Bioreactor scalability: laboratory-scale bioreactor design influences performance, ecology, and community physiology in expanded granular sludge bed bioreactors

Studies investigating the feasibility of new, or improved, biotechnologies, such as wastewater treatment digesters, inevitably start with laboratory-scale trials. However, it is rarely determined whether laboratory-scale results reflect full-scale performance or microbial ecology. The Expanded Granular Sludge Bed (EGSB) bioreactor, which is a high-rate anaerobic digester configuration, was used as a model to address that knowledge gap in this study. Two laboratory-scale idealizations of the EGSB—a one-dimensional and a three- dimensional scale-down of a full-scale design—were built and operated in triplicate under near-identical conditions to a full-scale EGSB. The laboratory-scale bioreactors were seeded using biomass obtained from the full-scale bioreactor, and, spent water from the distillation of whisky from maize was applied as substrate at both scales. Over 70 days, bioreactor performance, microbial ecology, and microbial community physiology were monitored at various depths in the sludge-beds using 16S rRNA gene sequencing (V4 region), specific methanogenic activity (SMA) assays, and a range of physical and chemical monitoring methods. SMA assays indicated dominance of the hydrogenotrophic pathway at full-scale whilst a more balanced activity profile developed during the laboratory-scale trials. At each scale, Methanobacterium was the dominant methanogenic genus present. Bioreactor performance overall was better at laboratory-scale than full-scale. We observed that bioreactor design at laboratory-scale significantly influenced spatial distribution of microbial community physiology and taxonomy in the bioreactor sludge-bed, with 1-D bioreactor types promoting stratification of each. In the 1-D laboratory bioreactors, increased abundance of Firmicutes was associated with both granule position in the sludge bed and increased activity against acetate and ethanol as substrates. We further observed that stratification in the sludge-bed in 1-D laboratory-scale bioreactors was associated with increased richness in the underlying microbial community at species (OTU) level and improved overall performance

Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells

__Background:__ It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta. __Methods:__ We used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta-producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells. __Results:__ We report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression. __Conclusions:__ These data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression

Validating flow cytometry as a method for quantifying Bdellovibrio predatory bacteria and Its prey for microbial ecology

Bdellovibrio bacteriovorus is a predatory, Gram-negative bacteria that feeds on many pathogenic bacteria and has been investigated as a possible solution for mitigating biofilms in different fields. The application depends on more fundamental ecological studies into the dynamics between Bdellovibrio and their prey. To do so requires an accurate, reliable, and, preferably rapid, way of enumerating the cells. Flow cytometry (FCM) is potentially a rapid, accurate, and inexpensive tool for this, but it has yet to be validated in the enumeration of Bdellovibrio. In this study, we developed a protocol to measure the number of Bdellovibrio in samples of various densities using FCM and compared the results with those of other methods: optical density (OD), PFU assay (PFU), and quantitative PCR (qPCR). We observed a strong correlation between values obtained using FCM and PFU (ρ = 0.923) and FCM and qPCR (ρ = 0.987). Compared to optical density there was a much weaker correlation (ρ = 0.784), which was to be expected given the well-documented uncertainty in converting optical density (OD) to cell numbers. The FCM protocol was further validated by demonstrating its ability to distinguish and count mixed populations of Bdellovibrio and the prey Pseudomonas. Thus, the accuracy of FCM as well as its speed and reproducibility make it a suitable alternative for measuring Bdellovibrio cell numbers, especially where many samples are required to capture the dynamics of predator-prey interactions

De novo extraction of microbial strains from metagenomes reveals intra-species niche partitioning

Background We introduce DESMAN for De novo Extraction of Strains from MetAgeNomes. Metagenome sequencing generates short reads from throughout the genomes of a microbial community. Increasingly large, multi-sample metagenomes, stratified in space and time are being generated from communities with thousands of species. Repeats result in fragmentary co-assemblies with potentially millions of contigs. Contigs can be binned into metagenome assembled genomes (MAGs) but strain level variation will remain. DESMAN identifies variants on core genes, then uses co-occurrence across samples to link variants into strain sequences and abundance profiles. These strain profiles are then searched for on non-core genes to determine the accessory genes present in each strain. Results We validated DESMAN on a synthetic twenty genome community with 64 samples. We could resolve the five E. coli strains present with 99.58% accuracy across core gene variable sites and their gene complement with 95.7% accuracy. Similarly, on real fecal metagenomes from the 2011 E. coli (STEC) O104:H4 outbreak, the outbreak strain was reconstructed with 99.8% core sequence accuracy. Application to an anaerobic digester metagenome time series reveals that strain level variation is endemic with 16 out of 26 MAGs (61.5%) examined exhibiting two strains. In almost all cases the strain proportions were not statistically different between replicate reactors, suggesting intra-species niche partitioning. The only exception being when the two strains had almost identical gene complement and, hence, functional capability. Conclusions DESMAN will provide a provide a powerful tool for de novo resolution of fine-scale variation in microbial communities. It is available as open source software from https://github.com/chrisquince/DESMAN

Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics. We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are ‘young’ and larger ones are ‘old’. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions. Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes

Solar Septic Tank: next generation sequencing reveals effluent microbial community composition as a useful index of system performance

