Implementation of anaerobic digestion for valorizing the organic fraction of municipal solid waste in developing countries: Technical insights from a systematic review.

Anaerobic digestion (AD) as a waste management strategy for the organic fraction of municipal waste (OFMSW) has received attention in developed countries for several decades, leading to the development of large-scale plants. In contrast, AD of OFMSW has only recently drawn attention in developing countries. This systematic review was carried out to investigate the implementation of AD to treat the OFMSW in developing countries, focusing on assessing pilot and full-scale AD plants reported in the last ten years. Studies that met the selection criteria were analyzed and data regarding operating parameters, feedstock characteristics, and biogas, digestate, and energy production were extracted. As outlined in this systematic review, AD plants located in developing countries are mostly one-stage mesophilic systems that treat OFMSW via mono-digestion, almost exclusively with the aim of producing electrical energy. Based on the analysis done throughout this systematic review, it was noted that there is a large difference in the maturity level of AD systems between developing and developed countries, mainly due to the economic capacity of developed countries to invest in sustainable waste management systems. However, the number of AD plants reported in scientific papers is significantly lower than the number of installed AD systems. Research articles regarding large-scale implementation of AD to treat OFMSW in developed countries were analyzed and compared with developing countries. This comparison identified practices used in plants in developed countries that could be utilized in the large-scale implementation and success of AD in developing countries. These practices include exploiting potential products with high market-values, forming partnerships with local industries to use industrial wastes as co-substrates, and exploring different biological and physical pretreatment technologies. Additionally, the analysis of capital and operational costs of AD plants showed that costs tend to be higher for developing countries due to their need to import of materials and equipment from developed countries. Technical, economical, and political challenges for the implementation of AD at a large-scale in developing countries are highlighted.

Emergent Lag Phase in Flux-Regulation Models of Bacterial Growth.

Lag phase is observed in bacterial growth during a sudden change in conditions: growth is inhibited whilst cells adapt to the environment. Bi-phasic, or diauxic growth is commonly exhibited by many species. In the presence of two sugars, cells initially grow by consuming the preferred sugar then undergo a lag phase before resuming growth on the second. Biomass increase is characterised by a diauxic growth curve: exponential growth followed by a period of no growth before a second exponential growth. Recent literature lacks a complete dynamic description, artificially modelling lag phase and employing non-physical representations of precursor pools. Here, we formulate a rational mechanistic model based on flux-regulation/proteome partitioning with a finite precursor pool that reveals core mechanisms in a compact form. Unlike earlier systems, the characteristic dynamics emerge as part of the solution, including the lag phase. Focussing on growth of Escherichia coli on a glucose-lactose mixture we show results accurately reproduce experiments. We show that for a single strain of E. coli, diauxic growth leads to optimised biomass yields. However, intriguingly, for two competing strains diauxic growth is not always the best strategy. Our description can be generalised to model multiple different microorganisms and investigate competition between species/strains.

Case Report: The effect of intravenous and oral antibiotics on the gut microbiome and breath volatile organic compounds over one year.

BACKGROUND: Antimicrobial resistance (AMR) is a global concern and better understanding of the gut microbiome, a known 'amplifier' of AMR, may allow future clinicians to tailor therapy to minimise this risk and offer a personalised medicine approach. To examine the gut microbiome, patients are required to provide faecal samples; more convenient and cheaper solutions need to be found. METHODS: As part of a pilot study looking at how routes of administration affect the gut microbiome in NHS patients undergoing routine clinical management for infections, we hypothesised that effects on the gut microbiome varied with the route and metabolism of antibiotic used, and these changes may be reflected in breath metabolites. We present a case report of a patient with an unusual clinical history, alongside breath metabolite and gut microbiome data taken before, during and after antibiotic therapy over a period of one year. RESULTS: We noted a shift in the dominant Bacteroides strain in the patient's gut microbiome between pre- and post-therapy samples, along with an alteration in the composition of breath metabolites. CONCLUSIONS: This study provides a framework for similar future work and highlights the need for further research on the relationships between changes in microbial gut communities and antimicrobial exposure, patient clinical status, and the metabolites of human breath.

The challenges of monitoring and manipulating anaerobic microbial communities.

