Dead in the water - Role of relic DNA and primer choice for targeted sequencing surveys of anaerobic sewage sludge intended for biological monitoring.

DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.

Evaluating novel biodegradable polymer matrix fertilizers for nitrogen-efficient agriculture.

Enhanced efficiency fertilizers (EEFs) can reduce nitrogen (N) losses in temperate agriculture but are less effective in the tropics. We aimed to design a new EEF and evaluate their performance in simple-to-complex tests with tropical soils and crops. We melt-extruded urea at different loadings into biodegradable polymer matrix composites using biodegradable polyhydroxyalkanoate (PHA) or polybutylene adipate-co-terephthalate (PBAT) polymers with urea distributed throughout the pellet. These contrast with commercially coated EEF that have a polymer-coated urea core. We hypothesized that matrix fertilizers would have an intermediate N release rate compared to fast release from urea or slow release from coated EEF. Nitrogen release rates in water and sand-soil columns confirmed that the matrix fertilizer formulations had a more progressive N release than a coated EEF. A more complex picture emerged from testing sorghum [Sorghum bicolor (L.) Moench] grown to maturity in large soil pots, as the different formulations resulted in minor differences in plant N accumulation and grain production. This confirms the need to consider soil interactions, microbial processes, crop physiology, and phenology for evaluating fertilizer performance. Promisingly, crop δ15N signatures emerged as an integrated measure of efficacy, tracking likely N conversions and losses. The three complementary evaluations combine the advantages of standardized high-throughput screening and more resource-intensive and realistic testing in a plant-soil system. We conclude that melt-blended biodegradable polymer matrix fertilizers show promise as EEF because they can be designed toward more abiotically or more microbially driven N release by selecting biopolymer type and N loading rate.

Impact of volatile solids destruction on the shear and solid-liquid separation behaviour of anaerobic digested sludge.

Systematic and comprehensive characterisation of shear and solid-liquid separation properties of sludge across a wide range of solids concentration and volatile solids destruction (VSD) is critical for design and optimization of the anaerobic digestion process. In addition, there is a need for studies at the psychrophilic temperature range as many unheated anaerobic digestion processes are operated under ambient conditions with minimal self-heating. In this study, two digesters were operated at different combinations of operating temperature (15-25 °C) and hydraulic retention time (16-32 d) to ensure a wide range of VSD in the range of 0.42-0.7 was obtained. For shear rheology, the viscosity increased 1.3 to 3.3 times with the increase of VSD from 43 % to 70 %, while other parameters (temperature, VS fraction) having a negligible impact. Analysis of a hypothetical digester indicated that there is an optimum VSD range 65-80 % where increase in viscosity due to the higher VSD is balanced by the decrease in solids concentration. For solid-liquid separation, a thickener model and a filtration model were used. No significant impact of VSD on the solids flux, underflow solids concentrations or specific solids throughput was observed in the thickener and filtration model. However, there was an increase in average cake solids concentration from 21 % to 31 % with increase of VSD from 55 % to 76 %, indicating better dewatering behaviour.

Lactic acid production from food waste at an anaerobic digestion biorefinery: effect of digestate recirculation and sucrose supplementation.

Low lactic acid (LA) yields from direct food waste (FW) fermentation restrict this production pathway. However, nitrogen and other nutrients within FW digestate, in combination with sucrose supplementation, may enhance LA production and improve feasibility of fermentation. Therefore, this work aimed to improve LA fermentation from FWs by supplementing nitrogen (0-400 mgN·L-1) as NH4Cl or digestate and dosing sucrose (0-150 g·L-1) as a low-cost carbohydrate. Overall, NH4Cl and digestate led to similar improvements in the rate of LA formation (0.03 ± 0.02 and 0.04 ± 0.02 h-1 for NH4Cl and digestate, respectively), but NH4Cl also improved the final concentration, though effects varied between treatments (5.2 ± 4.6 g·L-1). While digestate altered the community composition and increased diversity, sucrose minimised community diversion from LA, promoted Lactobacillus growth at all dosages, and enhanced the final LA concentration from 25 to 30 g·L-1 to 59-68 g·L-1, depending on nitrogen dosage and source. Overall, the results highlighted the value of digestate as a nutrient source and sucrose as both community controller and means to enhance the LA concentration in future LA biorefinery concepts.

Resource recovery using enriched purple phototrophic bacteria in an outdoor flat plate photobioreactor: Suspended vs. attached growth.

