Research

27 pages, 2865 KiB

Open AccessArticle

Diversity and Abundance of Microbial Communities in UASB Reactors during Methane Production from Hydrolyzed Wheat Straw and Lucerne

by Tong Liu, Anna Schnürer, Johanna Björkmalm, Karin Willquist and Emma Kreuger

Microorganisms 2020, 8(9), 1394; https://doi.org/10.3390/microorganisms8091394 - 11 Sep 2020

Cited by 12 | Viewed by 3089

Abstract

The use of straw for biofuel production is encouraged by the European Union. A previous study showed the feasibility of producing biomethane in upflow anaerobic sludge blanket (UASB) reactors using hydrolyzed, steam-pretreated wheat straw, before and after dark fermentation with Caldicellulosiruptor saccharolyticus, [...] Read more.

The use of straw for biofuel production is encouraged by the European Union. A previous study showed the feasibility of producing biomethane in upflow anaerobic sludge blanket (UASB) reactors using hydrolyzed, steam-pretreated wheat straw, before and after dark fermentation with Caldicellulosiruptor saccharolyticus, and lucerne. This study provides information on overall microbial community development in those UASB processes and changes related to acidification. The bacterial and archaeal community in granular samples was analyzed using high-throughput amplicon sequencing. Anaerobic digestion model no. 1 (ADM1) was used to predict the abundance of microbial functional groups. The sequencing results showed decreased richness and diversity in the microbial community, and decreased relative abundance of bacteria in relation to archaea, after process acidification. Canonical correspondence analysis showed significant negative correlations between the concentration of organic acids and three phyla, and positive correlations with seven phyla. Organic loading rate and total COD fed also showed significant correlations with microbial community structure, which changed over time. ADM1 predicted a decrease in acetate degraders after a decrease to pH ≤ 6.5. Acidification had a sustained effect on the microbial community and process performance. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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18 pages, 1719 KiB

Open AccessArticle

Microbial Consortiums of Hydrogenotrophic Methanogenic Mixed Cultures in Lab-Scale Ex-Situ Biogas Upgrading Systems under Different Conditions of Temperature, pH and CO

by Jun Xu, Fan Bu, Wenzhe Zhu, Gang Luo and Li Xie

Microorganisms 2020, 8(5), 772; https://doi.org/10.3390/microorganisms8050772 - 21 May 2020

Cited by 22 | Viewed by 3293

Abstract

In this study, hydrogenotrophic methanogenic mixed cultures taken from 13 lab-scale ex-situ biogas upgrading systems under different temperature (20–70 °C), pH (6.0–8.5), and CO (0–10%, v/v) variables were systematically investigated. High-throughput 16S rRNA gene sequencing was used to identify the [...] Read more.

In this study, hydrogenotrophic methanogenic mixed cultures taken from 13 lab-scale ex-situ biogas upgrading systems under different temperature (20–70 °C), pH (6.0–8.5), and CO (0–10%, v/v) variables were systematically investigated. High-throughput 16S rRNA gene sequencing was used to identify the microbial consortia, and statistical analyses were conducted to reveal the microbial diversity, the core functional microbes, and their correlative relationships with tested variables. Overall, bacterial community was more complex than the archaea community in all mixed cultures. Hydrogenotrophic methanogens Methanothermobacter, Methanobacterium, and Methanomassiliicoccus, and putative syntrophic acetate-oxidizing bacterium Coprothermobacter and Caldanaerobacter were found to predominate, but the core functional microbes varied under different conditions. Multivariable sensitivity analysis indicated that temperature (p < 0.01) was the crucial variable to determine the microbial consortium structures in hydrogenotrophic methanogenic mixed cultures. pH (0.01 < p < 0.05) significantly interfered with the relative abundance of dominant archaea. Although CO did not affect community (p > 0.1), some potential CO-utilizing syntrophic metabolisms might be enhanced. Understanding of microbial consortia in the hydrogenotrophic methanogenic mixed cultures related to environmental variables was a great advance to reveal the microbial ecology in microbial biogas upgrading process. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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15 pages, 2252 KiB

Open AccessArticle

A Rapid, Sensitive, Low-Cost Assay for Detecting Hydrogenotrophic Methanogens in Anaerobic Digesters Using Loop-Mediated Isothermal Amplification

by Anna M. Alessi, Bing Tao, Wei Zhang, Yue Zhang, Sonia Heaven, Charles J. Banks and James P. J. Chong

Microorganisms 2020, 8(5), 740; https://doi.org/10.3390/microorganisms8050740 - 15 May 2020

Cited by 4 | Viewed by 3121

Abstract

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea [...] Read more.