Mixed anaerobic microbial communities are a key component in valorization of waste biomass via anaerobic digestion. Similar microbial communities are important as soil and animal microbiomes and have played a critical role in shaping the planet as it is today. Understanding how individual species within communities interact with others and their environment is important for improving performance and potential applications of an inherently green technology. Here, the challenges associated with making measurements critical to assessing the status of anaerobic microbial communities are considered. How these measurements could be incorporated into control philosophies and augment the potential of anaerobic microbial communities to produce different and higher value products from waste materials are discussed. The benefits and pitfalls of current genetic and molecular approaches to measuring and manipulating anaerobic microbial communities and the challenges which should be addressed to realise the potential of this exciting technology are explored.

Dynamic changes in anaerobic digester metabolic pathways and microbial populations during acclimatisation to increasing ammonium concentrations.

Transitions in microbial community structure in response to increasing ammonia concentrations were determined by monitoring mesophilic anaerobic digesters seeded with a predominantly acetoclastic methanogenic community from a sewage sludge digester. Ammonia concentration was raised by switching the feed to source segregated domestic food waste and applying two organic loading rates (OLR) and hydraulic retention times (HRT) in paired digesters. One of each pair was dosed with trace elements (TE) known to be essential to the transition, with the other unsupplemented digester acting as a control. Samples taken during the trial were used to determine the metabolic pathway to methanogenesis using 14C labelled acetate. Partitioning of 14C between the product gases was interpreted via an equation to indicate the proportion produced by acetoclastic and hydrogenotrophic routes. Archaeal and selected bacterial groups were identified by 16S rRNA sequencing, to determine relative abundance and diversity. Acclimatisation for digesters with TE was relatively smooth, but OLR and HRT influenced both metabolic route and community structure. The 14C ratio could be used quantitatively and, when interpreted alongside archaeal community structure, showed that at longer HRT and lower loading Methanobacteriaceae were dominant and hydrogenotrophic activity accounted for 77% of methane production. At the higher OLR and shorter HRT, Methanosarcinaceae were dominant with the 14C ratio indicating simultaneous production of methane by acetoclastic and hydrogenotrophic pathways: the first reported observation of this in digestion under mesophilic conditions. Digesters without TE supplementation showed similar initial changes but, as expected failed to complete the transition to stable operation.

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production.

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

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

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.

A practical introduction to microbial molecular ecology through the use of isolation chips.

In the context of antimicrobial resistance as one of the most serious issues faced globally by health providers, we explored a practical introduction to molecular microbial ecology. We designed field work and practical experiments for third year members of a 4 year undergraduate Masters Program, in which the students employed traditional and novel isolation techniques to identify antimicrobial activities from soil dwelling microorganisms. Students gained experience in isolating DNA from complex microbial communities, amplifying 16S rRNA genes and applied richness/diversity indices as well as principal coordinate analyses to the interpretation of the data they obtained from high throughput sequencing. Our results confirmed that isolation chips facilitate the growth of a greater diversity and different species subset from the complex soil microorganism community than traditional plate spreading techniques. However, rarefaction of 16S rRNA amplicon sequencing data showed that the majority of observed species in soil remain unculturable by current methods. Based on the written reports produced by the students carrying out the work, we concluded that the described protocols are robust and informative, that these activities provide a good practical introduction to the theories and practice of molecular ecology and can be easily deployed to groups of six or more students in a cost-effective manner.

Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

Effects of N-glycosylation site removal in archaellins on the assembly and function of archaella in Methanococcus maripaludis.

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication.

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

SSuMMo: rapid analysis, comparison and visualization of microbial communities.

MOTIVATION: Next-generation sequencing methods are generating increasingly massive datasets, yet still do not fully capture genetic diversity in the richest environments. To understand such complicated and elusive systems, effective tools are needed to assist with delineating the differences found in and between community datasets. RESULTS: The Small Subunit Markov Modeler (SSuMMo) was developed to probabilistically assign SSU rRNA gene fragments from any sequence dataset to recognized taxonomic clades, producing consistent, comparable cladograms. Accuracy tests predicted >90% of genera correctly for sequences downloaded from public reference databases. Sequences from a next-generation sequence dataset, sampled from lean, overweight and obese individuals, were analysed to demonstrate parallel visualization of comparable datasets. SSuMMo shows potential as a valuable curatorial tool, as numerous incorrect and outdated taxonomic entries and annotations were identified in public databases. AVAILABILITY AND IMPLEMENTATION: SSuMMo is GPLv3 open source Python software, available at http://code.google.com/p/ssummo/. Taxonomy and HMM databases can be downloaded from http://bioltfws1.york.ac.uk/ssummo/. SUPPLEMENTARY INFORMATION: Supplemental materials are available at Bioinformatics Online.