Purple phototrophic bacteria (PPB) can produce single-cell protein from wastewater at high yields. Growing in a biofilm vs suspended can improve product quality and consistency. This study compares suspended and attached growths of enriched PPB cultures in an outdoor flat plate photobioreactor treating poultry-processing wastewater. Attached growth had lower VFA removal efficiencies (95 ± 2.7 vs 84 ± 6.4 %) due to light limitations and low substrate diffusion rates. Nevertheless, similar overall treatment performances and productivities were achieved (16 ± 2.2 and 18 ± 2.4 gCOD·m-2·d-1 for attached and suspended) at loading rates of 1.2-1.5 gCOD·L-1·d-1. Biofilms had higher quality than suspended biomass, with lower ash contents (6.9(0.6)% vs 57(16)%) and higher PPB abundances (0.45-0.67 vs 0.30-0.45). The biofilm (20-50 % of the total biomass) might be used as feed and the suspended fraction as fertiliser, improving the economics of the process. Semi-continuous PPB growth outdoors as biofilm is technically feasible, obtaining a superior product without jeopardising performance.

Expanding mechanistic models to represent purple phototrophic bacteria enriched cultures growing outdoors.

The economic feasibility of purple phototrophic bacteria (PPB) for resource recovery relies on using enriched-mixed cultures and sunlight. This work presents an extended Photo-Anaerobic Model (ePAnM), considering: (i) the diverse metabolic capabilities of PPB, (ii) microbial clades interacting with PPB, and (iii) varying environmental conditions. Key kinetic and stoichiometric parameters were either determined experimentally (with dedicated tests), calculated, or gathered from literature. The model was calibrated and validated using different datasets from an outdoors demonstration-scale reactor, as well as results from aerobic and anaerobic batch tests. The ePAnM was able to predict the concentrations of key compounds/components (e.g., COD, volatile fatty acids, and nutrients), as well as microbial communities (with anaerobic systems dominated by fermenters and PPB). The results underlined the importance of considering other microbial clades and varying environmental conditions. The model predicted a minimum hydraulic retention time of 0.5 d-1. A maximum width of 10 cm in flat plate reactors should not be exceeded. Simulations showed the potential of a combined day-anaerobic/night-aerobic operational strategy to allow efficient continuous operation.

Shear and solid-liquid separation behaviour of anaerobic digested sludge across a broad range of solids concentrations.

Due to the non-homogeneous and multiphase nature of anaerobic lagoon constituents, CFD modelling for process optimisation requires continuous functions for shear and solid-liquid separation properties across a large range of solids concentrations. Unfortunately, measurement of existing material properties of anaerobic sludges is limited to only shear or solid-liquid separation, or to a limited solids concentration. In this work, the shear properties of an anaerobic sludge were measured from 0.4 to 12.5 vol%, which corresponds to the solids concentrations seen in lagoons. The sludge showed Newtonian behaviour at 0.4 vol% and Herschel-Bulkley yield stress fluid behaviour for higher concentrations ranging from 0.5 to 12 vol%. We compared multiple approaches to determine relationships between the model fitting parameters of consistency, k, flow index, n, and shear yield stress, τy with solids volume fraction ϕ.The solid-liquid separation properties were measured from sedimentation and filtration experiments to obtain compressibility and permeability properties across all the above-mentioned concentrations, enabling development of hindered velocity sedimentation curves. Comparison to full-scale anaerobic digestate identified that the pilot lagoon sludge had faster sedimentation at a given solids concentration in comparison to the digestate. This is the first study on simultaneous rheological characterisation and solid-liquid separation behaviour of an anaerobic sludge across a wide range of concentrations, thus enabling CFD modelling of the hydrodynamics and performance of anaerobic lagoons.

Light attenuation in enriched purple phototrophic bacteria cultures: Implications for modelling and reactor design.