Septic tanks are widely deployed for off-grid sewage management but are typified by poor treatment performance, discharge of polluting effluents and the requirement for frequent de-sludging. The Solar Septic Tank (SST) is a novel septic tank design that uses passive heat from the sun to raise in-tank temperatures and improves solids degradation, resulting in a cleaner effluent. Treatment has been shown to exceed conventional systems, however, the underlying biology driving treatment in the system is poorly understood. We used next generation sequencing (Illumina Miseq (San Diego, CA, USA), V4 region 16S DNA) to monitor the microbiology in the sludge and effluent of two mature systems, a conventional septic tank and an SST, during four months of routine operation in Bangkok, Thailand, and evaluated the ecology against a suite of operating and performance data collected during the same time period. Significant differences were observed between the microbiome of the sludge and effluent in each system and the dominant taxa in each appeared persistent over time. Furthermore, variation in the microbial community composition in the system effluents correlated with effluent water quality and treatment performance parameters, including the removal of chemical and biochemical oxygen demand and the concentration of fecal and total coliforms in the effluent. Thus, we propose that a wide-scale survey of the biology underlying decentralised biotechnologies for sewage treatment such as the SST could be conducted by sampling system effluent rather than sampling sludge. This is advantageous as accessing sludge during sampling is both hazardous and potentially disruptive to the anaerobic methanogenic consortia underlying treatment in the systems

The role of shear dynamics in biofilm formation

There is growing evidence that individual bacteria sense and respond to changes in mechanical loading. However, the subtle responses of multispecies biofilms to dynamic fluid shear stress are not well documented because experiments often fail to disentangle any beneficial effects of shear stress from those delivered by convective transport of vital nutrients. We observed the development of biofilms with lognormally distributed microcolony sizes in drinking water on the walls of flow channels underflow regimes of increasing complexity. First, where regular vortices induced oscillating wall shear and simultaneously enhanced mass transport, which produced the thickest most extensive biofilms. Second, where unsteady uniform flow imposed an oscillating wall shear, with no enhanced transport, and where the biomass and coverage were only 20% smaller. Finally, for uniform steady flows with constant wall shear where the extent, thickness, and density of the biofilms were on average 60% smaller. Thus, the dynamics of shear stress played a significant role in promoting biofilm development, over and above its magnitude or mass transfer effects, and therefore, mechanosensing may prevail in complex multispecies biofilms which could open up new ways of controlling biofilm structure

Dry needling and exercise for chronic whiplash - a randomised controlled trial

Chronic whiplash is a common and costly problem. Sensory hypersensitivity is a feature of chronic whiplash that is associated with poor responsiveness to physical treatments such as exercise. Modalities such as dry-needling have shown some capacity to modulate sensory hypersensitivity, suggesting that when combined with advice and exercise, such an approach may be more effective in the management of chronic whiplash. The primary aim of this project is to investigate the effectiveness of dry-needling, advice and exercise for chronic whiplash

Monitoring schistosomiasis and sanitation interventions—the potential of environmental DNA

Transmission of schistosomiasis, a human parasitic disease, is intrinsically linked to inadequate water, sanitation, and hygiene (WASH) facilities and/or their use. The mainstay of control is population‐based chemotherapy. Globally, each year, 240 million people are estimated to require this preventative treatment. However, for long‐term, sustainable control of this disease, supplementary WASH improvements are required to prevent (re)infection of humans (provision of safe water) and transmission from humans to the environment (improved sanitation). While there is established methodology for monitoring transmission in human populations, presently methods for monitoring the impact of WASH interventions, in particular sanitation, on environmental transmission are lacking. Development of such becomes paramount as integrated control programs combine drug treatments with enhanced WASH facilities and behavior change interventions, with uptake likely correlated to a reduction in fecal matter, and schistosome eggs, in the environment but any impact on infection levels in humans taking longer to become apparent. This article reports and critiques the methods currently used to monitor schistosomiasis in freshwater and soil environments and explores how environmental DNA could be used to better understand and monitor environmental contamination in relation to sanitation. Stronger evidence is required to understand how different sanitation interventions serve to limit the environmental transmission of the parasite and their relative effectiveness in preventing disease

Growth and break-up of methanogenic granules suggests mechanisms for biofilm and community development

Methanogenic sludge granules are densely packed, small, spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. Each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion (AD) process. Information on exactly how methanogenic granules develop, and their eventual fate will be important for precision management of environmental biotechnologies. Granules from a full-scale bioreactor were size-separated into small (0.6–1 mm), medium (1– 1.4 mm), and large (1.4–1.8 mm) size fractions. Twelve laboratory-scale bioreactors were operated using either small, medium, or large granules, or unfractionated sludge. After >50 days of operation, the granule size distribution in each of the small, medium, and large bioreactor sets had diversified beyond—to both bigger and smaller than—the size fraction used for inoculation. Interestingly, extra-small (XS; <0.6 mm) granules were observed, and retained in all of the bioreactors, suggesting the continuous nature of granulation, and/or the breakage of larger granules into XS bits. Moreover, evidence suggested that even granules with small diameters could break. “New” granules from each emerging size were analyzed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae, and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration (MINT) analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules