The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 12, 2018
RESEARCH ARTICLE

Assessing Methanogenic Archaeal Community in Full Scale Anaerobic Sludge Digester Systems in Dubai, United Arab Emirates



Munawwar A. Khan1, *, Poojabahen G. Patel2, Arpitha G. Ganesh1, Naushad Rais2, Sultan M. Faheem2, Shams T. Khan3
1 Department of Life and Environmental Sciences, College of Natural and Health Sciences, Zayed University, P.O.Box: 19282, Dubai, UAE
2 School of Life Sciences, Manipal University, Dubai International Academic City, P.O.Box 345050, Dubai, UAE
3 Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, 2002002, UP. India

Abstract

Introduction:

Anaerobic digestion for methane production comprises of an exceptionally diverse microbial consortium, a profound understanding about which is still constrained. In this study, the methanogenic archaeal communities in three full-scale anaerobic digesters of a Municipal Wastewater Treatment Plant were analyzed by Fluorescence in situ hybridization and quantitative real-time Polymerase Chain Reaction (qPCR) technique.

Methods & Materials:

Fluorescence in situ hybridization (FISH) was performed to detect and quantify the methanogenic Archaea in the sludge samples whereas qPCR was carried out to support the FISH analysis. Multiple probes targeting domain archaea, different orders and families of Archaea were used for the studies.

Results and Discussion:

In general, the aceticlastic organisms (Methanosarcinaceae & Methanosaetaceae) were more abundant than the hydrogenotrophic organisms (Methanobacteriales, Methanomicrobiales, Methanobacteriaceae & Methanococcales). Both FISH and qPCR indicated that family Methanosaetaceae was the most abundant suggesting that aceticlastic methanogenesis is probably the dominant methane production pathway in these digesters.

Conclusion:

Future work involving high-throughput sequencing methods and correlating archaeal communities with the main operational parameters of anaerobic digesters will help to obtain a better understanding of the dynamics of the methanogenic archaeal community in wastewater treatment plants in United Arab Emirates (UAE) which in turn would lead to improved performance of anaerobic sludge digesters.

Keywords: Anaerobic digestion, Archaea, Fluorescence in situ hybridization, Methanogens, Quantitative Polymerase Chain Reaction, Sequencing methods, Hydrogenotrophic.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
First Page: 123
Last Page: 134
Publisher Id: TOMICROJ-12-123
DOI: 10.2174/1874285801812010123

Article History:

Received Date: 10/02/2018
Revision Received Date: 18/04/2018
Acceptance Date: 19/04/2018
Electronic publication date: 30/04/2018
Collection year: 2018

Article Metrics:

CrossRef Citations:
0

Total Statistics:

Full-Text HTML Views: 805
Abstract HTML Views: 408
PDF Downloads: 317
ePub Downloads: 388
Total Views/Downloads: 1918

Unique Statistics:

Full-Text HTML Views: 356
Abstract HTML Views: 169
PDF Downloads: 159
ePub Downloads: 166
Total Views/Downloads: 850
Geographical View

© 2018 Khan et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this authors at the Department of Life and Environmental Sciences, College of Natural and Health Sciences, Zayed University, P.O.Box: 19282, Dubai, UAE; Tel: +97144021825; E-mail: Munawwar.Khan@zu.ac.ae




1. INTRODUCTION

Anaerobic digestion plays an immensely significant role in the treatment of highly concentrated organic wastes. This process is beneficial in two simple yet important aspects: Firstly, the process is anaerobic thereby eliminating the need for aeration and rendering it as a cost-effective strategy. Secondly, in addition to diminishing the pollution load, anaerobic digestion converts complex organics into methane (biogas) [1Narihiro T, Sekiguchi Y. Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Curr Opin Biotechnol 2007; 18(3): 273-8.[http://dx.doi.org/10.1016/j.copbio.2007.04.003] [PMID: 17462878] ]. It is accomplished via the interaction between a diverse consortium of microorganisms, in four distinct stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis [2Hufnagel D, Chang S, Zhang J, Lu R. Analysis of methanogen communities in anaerobic membrane bioreactors and conventional anaerobic digester systems. JAAB 2015; 4(2): 1-9.]. The harmonized activity of the microbial consortia i.e., hydrolyzing/acidifying bacteria (acidogens) and methane generating Archaea (methanogens) is crucial from the perspective of process efficiency [3Kim J, Kim W, Lee C. Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters. J Environ Sci (China) 2013; 25(11): 2272-80.[http://dx.doi.org/10.1016/S1001-0742(12)60299-X] [PMID: 24552056] ]. Anaerobic digestion has been comprehensively explained in terms of process engineering but our knowledge about the microbial communities is still scarce which could be a major reason resulting in process failure in full scale anaerobic digestion facilities [4Connaughton S, Collins G, O’Flaherty V. Development of microbial community structure and actvity in a high-rate anaerobic bioreactor at 18°C. Water Res 2006; 40(5): 1009-17.[http://dx.doi.org/10.1016/j.watres.2005.12.026] [PMID: 16488461] ].