Flagella and pili are both necessary for efficient attachment of Methanococcus maripaludis to surfaces.

Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific role has been assigned as yet to pili. In this report, wild-type M. maripaludis cells are compared with mutants lacking either pili or flagella or both surface appendages in their ability to attach to a variety of surfaces including nickel, gold and molybdenum grids as well as glass, silicon and mica. Wild-type cells attached to varying degrees to all surfaces tested, except mica, via their flagella as observed by scanning electron microscopy. Large cables of flagella were found to leave the cell and to be unwound on the surface. In addition, such cables were often found to connect cells. In contrast, cells lacking either flagella or pili or both surface appendages were unable to attach efficiently to any surfaces. This indicates a second role for flagella in addition to swimming in M. maripaludis, as well as a first role for pili in this organism, namely in surface attachment.

Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency.

We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

Major players on the microbial stage: why archaea are important.

As microbiology undergoes a renaissance, fuelled in part by developments in new sequencing technologies, the massive diversity and abundance of microbes becomes yet more obvious. The Archaea have traditionally been perceived as a minor group of organisms forced to evolve into environmental niches not occupied by their more 'successful' and 'vigorous' counterparts, the bacteria. Here we outline some of the evidence gathered by an increasingly large and productive group of scientists that demonstrates not only that the Archaea contribute significantly to global nutrient cycling, but also that they compete successfully in 'mainstream' environments. Recent data suggest that the Archaea provide the major routes for ammonia oxidation in the environment. Archaea also have huge economic potential that to date has only been fully realized in the production of thermostable polymerases. Archaea have furnished us with key paradigms for understanding fundamentally conserved processes across all domains of life. In addition, they have provided numerous exemplars of novel biological mechanisms that provide us with a much broader view of the forms that life can take and the way in which micro-organisms can interact with other species. That this information has been garnered in a relatively short period of time, and appears to represent only a small proportion of what the Archaea have to offer, should provide further incentives to microbiologists to investigate the underlying biology of this fascinating domain.

Bacterial and eukaryotic systems collide in the three Rs of Methanococcus.

Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.

An archaeal order with multiple minichromosome maintenance genes.

In eukaryotes, a complex of six highly related minichromosome maintenance (MCM) proteins is believed to function as the replicative helicase. Until recently, systems for exploring the molecular mechanisms underlying eukaryotic MCM function have been biochemically intractable. To overcome this, molecular studies of MCM function have been carried out using MCM homologues from the archaea. Archaeal MCM systems studied to date possess a single functional MCM, which forms a homohexameric complex that displays DNA binding, ATPase and helicase activities. We have identified an archaeal order that possesses multiple MCM homologues. blast searches of available Methanococcales genomes reveal that members of this order possess between two and eight MCM homologues. Phylogenetic analysis suggests that an ancient duplication in the Methanococcales gave rise to two major groups of MCMs. One group contains Methanococcus maripaludis S2 McmD and possesses a conserved C-terminal insert similar to one observed in eukaryotic MCM3, while the other group contains McmA, -B and -C. Analysis of the genome context of MCMs in the latter group indicates that these genes could have arisen from phage-mediated events. When co-expressed in Escherichia coli, the four MCMs from M. maripaludis co-purify, indicating the formation of heteromeric complexes in vitro. The presence of homologues from both groups in all Methanococcales indicates that there could be functionally important differences between these proteins and that Methanococcales MCMs may therefore provide an interesting additional model for eukaryotic MCM function.

Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components.

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus DeltaH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus DeltaH. After incision at the 5'-side of a 2'-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3'-OH terminus and carry out repair synthesis generating a 5'-flap structure that is resolved by mthFEN, a 5'-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.