Light attenuation in enriched purple phototrophic bacteria (PPB) cultures has not been studied, and its understanding is critical for proper process modelling and reactor design, especially for scaled systems. This work evaluated the effect of different biomass concentrations, reactor configurations, wastewater matrices, and growth conditions, on the attenuation extent of near infra-red (NIR) and ultraviolet-visible (UV-VIS) light spectra. The results show that increased biomass concentrations lead to higher light attenuation, and that PPB absorb both VIS and NIR wavelengths, with both fractions of the spectrum being equally absorbed at biomass concentrations above 1,000 g COD·m-3. A flat plate configuration showed less attenuation compared with cylindrical reactors illuminated from the top, representative for open ponds. Neither a complex wastewater matrix nor the presence of polyhydroxyalkanoates (under nutrient limited conditions) affected light attenuation significantly. The pigment concentration (both bacteriochlorophyll and carotenoids) however, had a strong effect, with significant attenuation in the presence of pigments. Attenuation predictions using the Lambert-Beer law (excluding scattering) and the Schuster model (including scattering) indicated that light scattering had a minimal effect. A proposed mathematical model, based on the Lambert-Beer law and a Monod function for light requirements, allowed effective prediction of the kinetics of photoheterotrophic growth. This resulted in a half saturation coefficient of 4.6 W·m-2. Finally, the results showed that in dense outdoor PPB cultures (≥1,000 g COD·m-3), effective light penetration is only 5 cm, which biases design away from horizontal lagoons, and towards non-incident multi-panel systems such as flat plate reactors.

Characterising sedimentation velocity of primary waste water solids and effluents.

Sedimentation in waste water is a heavily studied topic, but mainly focused on hindered and compression settling in secondary sludge, a largely monodispersed solids, where bulk sedimentation velocity is effectively described by functions such as double Vesilind (Takacs). However, many waste water solids, including primary sludge and anaerobic digester effluent are polydispersed, for which application of velocity functions is not well understood. These systems are also subject to large concentration gradients, and poor availability of settling velocity functions has limited design and computational fluid dynamic (CFD) analysis of these units. In this work, we assess the use of various sedimentation functions in single and multi-dimensional domains, comparing model results against multiple batch settling tests at a range of high and low concentrations. Both solids concentration and sludge bed height (interface) over time are measured and compared. The method incorporates uncertainty analysis using Monte Carlo regression, DIRECT (dividing rectangles), and Newton optimisation. It was identified that a double Vesilind (Takacs) model was most effective in the dilute regime (<1%v/v), but could not effectively fit high solids concentrations (>1%v/v) without a substantial (50%) decrease in effective maximum sedimentation velocity (V0). Other parameters (Rh, Rp) did not change. A power law velocity model (Diehl) was significantly less predictive at low concentrations, and not significantly better at higher concentrations. The optimised model (with reduction in V0) was tested vs a standard (optimised) double Vesilind velocity model in a simple primary sedimentation unit, and resulted in deviation from -12% to +18% in solids capture prediction from underload to overload (washout) conditions, indicating that the effect is important in CFD based analysis of these systems.

Method development for PPB culture screening, pigment analysis with UPLC-UV-HRMS vs. spectrophotometric methods, and spectral decomposition-based analysis.

PPB carotenoids are usually measured through spectrophotometric analysis, measuring total carotenoids (TCs) which has low accuracy and cannot identify individual carotenoids or isomers. Here, we developed an ultra-performance liquid chromatography method with ultraviolet and high-resolution mass spectrometry detection (UPLC-UV-HRMS) to quantify neurosporene, lycopene, and bacteriochlorophyll a contents in PPB cultures. The method exhibited satisfactory recoveries for individual pigments (between 82.1% and 99.5%) and was applied to a range of mixed PPB cultures. The use of a C30 column also enabled the detection of three different isomers of lycopene. In addition, a method for anaerobic photoheterotrophic PPB cultivation to acquire live-cell spectrophotometric information was developed and tested by modifying a standard microbial culture microplate system. A rapid, and relatively low effort principal component analysis (PCA) based decomposition of the whole-cell spectra for pigment analysis in the microplates was also developed. Analysing whole-cell spectra via PCA allowed more accurate prediction of individual pigments compared to absorption methods, and can be done non-destructively, during live-cell growth, but requires calibration for new media and microbial matrices.

Creating value from purple phototrophic bacteria via single-cell protein production.

Mixed culture purple phototrophic bacteria (PPB) is a rapidly emerging technology for resource recovery from wastewaters. PPB biomass can be used as single-cell protein, with a high protein content complemented by value-added components (e.g. pigments and polyhydroxyalkanoates), merging functionalities within a single product. This has the potential to increase the value and impact the economic feasibility, justifying higher capital costs for PPB photobioreactors for real-life applications. Artificial illumination is prohibitively expensive, and naturally illuminated, outdoor units are a critical next step. However, information required for informed technoeconomic assessment of single-cell protein from PPB is still missing and can only be determined in dedicated larger-scale, outdoor systems. Larger scale units are also required to supply feed for larger cohort trials. Although data from microalgae research can be used as starting point to estimate costs, they cannot be translated directly for PPB, as the organisms and metabolic growth are fundamentally different.