The final stage of Anaerobic digestion i.e., methanogenesis is performed by obligate anaerobic Archaea and account for over 90% of CH4 generated on Earth [5Costa KC, Leigh JA. Metabolic versatility in methanogens. Curr Opin Biotechnol 2014; 29: 70-5.[http://dx.doi.org/10.1016/j.copbio.2014.02.012] [PMID: 24662145] ]. Methanogenesis is accomplished via three different pathways namely a) hydrogenotrophic (H2/CO2 used for CH4 synthesis) b) acetoclastic (involves transfer of methyl group from acetate to tetrahydrosarcinapterin and finally to coenzyme M (CoM)) c) methylotrophic methanogenesis (production of methyl CoM by utilizing methyl groups from methanol and methylamines (mono-, di-, and trimethylamine). These three pathways converge at a common final step in which methyl CoM is converted into methane by an enzymatic complex ubiquitous in all methanogens i.e., methyl coenzyme M reductase [6Borrel G, O’Toole PW, Harris HM, Peyret P, Brugère JF, Gribaldo S. Phylogenomic data support a seventh order of Methylotrophic methanogens and provide insights into the evolution of Methanogenesis. Genome Biol Evol 2013; 5(10): 1769-80.[http://dx.doi.org/10.1093/gbe/evt128] [PMID: 23985970] ]. These methanogens categorized into five main orders within the Archaeal domain i.e., Methanobacteriales, Methanopyrales, Methanomicrobiales, Methanosarcinales and Methanococcales are able to use only a minimal number of thermodynamically suitable substrates for methane production [7Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 2005; 89(6): 670-9.[http://dx.doi.org/10.1002/bit.20347] [PMID: 15696537] , 8Yu Y, Kim J, Hwang S. Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnol Bioeng 2006; 93(3): 424-33.[http://dx.doi.org/10.1002/bit.20724] [PMID: 16196054] ]. Methanogenic archaea have proven to be extremely challenging in terms of cultivation in the laboratory due to their requirement of very peculiar environmental conditions [9Sekiguchi Y. Yet-to-be cultured microorganisms relevant to methane fermentation processes. Microbes Environ 2006; 21(1): 1-15.[http://dx.doi.org/10.1264/jsme2.21.1] , 10Sakai S, Imachi H, Sekiguchi Y, et al. Cultivation of methanogens under low-hydrogen conditions by using the coculture method. Appl Environ Microbiol 2009; 75(14): 4892-6.[http://dx.doi.org/10.1128/AEM.02835-08] [PMID: 19465530] ].

There have been quite a few culture-independent techniques employed for the investigation of the methanogenic consortia like: a) Fluorescence In situ Hybridization (FISH) [11Díaz EE, Stams AJ, Amils R, Sanz JL. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater. Appl Environ Microbiol 2006; 72(7): 4942-9.[http://dx.doi.org/10.1128/AEM.02985-05] [PMID: 16820491] -13Zahedi S, Solera R, Micolucci F, Cavinato C, Bolzonella D. Changes in microbial community during hydrogen and methane production in two-stage thermophilic anaerobic co-digestion process from biowaste. Waste Manag 2016; 49: 40-6.[http://dx.doi.org/10.1016/j.wasman.2016.01.016] [PMID: 26810032] ] b) Terminal Restriction Fragment Length Polymorphism (T-RFLP) [14Akuzawa M, Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y. Distinctive responses of metabolically active microbiota to acidification in a thermophilic anaerobic digester. Microb Ecol 2011; 61(3): 595-605.[http://dx.doi.org/10.1007/s00248-010-9788-1] [PMID: 21240482] ] c) community fingerprinting by Denaturing Gradient Gel Electrophoresis (DGGE) [15Watanabe T, Asakawa S, Nakamura A, Nagaoka K, Kimura M. DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. FEMS Microbiol Lett 2004; 232(2): 153-63.[http://dx.doi.org/10.1016/S0378-1097(04)00045-X] [PMID: 15033234] ] d) real-time quantitative PCR (qPCR) [16Sawayama S, Tsukahara K, Yagishita T. Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester. Bioresour Technol 2006; 97(1): 69-76.[http://dx.doi.org/10.1016/j.biortech.2005.02.011] [PMID: 16154504] -18Koo T, Shin SG, Lee J, et al. Identifying methanogen community structures and their correlations with performance parameters in four full-scale anaerobic sludge digesters. Bioresour Technol 2017; 228: 368-73.[http://dx.doi.org/10.1016/j.biortech.2016.12.118] [PMID: 28087103] ] e) Single Strand Conformation Polymorphism (SSCP) [19Delbès C, Moletta R, Godon J. Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem. FEMS Microbiol Ecol 2001; 35(1): 19-26.[http://dx.doi.org/10.1016/S0168-6496(00)00107-0] [PMID: 11248386] ]. As suggested in the past studies conducted in several countries, anaerobic sludge digester (ASD) treatment systems in the UAE are susceptible to several operational problems, potentially reducing their overall efficiency. Excessive competition and growth of problematic microbial community members are widely recognized as the main cause of reduced efficiency of ASD systems. On the other hand, the fluctuations of operational parameters of ASD systems might also affect stability and balance of microbial communities. Therefore, more detailed and fundamental understanding of microbial populations is required for effective long-term control of ASD conditions. Molecular techniques have shown promise for gaining a better understanding of microbial community members in ASD treatment systems and quantitative data provided by molecular techniques such, as FISH [11Díaz EE, Stams AJ, Amils R, Sanz JL. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater. Appl Environ Microbiol 2006; 72(7): 4942-9.[http://dx.doi.org/10.1128/AEM.02985-05] [PMID: 16820491] -13Zahedi S, Solera R, Micolucci F, Cavinato C, Bolzonella D. Changes in microbial community during hydrogen and methane production in two-stage thermophilic anaerobic co-digestion process from biowaste. Waste Manag 2016; 49: 40-6.[http://dx.doi.org/10.1016/j.wasman.2016.01.016] [PMID: 26810032] ] and Q-PCR [16Sawayama S, Tsukahara K, Yagishita T. Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester. Bioresour Technol 2006; 97(1): 69-76.[http://dx.doi.org/10.1016/j.biortech.2005.02.011] [PMID: 16154504] -18Koo T, Shin SG, Lee J, et al. Identifying methanogen community structures and their correlations with performance parameters in four full-scale anaerobic sludge digesters. Bioresour Technol 2017; 228: 368-73.[http://dx.doi.org/10.1016/j.biortech.2016.12.118] [PMID: 28087103] ] have been successfully utilized in several studies to validate engineered models and to optimize biogas production and for this reason these two molecular approaches have been used in the current study.

There have been many studies dealing with the characterization of bacterial communities in activated sludge systems [20Juretschko S, Loy A, Lehner A, Wagner M. The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach. Syst Appl Microbiol 2002; 25(1): 84-99.[http://dx.doi.org/10.1078/0723-2020-00093] [PMID: 12086193] -22Bouchez T, Jacob P, d’Hugues P, Durand A. Acidophilic microbial communities catalyzing sludge bioleaching monitored by fluorescent in situ hybridization. Antonie van Leeuwenhoek 2006; 89(3-4): 435-42.[http://dx.doi.org/10.1007/s10482-005-9052-8] [PMID: 16622787] ] but not much is known about the archaeal communities present in sludge digester operating in the UAE. As per our knowledge, there hasn’t been any study published so far regarding the community structure of methanogenic Archaea inhabiting the anaerobic digesters of full-scale Wastewater Treatment Plant (WWTP) in the UAE. Therefore, this study attempts to provide insights into the methanogenic consortia and analyze the different methanogenic groups present.