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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15 pages, 1987 KiB

Open AccessArticle

A Modular Metagenomics Pipeline Allowing for the Inclusion of Prior Knowledge Using the Example of Anaerobic Digestion

by Daniela Becker, Denny Popp, Hauke Harms and Florian Centler

Microorganisms 2020, 8(5), 669; https://doi.org/10.3390/microorganisms8050669 - 05 May 2020

Cited by 1 | Viewed by 3784

Abstract

Metagenomics analysis revealing the composition and functional repertoire of complex microbial communities typically relies on large amounts of sequence data. Numerous analysis strategies and computational tools are available for their analysis. Fully integrated automated analysis pipelines such as MG-RAST or MEGAN6 are user-friendly [...] Read more.

Metagenomics analysis revealing the composition and functional repertoire of complex microbial communities typically relies on large amounts of sequence data. Numerous analysis strategies and computational tools are available for their analysis. Fully integrated automated analysis pipelines such as MG-RAST or MEGAN6 are user-friendly but not designed for integrating specific knowledge on the biological system under study. In order to facilitate the consideration of such knowledge, we introduce a modular, adaptable analysis pipeline combining existing tools. We applied the novel pipeline to simulated mock data sets focusing on anaerobic digestion microbiomes and compare results to those obtained with established automated analysis pipelines. We find that the analysis strategy and choice of tools and parameters have a strong effect on the inferred taxonomic community composition, but not on the inferred functional profile. By including prior knowledge, computational costs can be decreased while improving result accuracy. While automated off-the-shelf analysis pipelines are easy to apply and require no knowledge on the microbial system under study, custom-made pipelines require more preparation time and bioinformatics expertise. This extra effort is minimized by our modular, flexible, custom-made pipeline, which can be adapted to different scenarios and can take available knowledge on the microbial system under study into account. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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18 pages, 2180 KiB

Open AccessEditor’s ChoiceArticle

Microbial Resource Management for Ex Situ Biomethanation of Hydrogen at Alkaline pH

by Washington Logroño, Denny Popp, Sabine Kleinsteuber, Heike Sträuber, Hauke Harms and Marcell Nikolausz

Microorganisms 2020, 8(4), 614; https://doi.org/10.3390/microorganisms8040614 - 24 Apr 2020

Cited by 38 | Viewed by 4874

Abstract

Biomethanation is a promising solution to convert H₂ (produced from surplus electricity) and CO₂ to CH₄ by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H₂/CO₂. We enriched a [...] Read more.

Biomethanation is a promising solution to convert H₂ (produced from surplus electricity) and CO₂ to CH₄ by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H₂/CO₂. We enriched a hydrogenotrophic microbiota on CO₂ and H₂ as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH₄. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH₄ (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH₄ from H₂/CO₂ by adapting the media composition, the reducing agent and the stirring intensity. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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18 pages, 2489 KiB

Open AccessArticle

Effect of Cobalt, Nickel, and Selenium/Tungsten Deficiency on Mesophilic Anaerobic Digestion of Chemically Defined Soluble Organic Compounds

by Luka Šafarič, Sepehr Shakeri Yekta, Bo H. Svensson, Anna Schnürer, David Bastviken and Annika Björn

Microorganisms 2020, 8(4), 598; https://doi.org/10.3390/microorganisms8040598 - 20 Apr 2020

Cited by 20 | Viewed by 3476

Abstract

Trace elements (TEs) are vital for anaerobic digestion (AD), due to their role as cofactors in many key enzymes. The aim of this study was to evaluate the effects of specific TE deficiencies on mixed microbial communities during AD of soluble polymer-free substrates, [...] Read more.