Outdoor demonstration-scale flat plate photobioreactor for resource recovery with purple phototrophic bacteria.

To make purple phototrophic bacteria (PPB)-based technologies a reality for resource recovery, research must be demonstrated outdoors, using scaled reactors. In this study, a 10 m long PPB-enriched flat plate photobioreactor (FPPBR) with a volume of 0.95 m3 was operated for 253 days, fed with poultry processing wastewater. Different operational strategies were tested, including varying influent types, retention times, feeding strategies, and anaerobic/aerobic conditions in a novel mixed metabolic mode concept. The overall results show that regardless of the fermented wastewater fed (raw or after solid removal via dissolved air flotation) and the varying environmental conditions (e.g., light exposure and temperatures), the FPPBR provided effective volatile fatty acids (VFAs), N, and P removals (average efficiencies of >90%, 34-77%, and 28-45%, respectively). The removal of N and P was limited by the availability of biodegradable COD. Biomass (C, N and P) could be harvested at ∼90% VS/TS ratio, 58% crude protein content and a suitable amino acid profile for potential feed applications. During fully anaerobic operation with semicontinuous/day-only feeding, the FPPBR showed biomass productivities between 25 and 84 g VS m-2 d-1 (high due to solid influx; the productivities estimated from COD removal rates were 6.0-24 g VS•m-2•d-1 (conservative values)), and soluble COD removal rates of up to 1.0 g•L-1•d-1 (overall average of 0.34 ± 0.16 g•L-1•d-1). Under these conditions, the relative abundance of PPB in the harvested biomass was up to 56%. A minimum overall HRT of 2-2.4 d (1.0-1.2 d when only fed during the day) is recommended to avoid PPB washout, assuming no biomass retention. A combined daily-illuminated-anaerobic/night-aerobic operation (supplying air during night-time) exploiting photoheterotrophy during the day and aerobic chemoheterotrophy of the same bacteria at night improved the overall removal performance, avoiding VFA accumulation during the night. However, while enabling enhanced treatment, this resulted in a lower relative abundance of PPB and reduced biomass productivities, highlighting the need to balance resource recovery and treatment goals.

Naturally illuminated photobioreactors for resource recovery from piggery and chicken-processing wastewaters utilising purple phototrophic bacteria.

Resource recovery from wastewater, preferably as high value products, has become an integral part of modern wastewater treatment. This work presents the potential to produce single cell protein (SCP) from pre-settled piggery wastewater (PWW) and meat chicken processing wastewater (CWW), utilising anaerobic purple phototrophic bacteria (PPB). PPB were grown as biofilm in outdoors 60 L, 80 L and 100 L flat-plate reactors, operated in sequential batch mode. PPB biofilm was recovered from reactor walls at a total solid (TS) content ∼90 g•L - 1, and the harvested biomass (depending on the wastewater) had a consistent quality, with high protein contents (50-65%) and low ash, potentially applicable as SCP. The COD, N and P removal efficiencies were 71±5.3%, 22±6.6%, 65±5.6% for PWW and 78±1.8%, 67±2.7% and 37±4.0% for CWW, respectively, with biofilm areal productivities up to 14 g TS•m - 2•d - 1. This was achieved at ammonium-N concentrations over 1.0 g•L - 1 and temperatures up to 55 °C and down to 6 °C (daily fluctuations of 20-30 °C). The removal performances and biomass productivities were mostly dependent on the bioavailable COD in the form of volatile fatty acids (VFA). At sufficient VFA availability, the irradiance became limiting, capping biofilm formation. Harvesting of the suspended fraction resulted in increased productivities and recovery efficiencies, but lowered the product quality (e.g., containing undesired inerts). The optimum between quantity and quality of product is dependent on the wastewater characteristics (i.e., organic degradable fraction) and potential pre-treatment. This study shows the potential to utilise sunlight to treat agri-industrial wastewaters while generating protein-rich PPB biomass to be used as a feed, feed additive or feed supplement.

Kinetics of aerobic cellulose degradation in raw municipal wastewater.