2. MATERIALS AND METHODS

2.1. Anaerobic Digester Sampling

The waste sludge samples were collected from the Jebel Ali Wastewater Treatment Plant (JASTP), Dubai, UAE on a monthly basis for a period of five months. JASTP is one of the two-wastewater treatment facilities in the emirate of Dubai with the capacity of treating about 375,000 cubic meters of mostly domestic wastewater on daily basis. JASTP utilize activated sludge process coupled with advanced level nitrogen removal stages. The treated effluent from biological stages undergoes further treatment by sand filtration and UV based disinfection. The large quantities of sludge produced during various stages of wastewater treatment process pass through full-scale sludge digesters where anaerobic digestion of sludge take place during which sludge is stabilized and part of solids are converted to methane gas. The tertiary treated effluent is reused in irrigation, and treated sludge is converted to manure for use as soil conditioner and fertilizer.

There are five full-scale continuous stirred type anaerobic digesters in total but for this project the samples were collected from anaerobic digesters 1, 3 & 5. These three digesters were chosen for this study as per recommendation from the treatment plant officials based on construction timing of the digesters. The digester 3 is the oldest and digester 5 (the newest) and digester 1 was constructed sometime between to the 3 and 5. The operating physicochemical conditions data of the digesters was provided by Jebel Ali Sewage Treatment Plant laboratory, Dubai, UAE Table 1. The samples were directly taken from anaerobic digesters (1, 3 & 5) into an autoclaved plastic bottle with 1 liter volume. The sample bottles were placed in an icebox and brought to the laboratory. The collected samples were stored at 4oC until DNA extraction and fixation of biomass for qPCR and FISH analysis, respectively.

Table 1
Operational physicochemical conditions of the anaerobic sludge digesters.


2.2. Fluorescence in situ Hybridization

The samples were fixed within 24 hours of sampling with paraformaldehyde; incubated for 3-4 hours and then washed thrice with 1X PBS (Phosphate Buffer Saline) and stored at -20oC. FISH is a method basically used to quantify the presence and relative abundance of targeted microbes within the sample. It includes three major steps firstly, the hybridization of the cells with the probes, followed by washing off the excessive probe finally the visualization of the hybridized cells under the fluorescence microscope as per the standard procedure described earlier (26-28).

For the hybridization, teflon printed slides with wells of 8mm diameter from Vermicon VIT identification technology (Munich, Germany) were used. The slides were cleaned with acid alcohol (1% HCl in 70% EtOH) and placed in 0.01% Poly-L-Lysine solution for adhering to the fixed cells.

1 – 3μl of the sample was applied to a coated slide and air-dried. These cells were then dehydrated using a graded ethanol series (50%, 80%, 96% for 3 minutes each) and air dried for 5 minutes. Subsequently, the probe mix was prepared by adding 9μl of the hybridization solution and 1μl of the respective working probe. The remainder of the hybridization solution was loaded onto the tray of the polypropylene hybridization chamber and placed in the oven at 46oC. The probe mix was applied to the respective well on the slide. The slide was then placed in the hybridization chamber with the tray containing hybridization solution. The chamber was tightly sealed and incubated at 46oC for 3 – 4 hours. The different probes targeted for 16S rRNA used for this study are listed in Table 2. All the oligonucleotide probes were labeled with cyanine dye Cy3.

The washing solutions were prepared that accompanied the hybridization solutions used earlier. The washing solution was pre-warmed at 48oC. The slide was rinsed with 1ml of pre-warmed washing solution. Then slides were kept immersed in the washing solution and incubated at 48oC for 10-15 mins. Post incubation, the slide was rinsed with ddH2O at room temperature, air dried for a few minutes and visualized under the fluorescence microscope. Prior to visualization, 3μl of 10 mg/l DAPI (4',6-diamidino-2-phenylindole) was added to each well. The slide was then visualized under the fluorescence microscope system Olympus BX51 Series which was connected to a DP72 digital camera. Images were captured using the DP2-BSW Software. Images taken from the microscope were subjected to MetaMorph software (Version 7.10.0.119 to count the number of cells present in each image. For each sample minimum of at least 10 images of same DAPI and CY3 images were taken and then considered the average of all to calculate the total number of cells versus the % of hybridized cells.

Table 2
Oligonucleotide probes used in this study.


2.3. Quantitative Polymerase Chain Reaction (qPCR)

Genomic DNA was extracted from the sludge samples using Power Soil DNA Extraction Kit (MO BIO Laboratories). The samples were vortexed and then subsequently centrifuged to get rid of maximum water content. DNA was then extracted from 0.25g of the obtained pellet according to the manufacturer’s protocol. The solution containing the extracted DNA was stored at -20oC to -80oC. The A260/A280 and A260/A230 ratios were utilized to determine the purity and concentration of the extracted DNA using a Nano-Drop 2000c spectrophotometer (Thermo Fisher Scientific, USA). Quantitative PCR (qPCR) was employed to relatively quantify the presence of the respective methanogenic archaeal members relative to an endogenous control using the Comparative CT method (∆∆CT). The endogenous control used in this study was archaeal bacterial DNA extracted from the digester sludge and amplified using archaeal domain specific primer and probe sets described in Table 3. The qPCR amplifications were performed in 20μl reactions. Each reaction contained 1μl of 20X stock assay (5μl of 10μM forward/reverse primer, 5μl of respective 5 μM TaqMan probe, 85μl of PCR-grade pure water), 10μl of TaqMan Master Mix, 8μl of PCR grade pure water and 1μl of extracted template DNA. Two-step amplification of the target DNA, combining the annealing and the extension steps, was performed applying the following conditions an initial 10-min incubation at 95oC for denaturation & Taq DNA polymerase activation followed by 40 cycles of denaturation at 95oC for 15s; and simultaneous annealing and extension at 60oC for 1min. Methanogens, the key players responsible for methanogenesis, were investigated at the domain, 4 different order and 2 family levels covering majority of the methanogenic archaea present in anaerobic digester systems to obtain a comprehensive insight into their community structures. Real-Time PCR was performed using StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA) with seven primer and probe sets listed in the Table (3).