Trace elements (TEs) are vital for anaerobic digestion (AD), due to their role as cofactors in many key enzymes. The aim of this study was to evaluate the effects of specific TE deficiencies on mixed microbial communities during AD of soluble polymer-free substrates, thus focusing on AD after hydrolysis. Three mesophilic (37 °C) continuous stirred-tank biogas reactors were depleted either of Co, Ni, or a combination of Se and W, respectively, by discontinuing their supplementation. Ni and Se/W depletion led to changes in methane kinetics, linked to progressive volatile fatty acid (VFA) accumulation, eventually resulting in process failure. No significant changes occurred in the Co-depleted reactor, indicating that the amount of Co present in the substrate in absence of supplementation was sufficient to maintain process stability. Archaeal communities remained fairly stable independent of TE concentrations, while bacterial communities gradually changed with VFA accumulation in Ni- and Se-/W-depleted reactors. Despite this, the communities remained relatively similar between these two reactors, suggesting that the major shifts in composition likely occurred due to the accumulating VFAs. Overall, the results indicate that Ni and Se/W depletion primarily lead to slower metabolic activities of methanogenic archaea and their syntrophic partners, which then has a ripple effect throughout the microbial community due to a gradual accumulation of intermediate fermentation products. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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21 pages, 4278 KiB

Open AccessArticle

Effect of Inoculum Pretreatment on the Composition of Microbial Communities in Anaerobic Digesters Producing Volatile Fatty Acids

by Lucia Blasco, Minna Kahala, Elina Tampio, Markku Vainio, Satu Ervasti and Saija Rasi

Microorganisms 2020, 8(4), 581; https://doi.org/10.3390/microorganisms8040581 - 17 Apr 2020

Cited by 31 | Viewed by 2642

Abstract

Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and can be produced through the fermentation of organic wastes. VFAs have become an anticipated resource- and cost-effective way to replace fossil resources with higher added value and more [...] Read more.

Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and can be produced through the fermentation of organic wastes. VFAs have become an anticipated resource- and cost-effective way to replace fossil resources with higher added value and more versatile fuels and chemicals. However, there are still challenges in the production of targeted compounds from diverse and complex biomasses, such as urban biowastes. In this study, the aim was to modulate the microbial communities through inoculum treatment to enhance the production of green chemicals. Thermal and freeze-thaw treatments were applied to the anaerobic digester inoculum to inhibit the growth of methanogens and to enhance the performance of acidogenic and acetogenic bacteria. VFA fermentation after different inoculum treatments was studied in batch scale using urban biowaste as the substrate and the process performance was assessed with chemical and microbial analyses. Inoculum treatments, especially thermal treatment, were shown to increase VFA yields, which were also correlating with the dynamics of the microbial communities and retention times of the test. There was a strong correlation between VFA production and the relative abundances of the microbial orders Clostridiales (families Ruminococcaceae, Lachnospiraceae and Clostridiaceae), and Lactobacillales. A syntrophic relationship of these taxa with members of the Methanobacteriales order was also presumed. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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13 pages, 1259 KiB

Open AccessArticle

Enhanced Anaerobic Digestion of Long Chain Fatty Acid by Adding Magnetite and Carbon Nanotubes

by Alsayed Mostafa, Seongwon Im, Young-Chae Song, Seoktae Kang and Dong-Hoon Kim

Microorganisms 2020, 8(3), 333; https://doi.org/10.3390/microorganisms8030333 - 27 Feb 2020

Cited by 39 | Viewed by 3759

Abstract

This study investigated the impact of stimulating direct interspecies electron transfer (DIET), by supplementing nano-sized magnetite (nFe₃O₄, 0.5 g Fe/g VSS) and carbon nanotubes (CNT, 1 g/L), in anaerobic digestion of oleic acid (OA) at various concentrations (0.10–4.00 g [...] Read more.