Cellulose contributes approximately one third of the influent suspended solids to wastewater treatment plants and is a key target for resource recovery. This study investigated the temperature impact on biological aerobic degradation of cellulose in laboratory-scale sequencing batch reactors (SBR) at four different temperatures (10-33 °C) and two different solids retention times (SRT) of 15 days and 3 days. The degradation efficiency of cellulose was observed to increase with temperature and was slightly dependent on SRT (80%-90% at an SRT of 15 days, and 78%-85% at an SRT of 3 days). Hydrolysis followed 1st order kinetics, rather than the biomass dependent Contois kinetics (default in the activated sludge models), with a hydrolysis coefficient at 20 °C of 1.14 ± 0.01 day-1.

The role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge.

The use of next-generation diagnostic tools to optimise the anaerobic digestion of municipal sewage sludge has the potential to increase renewable natural gas recovery, improve the reuse of biosolid fertilisers and help operators expand circular economies globally. This review aims to provide perspectives on the role of microbial ecology in improving digester performance in wastewater treatment plants, highlighting that a systems biology approach is fundamental for monitoring mesophilic anaerobic sewage sludge in continuously stirred reactor tanks. We further highlight the potential applications arising from investigations into sludge ecology. The principal limitation for improvements in methane recoveries or in process stability of anaerobic digestion, especially after pre-treatment or during co-digestion, are ecological knowledge gaps related to the front-end metabolism (hydrolysis and fermentation). Operational problems such as stable biological foaming are a key problem, for which ecological markers are a suitable approach. However, no biomarkers exist yet to assist in monitoring and management of clade-specific foaming potentials along with other risks, such as pollutants and pathogens. Fundamental ecological principles apply to anaerobic digestion, which presents opportunities to predict and manipulate reactor functions. The path ahead for mapping ecological markers on process endpoints and risk factors of anaerobic digestion will involve numerical ecology, an expanding field that employs metrics derived from alpha, beta, phylogenetic, taxonomic, and functional diversity, as well as from phenotypes or life strategies derived from genetic potentials. In contrast to addressing operational issues (as noted above), which are effectively addressed by whole population or individual biomarkers, broad improvement and optimisation of function will require enhancement of hydrolysis and acidogenic processes. This will require a discovery-based approach, which will involve integrative research involving the proteome and metabolome. This will utilise, but overcome current limitations of DNA-centric approaches, and likely have broad application outside the specific field of anaerobic digestion.

Hydrodynamic analysis of full-scale in-situ biogas upgrading in manure digesters.

The addition of hydrogen to anaerobic digesters is an emerging technique for the sustainable upgrading of biogas to biomethane with renewable electricity. However, it is critically dependent on the effective gas-liquid transfer of hydrogen, which is a sparingly soluble gas. Very little is known about the impact of liquid and gas flow and bubble size on gas-liquid transfer during H2 injection in full-scale anaerobic digesters. A computational fluid dynamic model was developed using a two-fluid approach for non-Newtonian liquid in the open-source computational fluid dynamics (CFD) platform, OpenFOAM. The newly developed model was validated against published experimental data-sets of a gas-mixed, laboratory-scale anaerobic digester, with good agreement between the numerical and experimental velocity fields. The hydrodynamics of the full-scale in-situ biomethanation system using venturi ejectors for H2 injection was then simulated to investigate gas-liquid dynamics, including gas-liquid mass transfer, at different operational conditions. Gas-liquid mixing is mainly controlled by the gas-plumes interaction, which promotes turbulence at the interaction zone, resulting in increasing gas bubbles mixing with the liquid and the gas-liquid interfacial area. However, beyond the plume interaction zone, the digester had flow short-circuiting and inactive zones. It was found that, due to this short-circuiting behaviour, an increase in gas flow-rate may not be an effective option in reducing inactive zones, although it can increase the gas-liquid interfacial area. Comparative analysis of the impact of gas flow and bubble size indicated that gas flow had a linear effect on both kLa and gas holdup, but that bubble size had a non-linear impact, with higher kLa values achieved at bubble sizes less than 2 mm. Comparison against measured data in the same system indicated the predicted kLa values were at the same level as measured kLa, at a bubble size of 2 mm.

Uncertainty analysis of rising sewer models with respect to input parameters and model structure using Monte Carlo simulations and computational fluid dynamics.