Table 3
Primer and probe sets used for qPCR.


2.4. Result & Discussion

2.4.1. Profiling of Archaeal Community Composition by FISH Technique

The JASTP consists of five full-scale anaerobic digesters. For this study, sludge samples were obtained from AD 1, 3 & 5. The three digesters in sequence of their age, newest to oldest, are AD5, AD1 & AD3. Under optimal hybridization conditions, methanogenic Archaea were specifically visualized and detected using the corresponding probes labeled with Cy3. Fig. (1) displays the epifluorescence micrograph showing in situ hybridization with probe ARC915. In each respective sludge sample, the active Archaeal populations were observed with respect to 4’, 6-diamidino-2-phenylindole (DAPI) [26Manz W, Amann R, Ludwig W, Wagner M, Schleifer K. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: Problems and solutions. Syst Appl Microbiol 1992; 15(4): 593-600.[http://dx.doi.org/10.1016/S0723-2020(11)80121-9] -28Daims H, Stoecker K, Wagner M. Fluorescence in situ hybridization for the detection of prokaryotes.Advanced methods in molecular microbial ecology 2005; 213-39.].

Fig. (1)
Epifluorescence micrograph showing in situ hybridization with probe ARC915 – Cy3; Bar = 10μm.


Yellow color oval represents Coccus, blue color for clumps of filaments green color represents rod-shaped cell.

Samples were hybridized with the universal Archaeal probe ARC915 as well as with order-, family- and genus-specific 16S rRNA oligonucleotide probes. A large majority of the Archaeal community gave positive hybridization signals with the ARC915 probe. From the photomicrographs it was observed that 49.73%, 47.72%, 54.13% of cells belonged to the archaea for the anaerobic digester AD1, AD3, and AD5 respectively when compared with the total number of cells. This probe identified various archaeal morphologies like cocci, rod-shaped cells and clumps of long filaments as shown in Fig. (2).

Fig. (2)
The proportions contributed by various methanogenic Archaea versus total Archaeal population. (A) Anaerobic Digester-1 (AD1) (B) Anaerobic Digester-3 (AD3) (C) Anaerobic Digester-5 (AD5)


For samples taken from anaerobic digester 1, 3 and 5, probes targeting the subgroups of class Methanomicrobia were utilized: MG1200b, MS1414, MS821, MX1361 and MX825. Two probes, i.e., MX1361 and MX825 were employed to target the family Methanosaetaceae to compare the result of hybridized cells targeted by these two probes. Upon comparison, it was observed that overall, MX825 hybridized with a greater percentage of the target Methanosaetaceae cells than probe MX1361. This observation is supported by the finding of the study conducted by Raskin et al. 1996 [23Raskin L, Stromley JM, Rittmann BE, Stahl DA. Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 1994; 60(4): 1232-40.[PMID: 7517128] ], wherein it is stated that probe MX825 should be used only for the characterization of microbial communities under mesophilic conditions. According to Crocetti et al. 2006 [24Crocetti G, Murto M, Björnsson L. An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH). J Microbiol Methods 2006; 65(1): 194-201.[http://dx.doi.org/10.1016/j.mimet.2005.07.007] [PMID: 16126291] ], the accurate specificity of probe MX1361 cannot be appreciated due to the fact that GenBank houses comparatively scarce sequence data for MX1361 target position.

In both AD1 and AD3, the family Methanosaetaceae (MX825) and in AD5, the family Methanosarcinaceae was the predominant Archaeal methanogens contributing 17%, 17% and 16% respectively Fig. (2) among the total Archaeal population. The existence of the Methanosaetaceae family members has been extensively reported in anaerobic bioreactors. In addition, the presence of the species Methanosaeta has been linked to a more stable and consistent bioreactor operation [29Chan OC, Liu WT, Fang HH. Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene. Water Sci Technol 2001; 43(1): 77-82.[PMID: 11379115] -31Hulshoff Pol LW, de Castro Lopes SI, Lettinga G, Lens PN. Anaerobic sludge granulation. Water Res 2004; 38(6): 1376-89.[http://dx.doi.org/10.1016/j.watres.2003.12.002] [PMID: 15016515] ]. In all the samples, the MX825 and MX1361 probes identified rod-shaped cells either present individually or forming a chain of rods. Within the granule, these rods appear to play an instrumental role in contributing to a network thereby facilitating the association of other bacteria to this network [32Bialek K, Kim J, Lee C, Collins G, Mahony T, O’Flaherty V. Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors. Water Res 2011; 45(3): 1298-308.[http://dx.doi.org/10.1016/j.watres.2010.10.010] [PMID: 21093011] ]. Filamentous like aggregations of Methanosaeta cells were also observed. Garrity and Holt, in 2001 [33Garrity G, Holt J. Phylum AII. Euryarchaeota.Bergey’s Manual of Systematic Bacteriology 2nd ed. 2nd ed.2011; 211-94.] have reported the formation of filaments comprising of 10-300 cells by Methanosaeta species. According to Zitomer [34Zitomer D, Maki J, Venkiteshwaran K, Bocher B. Relating Anaerobic Digestion Microbial Community and Process Function.Microbiol Insights2016; 37], the filamentous morphology of Methanosaeta aid in the process of granule formation wherein the filaments serve as binders to help hold the granule together however granulation process is not relevant as all digesters were continuously stirred type reactors. In some of the samples, quite a few randomly distributed cocci were also noticed. It is difficult to confirm whether these cells belong to the targeted family since only rod shaped and filamentous morphology has been observed previously for the family Methanosaetaceae members.

On the other hand, probes MS1414 and MS821 targeting family Methanosarcinaceae and genus Methanosarcina respectively identified numerous coccoidal cells. These cells assumed various patterns like pairs, chain and irregular clumps. According to Demirel and Scherer [35Demirel B, Scherer P. The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review. Rev Environ Sci Biotechnol 2008; 7(2): 173-90.[http://dx.doi.org/10.1007/s11157-008-9131-1] ], existence of these cells in clumps aid in their protection from harmful chemical agents. Formation of irregular cell clumps by members of the genus Methanosarcina increases their tolerance to high concentrations of toxic ionic agents and pH fluctuations [36Conklin A, Stensel HD, Ferguson J. Growth kinetics and competition between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion. Water Environ Res 2006; 78(5): 486-96.[http://dx.doi.org/10.2175/106143006X95393] [PMID: 16752610] ]. The second populous Archaeal group belonged to genus Methanosarcina (MS821) in AD1 and AD3 (15% each) whereas in AD5 it belonged to order Methanomicrobiales (15%) from the total archaeal population.

