 221: 766-775. 10.1007/s00425-005-1486-8View ArticleGoogle Scholar
- Chen MY, Lee DJ, Tay JH, Show KY: Staining of extracellular polymeric substances and cells in bioaggregates. Appl Microbiol Biotechnol 2007, 75: 467-474. 10.1007/s00253-006-0816-5View ArticleGoogle Scholar
- Qu X, Mazeas L, Vavilin VA, Epissard J, Lemunier M, Mouchel JM, He PJ, Bouchez T: Combined monitoring of changes in delta δ 13 CH 4 and archaeal community structure during mesophilic methanization of municipal solid waste. FEMS Microbiol Ecol 2009, 68: 236-245. 10.1111/j.1574-6941.2009.00661.xView ArticleGoogle Scholar
- Hammer Ø, Harper DAT, Ryan PD: PAST: Paleontological statistics software package for education and data analysis. Palaeontol Electron 2001, 4: 1-9.Google Scholar
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