This study investigated the impact of stimulating direct interspecies electron transfer (DIET), by supplementing nano-sized magnetite (nFe₃O₄, 0.5 g Fe/g VSS) and carbon nanotubes (CNT, 1 g/L), in anaerobic digestion of oleic acid (OA) at various concentrations (0.10–4.00 g chemical oxygen demand(COD)/L). Both supplementations could enhance CH₄ production, and its beneficial impact increased with increased OA concentration. The biggest improvements of 114% and 165% compared to the control were achieved by nFe₃O₄ and CNT, respectively, at OA of 4 g COD/L. The enhancement can be attributed to the increased sludge conductivity: 7.1 ± 0.5 (control), 12.5 ± 0.8 (nFe₃O₄-added), and 15.7 ± 1.1 µS/cm (CNT-supplemented). Dissolved iron concentration, released from nFe₃O₄, seemed to have a negligible role in improving CH₄ production. The excretion of electron shuttles, i.e., humic-like substances and protein-like substances, were found to be stimulated by supplementing nFe₃O₄ and CNT. Microbial diversity was found to be simplified under DIET-stimulating conditions, whereby five genera accounted for 88% of the total sequences in the control, while more than 82% were represented by only two genera (Methanotrix concilli and Methanosarcina flavescens) by supplementing nFe₃O₄ and CNT. In addition, the abudance of electro-active bacteria such as Syntrophomonas zehnderi was significantly increased from 17% to around 45%. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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22 pages, 3095 KiB

Open AccessArticle

pH and Phosphate Induced Shifts in Carbon Flow and Microbial Community during Thermophilic Anaerobic Digestion

by Nina Lackner, Andreas O. Wagner, Rudolf Markt and Paul Illmer

Microorganisms 2020, 8(2), 286; https://doi.org/10.3390/microorganisms8020286 - 20 Feb 2020

Cited by 14 | Viewed by 3274

Abstract

pH is a central environmental factor influencing CH₄ production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0–8.5, phosphate buffered) and the application of a phosphate buffer per [...] Read more.

pH is a central environmental factor influencing CH₄ production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0–8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH₄ production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH₄ production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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8 pages, 1529 KiB

Open AccessArticle

Isolation of an Obligate Mixotrophic Methanogen That Represents the Major Population in Thermophilic Fixed-Bed Anaerobic Digesters

by Misa Nagoya, Atsushi Kouzuma, Yoshiyuki Ueno and Kazuya Watanabe

Microorganisms 2020, 8(2), 217; https://doi.org/10.3390/microorganisms8020217 - 06 Feb 2020

Cited by 1 | Viewed by 2667

Abstract

Methanothermobacter Met2 is a metagenome-assembled genome (MAG) that encodes a putative mixotrophic methanogen constituting the major populations in thermophilic fixed-bed anaerobic digesters. In order to characterize its physiology, the present work isolated an archaeon (strain Met2-1) that represents Met2-type methanogens by using a [...] Read more.

Methanothermobacter Met2 is a metagenome-assembled genome (MAG) that encodes a putative mixotrophic methanogen constituting the major populations in thermophilic fixed-bed anaerobic digesters. In order to characterize its physiology, the present work isolated an archaeon (strain Met2-1) that represents Met2-type methanogens by using a combination of enrichments under a nitrogen atmosphere, colony formation on solid media and limiting dilution under high partial pressures of hydrogen. Strain Met2-1 utilizes hydrogen and carbon dioxide for methanogenesis, while the growth is observed only when culture media are additionally supplemented with acetate. It does not grow on acetate in the absence of hydrogen. The results demonstrate that Methanothermobacter sp. strain Met2-1 is a novel methanogen that exhibits obligate mixotrophy. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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18 pages, 1450 KiB

Open AccessArticle

Microbial and Phenyl Acid Dynamics during the Start-up Phase of Anaerobic Straw Degradation in Meso- and Thermophilic Batch Reactors

by Eva Maria Prem, Rudolf Markt, Nina Lackner, Paul Illmer and Andreas Otto Wagner

Microorganisms 2019, 7(12), 657; https://doi.org/10.3390/microorganisms7120657 - 05 Dec 2019

Cited by 13 | Viewed by 3020

Abstract

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts [...] Read more.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation. Full article

(This article belongs to the Special Issue Anaerobic Digestion)

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