Previous culture-independent studies have shown the families Methanosarcinaceae and Methanosaetaceae (in the order Methanosarcinales) and the order Methanomicrobiales to be very common in anaerobic digestion [37Steinberg LM, Regan JM. Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge. Appl Environ Microbiol 2008; 74(21): 6663-71.[http://dx.doi.org/10.1128/AEM.00553-08] [PMID: 18776026] ]. All members of the order Methanomicrobiales produce methane by CO2 reduction with H2 whereas the order Methanosarcinales use acetate as the sole substrate to carry out methanogenesis [38Timmis K. Handbook of hydrocarbon and lipid microbiology 1st ed. 1st ed.2010.[http://dx.doi.org/10.1007/978-3-540-77587-4] ]. Moreover, acetate is a more common substrate than CO2 methanogenesis since wastewater usually contain high levels of organic acids [39Microbewiki.kenyon.edu. 2017. The Role of Methanogens in Waste Water Treatment - MicrobeWiki.[online]Available https://microbewiki.kenyon.edu/index.php/The_Role_of_Methanogens_in_Waste_Water_Treatment]. This could be a plausible explanation for their prevalence in all the three anaerobic digesters.

The order Methanomicrobiales exhibited a nearly similar hybridization percentage i.e., 12% and 11% in AD1 and AD3 respectively. Only in digester 5, the percentage shot up to 15% suggesting a greater abundance in this digester. The members of this order targeted by oligonucleotide probe MG1200b displayed diverse morphologies including small cocci and rods and many filamentous shaped Archaeal cells. This morphological observation is in line with the literature discussed by Seckbach [40Seckbach J. Journey to Diverse Microbial Worlds Dordrecht 2013.], wherein this order was reported to include rods, cocci, irregular cocci, ring- or corpuscle-shaped organisms, plates and spirals.

The predominance of class Methanomicrobia is associated with the abundant methanogens in the sample, in which numerous Methanosarcinale members were detected. Oligonucleotide probes MB1175 and MB310 were used to target the Methanobacteria subgroups at the order and family level respectively. MB1175 succeeded in hybridizing with 10%, 14% and 13% of the target cells whereas MB310 hybridized with 12%, 10% and 8% of the target cells belonging to family Methanobacteriaceae in AD1, AD3 and AD5 out of the total archaeal population. These numbers make it quite evident that the methanogenic population contributed by the class Methanobacteria was comparatively lower than class Methanomicrobia. Members of the order Methanobacteriales are generally hydrogenotrophic, which utilize a narrow range of substrates for methanogenesis, which include H2, CO2 and formate [38Timmis K. Handbook of hydrocarbon and lipid microbiology 1st ed. 1st ed.2010.[http://dx.doi.org/10.1007/978-3-540-77587-4] ]. The cells identified by the above-mentioned probes displayed two main morphologies: a) cocci shaped cells existing alone or in pairs (diplococci) b) rod-shaped cells forming chains & clumps of filaments. Seckbach and Boone [40Seckbach J. Journey to Diverse Microbial Worlds Dordrecht 2013., 41Boone D, Castenholz R. The Archaea and the deeply branching and phototrophic bacteria.Bergey’s Manual of Systematic Bacteriology 2nd ed. 2nd ed.2001; 1-721.[http://dx.doi.org/10.1007/978-0-387-21609-6] ] described the order Methanobacteriales to mainly consist of short rods. However, we have observed the presence of few coccoidal cells existing either alone or in pairs.

The MC1109 probe targeting Methanococcales detected 10% each for AD1 and AD3; AD5 showed 7% of the target irregular cocci cells among the total archaeal cells. This suggests that very few cells of this order were present indicating that Methanococcale type-methanogens were not frequent in mesophilic digesters. Members of this order use H2 or formate to reduce CO2 for methanogenesis [38Timmis K. Handbook of hydrocarbon and lipid microbiology 1st ed. 1st ed.2010.[http://dx.doi.org/10.1007/978-3-540-77587-4] ].

Overall upon close observation, it is quite evident that the Methanosaetacea, Methanosarcinacea and Methanomicrobia subgroups occupied the top positions in terms of dominance in nearly all the three anaerobic digesters. The presence of aceticlastic methanogens outweighs the hydrogenotrophic methanogens in nearly all the three anaerobic digesters suggesting that the aceticlastic pathway is probably the favorable route for methanogenesis in these digesters. Moreover, aceticlastic methanogens have been reported to be responsible for approximately 70% of the methane produced in anaerobic digesters [42Jetten M. Methanogenesis from acetate: a comparison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina spp. FEMS Microbiol Lett 1992; 88(3-4): 181-97.[http://dx.doi.org/10.1111/j.1574-6968.1992.tb04987.x] , 43Ahring BK. Methanogenesis in thermophilic biogas reactors. Antonie van Leeuwenhoek 1995; 67(1): 91-102.[http://dx.doi.org/10.1007/BF00872197] [PMID: 7741531] ].

2.4.2. Analysis of Methanogenic Community by Real-Time Quantitative PCR

Quantitative real time polymerase chain reaction (qPCR) has gained popularity in recent years as a method for determining microbial populations in methanogenic systems [17Williams J, Williams H, Dinsdale R, Guwy A, Esteves S. Monitoring methanogenic population dynamics in a full-scale anaerobic digester to facilitate operational management. Bioresour Technol 2013; 140: 234-42.[http://dx.doi.org/10.1016/j.biortech.2013.04.089] [PMID: 23707910] , 18Koo T, Shin SG, Lee J, et al. Identifying methanogen community structures and their correlations with performance parameters in four full-scale anaerobic sludge digesters. Bioresour Technol 2017; 228: 368-73.[http://dx.doi.org/10.1016/j.biortech.2016.12.118] [PMID: 28087103] , 44Bartell RD, Matson E, Mueller-Spitz S, Kleinheinz TG. Investigation of methanosarcinales and methanomicrobiales presence within a dry Anaerobic digester. J Microbiol Res (Rosemead Calif) 2015; 5(3): 101-8.]. In this study, quantitative real time PCR was applied for the relative quantification of predominant methanogenic Archaea at the level of domain, order and family by the Comparative CT method. qPCR was performed for sludge samples of 3 months i.e., November, December and January. For this comparative CT study, the Archaeal 16S rRNA was chosen as the endogenous control.

To use qPCR to quantify rRNA, the computer software connected to the instrument constructs the amplification plot for detecting increase in fluorescence emission. The fluorescence emission generated and detected as a threshold cycle (CT) value. The CT value is defined as the actual PCR cycle when the intensity of the fluorescent signal increases to above the background threshold and is proportional to the initial copy number of the target gene. For use of the same primer and probe sets for the target group, the higher the CT value, the lower the initial rRNA concentration is likely to be [7Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 2005; 89(6): 670-9.[http://dx.doi.org/10.1002/bit.20347] [PMID: 15696537] ]. For all the samples analyzed, the amplification plot and CT values obtained for the targeted orders and families are depicted in the Fig. (3).

Fig. (3)
CT values of probes targeted at mehanogenic community in sludge digesters over sampling period.


In the month of November, AD1 and AD3 had exactly the same trend of Archaeal population i.e., Methanosaetaceae (MST) followed by Methanobacteriales (MBL), Methanomicrobiales (MMB), Methanosarcinales (MSL) and Methanosarcinaceae (MSC). However, in AD5, order Methanomicrobiales and Methanosarcinales were detected more than Methanobacteriales.

In the month of December, AD3 and AD5 consisted of methanogenic Archaea that populated the digesters in the same sequence i.e., Methanosaetaceae followed by Methanomicrobiales, Methanosarcinales, Methanobacteriales and Methanosarcinaceae. AD1 also displayed a similar trend with slight variations: a) MMB was slightly higher in abundance than MST b) MBL was the least abundant. In December and January, the archaeal communities in a similar range populated AD3 and AD5. AD1 showed a slight difference with Methanobacteriales dominating over Methanosarcinales. This indicates that overall the family Methanosaetaceae was the predominant taxon whereas the family Methanosarcinaceae was the least abundant throughout the months of November, December and January. Comparing the two-targeted orders, Methanosarcinales prevailed over Methanomicrobiales although the latter wasn’t far behind in terms of CT value. The MCL primer and probe set failed to detect the members of the order Methanococcales in this trial. This could be either because the members of this order were below the detection limit of this technique or probably due to their growth requirement of high salt conditions (0.3-9.4% (w/v) NaCl) that are not normally found in anaerobic reactors [41Boone D, Castenholz R. The Archaea and the deeply branching and phototrophic bacteria.Bergey’s Manual of Systematic Bacteriology 2nd ed. 2nd ed.2001; 1-721.[http://dx.doi.org/10.1007/978-0-387-21609-6] ]. The results obtained using the two molecular techniques i.e., FISH and qPCR were compared. According to qPCR, at the order level, Methanomicrobiales seems to be predominant in the months of December and January in all the three anaerobic digesters whereas in November AD1 and AD3 were dominated by order Methanobacteriales. However, FISH results indicate that Methanomicrobiales dominated AD1 and AD5 during the three months whereas order Methanobacteriales prevailed in AD3. The increased abundance of Methanomicrobiales might be hypothetically correlated with the presence of more diverse bacterial communities [32Bialek K, Kim J, Lee C, Collins G, Mahony T, O’Flaherty V. Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors. Water Res 2011; 45(3): 1298-308.[http://dx.doi.org/10.1016/j.watres.2010.10.010] [PMID: 21093011] ].

Both FISH and qPCR data suggested that at the family level, barring AD1 in December, Methanosaetaceae was the most abundant throughout the three-month period in nearly all the digesters while Methanosarcinaceae was the least prevalent. Most of the Methanosaetaceae family members survive best at pH between 6.6 – 7.5 and temperature between 30oC – 40oC. The operational parameters of the anaerobic digesters under study seem to suit their growth requirements and favor their proliferation causing them to be prevailing in these digesters. The findings of the study conducted by Karakashev and colleagues [45Karakashev D, Batstone DJ, Angelidaki I. Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors. Appl Environ Microbiol 2005; 71(1): 331-8.[http://dx.doi.org/10.1128/AEM.71.1.331-338.2005] [PMID: 15640206] ] indicated that the methanogenic diversity was broader in plants operating at mesophilic ranges than the thermophilic plants. The research study conducted by Liu et.al. in 2002 [46Liu Y, Xu H, Show K, Tay J. Anaerobic granulation technology for wastewater treatment. World J Microbiol Biotechnol 2002; 18(2): 99-113.[http://dx.doi.org/10.1023/A:1014459006210] ], proposed that abundant Methanosaeta spp. improves granulation and consequently leads to more stable reactor performance. However, granulation process is not relevant to the observed predominance of Methanosaetaceae members as studied anaerobic digesters were continuously stirred tank type reactors. Overall, the results obtained by the two molecular techniques seem to agree at the family level but not much at the order level. Previous studies suggest that qPCR results are considered to be more efficient and superior to FISH technique [47Ramdhani N, Kumari S, Bux F. Comparison of quantitative PCR and fluorescent in situ hybridization techniques to quantify nitrobacter from wastewater samples. IWA World Water Congress Secretariat 2014.Lisbon, Portugal. 2014.].

CONCLUSION

The aim of this study was to evaluate the methanogenic archaeal community structure of three full-scale anaerobic digester systems of a municipal wastewater treatment plant in Dubai, UAE. The fluorescence in situ hybridization and quantitative real-time PCR was used for in-situ identification and quantification of the methanogenic archaeal community in the anaerobic digesters. The archaeal populations were targeted at the domain, order and family level. All the three anaerobic digesters showed almost similar type of Archaeal community. The results of this study suggest the dominance of the family Methanosaetaceae in all the digesters. These results suggest that the methane in these digesters is produced through aceticlastic methanogenesis. Further work to obtain in-depth understanding of the relationships between archaeal communities, their functional gene under the influence of key operational physico-chemical parameters using high-throughput sequencing methods will help to provide better understanding of the dynamics of the methanogenic archaeal community in wastewater treatment plants in UAE and this knowledge will help to improve the performance of anaerobic digesters.

LIST OF ABBREVIATIONS

WWTP = Wastewater Treatment Plant
JASTP = Jebel Ali Sewage Treatment Plant
FISH = Fluorescence in situ hybridization
qPCR = real-time quantitative Polymerase Chain Reaction
AD = Anaerobic Digester
CT = Threshold cycle
T-RFLP = Terminal Restriction Fragment Length Polymorphism
DGGE = Denaturing Gradient Gel Electrophoresis
SSCP = Single Strand Conformation Polymorphism

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

This research was supported by Zayed University Research Cluster Project: Activity code: R16092 awarded to the first author (PI of the cluster research project). We thank Engineer Rashed Mohammad Karkain from Jebel Ali Wastewater Treatment Plant for providing samples of anaerobic digesters used in this study.

REFERENCES

[1] Narihiro T, Sekiguchi Y. Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Curr Opin Biotechnol 2007; 18(3): 273-8.[http://dx.doi.org/10.1016/j.copbio.2007.04.003] [PMID: 17462878]
[2] Hufnagel D, Chang S, Zhang J, Lu R. Analysis of methanogen communities in anaerobic membrane bioreactors and conventional anaerobic digester systems. JAAB 2015; 4(2): 1-9.
[3] Kim J, Kim W, Lee C. Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters. J Environ Sci (China) 2013; 25(11): 2272-80.[http://dx.doi.org/10.1016/S1001-0742(12)60299-X] [PMID: 24552056]
[4] Connaughton S, Collins G, O’Flaherty V. Development of microbial community structure and actvity in a high-rate anaerobic bioreactor at 18°C. Water Res 2006; 40(5): 1009-17.[http://dx.doi.org/10.1016/j.watres.2005.12.026] [PMID: 16488461]
[5] Costa KC, Leigh JA. Metabolic versatility in methanogens. Curr Opin Biotechnol 2014; 29: 70-5.[http://dx.doi.org/10.1016/j.copbio.2014.02.012] [PMID: 24662145]
[6] Borrel G, O’Toole PW, Harris HM, Peyret P, Brugère JF, Gribaldo S. Phylogenomic data support a seventh order of Methylotrophic methanogens and provide insights into the evolution of Methanogenesis. Genome Biol Evol 2013; 5(10): 1769-80.[http://dx.doi.org/10.1093/gbe/evt128] [PMID: 23985970]
[7] Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 2005; 89(6): 670-9.[http://dx.doi.org/10.1002/bit.20347] [PMID: 15696537]
[8] Yu Y, Kim J, Hwang S. Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnol Bioeng 2006; 93(3): 424-33.[http://dx.doi.org/10.1002/bit.20724] [PMID: 16196054]
[9] Sekiguchi Y. Yet-to-be cultured microorganisms relevant to methane fermentation processes. Microbes Environ 2006; 21(1): 1-15.[http://dx.doi.org/10.1264/jsme2.21.1]
[10] Sakai S, Imachi H, Sekiguchi Y, et al. Cultivation of methanogens under low-hydrogen conditions by using the coculture method. Appl Environ Microbiol 2009; 75(14): 4892-6.[http://dx.doi.org/10.1128/AEM.02835-08] [PMID: 19465530]
[11] Díaz EE, Stams AJ, Amils R, Sanz JL. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater. Appl Environ Microbiol 2006; 72(7): 4942-9.[http://dx.doi.org/10.1128/AEM.02985-05] [PMID: 16820491]
[12] Ennouri H, Miladi B, Diaz SZ, et al. Effect of thermal pretreatment on the biogas production and microbial communities balance during anaerobic digestion of urban and industrial waste activated sludge. Bioresour Technol 2016; 214: 184-91.[http://dx.doi.org/10.1016/j.biortech.2016.04.076] [PMID: 27132226]
[13] Zahedi S, Solera R, Micolucci F, Cavinato C, Bolzonella D. Changes in microbial community during hydrogen and methane production in two-stage thermophilic anaerobic co-digestion process from biowaste. Waste Manag 2016; 49: 40-6.[http://dx.doi.org/10.1016/j.wasman.2016.01.016] [PMID: 26810032]
[14] Akuzawa M, Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y. Distinctive responses of metabolically active microbiota to acidification in a thermophilic anaerobic digester. Microb Ecol 2011; 61(3): 595-605.[http://dx.doi.org/10.1007/s00248-010-9788-1] [PMID: 21240482]
[15] Watanabe T, Asakawa S, Nakamura A, Nagaoka K, Kimura M. DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. FEMS Microbiol Lett 2004; 232(2): 153-63.[http://dx.doi.org/10.1016/S0378-1097(04)00045-X] [PMID: 15033234]
[16] Sawayama S, Tsukahara K, Yagishita T. Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester. Bioresour Technol 2006; 97(1): 69-76.[http://dx.doi.org/10.1016/j.biortech.2005.02.011] [PMID: 16154504]
[17] Williams J, Williams H, Dinsdale R, Guwy A, Esteves S. Monitoring methanogenic population dynamics in a full-scale anaerobic digester to facilitate operational management. Bioresour Technol 2013; 140: 234-42.[http://dx.doi.org/10.1016/j.biortech.2013.04.089] [PMID: 23707910]
[18] Koo T, Shin SG, Lee J, et al. Identifying methanogen community structures and their correlations with performance parameters in four full-scale anaerobic sludge digesters. Bioresour Technol 2017; 228: 368-73.[http://dx.doi.org/10.1016/j.biortech.2016.12.118] [PMID: 28087103]
[19] Delbès C, Moletta R, Godon J. Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem. FEMS Microbiol Ecol 2001; 35(1): 19-26.[http://dx.doi.org/10.1016/S0168-6496(00)00107-0] [PMID: 11248386]
[20] Juretschko S, Loy A, Lehner A, Wagner M. The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach. Syst Appl Microbiol 2002; 25(1): 84-99.[http://dx.doi.org/10.1078/0723-2020-00093] [PMID: 12086193]
[21] Figuerola EL, Erijman L. Bacterial taxa abundance pattern in an industrial wastewater treatment system determined by the full rRNA cycle approach. Environ Microbiol 2007; 9(7): 1780-9.[http://dx.doi.org/10.1111/j.1462-2920.2007.01298.x] [PMID: 17564611]
[22] Bouchez T, Jacob P, d’Hugues P, Durand A. Acidophilic microbial communities catalyzing sludge bioleaching monitored by fluorescent in situ hybridization. Antonie van Leeuwenhoek 2006; 89(3-4): 435-42.[http://dx.doi.org/10.1007/s10482-005-9052-8] [PMID: 16622787]
[23] Raskin L, Stromley JM, Rittmann BE, Stahl DA. Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 1994; 60(4): 1232-40.[PMID: 7517128]
[24] Crocetti G, Murto M, Björnsson L. An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH). J Microbiol Methods 2006; 65(1): 194-201.[http://dx.doi.org/10.1016/j.mimet.2005.07.007] [PMID: 16126291]
[25] Kim TG, Jeong SY, Cho KS. Development of droplet digital PCR assays for methanogenic taxa and examination of methanogen communities in full-scale anaerobic digesters. Appl Microbiol Biotechnol 2015; 99(1): 445-58.[http://dx.doi.org/10.1007/s00253-014-6007-x] [PMID: 25117548]
[26] Manz W, Amann R, Ludwig W, Wagner M, Schleifer K. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: Problems and solutions. Syst Appl Microbiol 1992; 15(4): 593-600.[http://dx.doi.org/10.1016/S0723-2020(11)80121-9]
[27] Amann R, Fuchs BM, Behrens S. The identification of microorganisms by fluorescence in situ hybridisation. Curr Opin Biotechnol 2001; 12(3): 231-6.[http://dx.doi.org/10.1016/S0958-1669(00)00204-4] [PMID: 11404099]
[28] Daims H, Stoecker K, Wagner M. Fluorescence in situ hybridization for the detection of prokaryotes.Advanced methods in molecular microbial ecology 2005; 213-39.
[29] Chan OC, Liu WT, Fang HH. Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene. Water Sci Technol 2001; 43(1): 77-82.[PMID: 11379115]
[30] Batstone DJ, Keller J, Blackall LL. The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass. Water Res 2004; 38(6): 1390-404.[http://dx.doi.org/10.1016/j.watres.2003.12.003] [PMID: 15016516]
[31] Hulshoff Pol LW, de Castro Lopes SI, Lettinga G, Lens PN. Anaerobic sludge granulation. Water Res 2004; 38(6): 1376-89.[http://dx.doi.org/10.1016/j.watres.2003.12.002] [PMID: 15016515]
[32] Bialek K, Kim J, Lee C, Collins G, Mahony T, O’Flaherty V. Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors. Water Res 2011; 45(3): 1298-308.[http://dx.doi.org/10.1016/j.watres.2010.10.010] [PMID: 21093011]
[33] Garrity G, Holt J. Phylum AII. Euryarchaeota.Bergey’s Manual of Systematic Bacteriology 2nd ed. 2nd ed.2011; 211-94.
[34] Zitomer D, Maki J, Venkiteshwaran K, Bocher B. Relating Anaerobic Digestion Microbial Community and Process Function.Microbiol Insights2016; 37
[35] Demirel B, Scherer P. The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review. Rev Environ Sci Biotechnol 2008; 7(2): 173-90.[http://dx.doi.org/10.1007/s11157-008-9131-1]
[36] Conklin A, Stensel HD, Ferguson J. Growth kinetics and competition between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion. Water Environ Res 2006; 78(5): 486-96.[http://dx.doi.org/10.2175/106143006X95393] [PMID: 16752610]
[37] Steinberg LM, Regan JM. Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge. Appl Environ Microbiol 2008; 74(21): 6663-71.[http://dx.doi.org/10.1128/AEM.00553-08] [PMID: 18776026]
[38] Timmis K. Handbook of hydrocarbon and lipid microbiology 1st ed. 1st ed.2010.[http://dx.doi.org/10.1007/978-3-540-77587-4]
[39] Microbewiki.kenyon.edu. 2017. The Role of Methanogens in Waste Water Treatment - MicrobeWiki.[online]Available https://microbewiki.kenyon.edu/index.php/The_Role_of_Methanogens_in_Waste_Water_Treatment
[40] Seckbach J. Journey to Diverse Microbial Worlds Dordrecht 2013.
[41] Boone D, Castenholz R. The Archaea and the deeply branching and phototrophic bacteria.Bergey’s Manual of Systematic Bacteriology 2nd ed. 2nd ed.2001; 1-721.[http://dx.doi.org/10.1007/978-0-387-21609-6]
[42] Jetten M. Methanogenesis from acetate: a comparison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina spp. FEMS Microbiol Lett 1992; 88(3-4): 181-97.[http://dx.doi.org/10.1111/j.1574-6968.1992.tb04987.x]
[43] Ahring BK. Methanogenesis in thermophilic biogas reactors. Antonie van Leeuwenhoek 1995; 67(1): 91-102.[http://dx.doi.org/10.1007/BF00872197] [PMID: 7741531]
[44] Bartell RD, Matson E, Mueller-Spitz S, Kleinheinz TG. Investigation of methanosarcinales and methanomicrobiales presence within a dry Anaerobic digester. J Microbiol Res (Rosemead Calif) 2015; 5(3): 101-8.
[45] Karakashev D, Batstone DJ, Angelidaki I. Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors. Appl Environ Microbiol 2005; 71(1): 331-8.[http://dx.doi.org/10.1128/AEM.71.1.331-338.2005] [PMID: 15640206]
[46] Liu Y, Xu H, Show K, Tay J. Anaerobic granulation technology for wastewater treatment. World J Microbiol Biotechnol 2002; 18(2): 99-113.[http://dx.doi.org/10.1023/A:1014459006210]
[47] Ramdhani N, Kumari S, Bux F. Comparison of quantitative PCR and fluorescent in situ hybridization techniques to quantify nitrobacter from wastewater samples. IWA World Water Congress Secretariat 2014.Lisbon, Portugal. 2014.

Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


Browse Contents



Advertisements


Webmaster Contact: info@benthamopen.com
Copyright © 2018 Bentham Open