Modelling conversion processes in sewers can help minimize odour and pipe corrosion issues, but model uncertainties and errors must be understood. In this study, the Wastewater Aerobic/Anaerobic Transformation in Sewers (WATS) model is implemented in two different frameworks; 1-D (CSTR-in-series) and computational fluid dynamics (CFD) to study the uncertainties due to model parameters and its mathematical form. The 1-D model is used to conduct uncertainty/sensitivity analysis using Monte Carlo simulations. Time-averaged outputs were represented using a general linearized model to quantify the importance of specific parameters. The sulfide formation rate per unit area of the biofilm is the most influential parameter. Parameters controlling anaerobic hydrolysis and fermentation are also significant. Uncertainty due to model structure is studied using CFD to explore the influences of non-homogeneous surface reactions and solids settling. These showed that the 1-D model provides a reasonable characterisation of the process for simple flows in pressure mains.

Purple phototrophic bacteria are outcompeted by aerobic heterotrophs in the presence of oxygen.

There is an ongoing debate around the effect of microaerobic/aerobic conditions on the wastewater treatment performance and stability of enriched purple phototrophic bacteria (PPB) cultures. It is well known that oxygen-induced oxidative conditions inhibit the synthesis of light harvesting complexes, required for photoheterotrophy. However, in applied research, several publications have reported efficient wastewater treatment at high dissolved oxygen (DO) levels. This study evaluated the impact of different DO concentrations (0-0.25 mg·L-1, 0-0.5 mg·L-1 and 0-4.5 mg·L-1) on the COD, nitrogen and phosphorus removal performances, the biomass yields, and the final microbial communities of PPB-enriched cultures, treating real wastewaters (domestic and poultry processing wastewater). The results show that the presence of oxygen suppressed photoheterotrophic growth, which led to a complete pigment and colour loss in a matter of 20-30 h after starting the batch. Under aerobic conditions, chemoheterotrophy was the dominant catabolic pathway, with wastewater treatment performances similar to those achieved in common aerobic reactors, rather than those corresponding to phototrophic systems (i.e. considerable total COD decrease (45-57% aerobically vs. ± 10% anaerobically). This includes faster consumption of COD and nutrients, lower nutrient removal efficiencies (50-58% vs. 72-99% for NH4+-N), lower COD:N:P substrate ratios (100:4.5-5.0:0.4-0.8 vs. 100:6.7-12:0.9-1.2), and lower apparent biomass yields (0.15-0.31 vs. 0.8-1.2 g CODbiomass·g CODremoved-1)). The suppression of photoheterotrophy inevitably resulted in a reduction of the relative PPB abundances in all the aerated tests (below 20% at the end of the tests), as PPB lost their main competitive advantage against competing aerobic heterotrophic microbes. This was explained by the lower aerobic PPB growth rates (2.4 d-1 at 35 °C) when compared to common growth rates for aerobic heterotrophs (6.0 d-1 at 20 °C). Therefore, PPB effectively outcompete other microbes under illuminated-anaerobic conditions, but not under aerobic or even micro-aerobic conditions, as shown by continuously aerated tests controlled at undetectable DO levels. While their aerobic heterotrophic capabilities provide some resilience, at non-sterile conditions PPB cannot dominate when growing chemoheterotrophically, and will be outcompeted.

Purple phototrophic bacteria granules under high and low upflow velocities.

The application of granular biomass has enabled energy efficient, high-rate wastewater treatment systems. While initially designed for high-strength wastewater treatment, granular systems can also play a major role in resource recovery. This study focused on the formation of purple phototrophic bacteria (PPB) granular biomass during synthetic wastewater treatment. Liquid upflow velocity was applied as the driving force for granulation. Separate reactors were operated at either low (2-5m h-1) or high (6-9m h-1) upflow velocities, with sludge retention times (SRTs) ranging from 5-15d. Reactors produced anaerobic, photo-granules within ~50d. The sludge volume index (SVI30) of the granules was 10mL g-1 and average settling rates were greater than 30m h-1, both metrics being similar to existing granular technologies. Granule sizes of 2-3mm were recorded, however the particle size distribution was bimodal with a large floc fraction (70-80% volume fraction). The extracellular polymeric substance (EPS) and alginate-like extract (ALE) contents were similar to those in aerobic granular biomass. Fluorescence in-situ hybridisation (FISH) imaging identified PPB bacteria dispersed throughout the granules with very few methanogens and an active core. Outer layer morphology was substantially different in the two reactors. The high-upflow reactor had an outer layer of Chromatiales and an inner layer of Rhodobacteriales, while the low-upflow reactor had lower abundances of both, and limited layering. According to 16s gene sequencing, PPB were a similar fraction of the microbial community in both reactors (40-70%), but the high upflow granules were dominated by Chromatiales (supporting FISH results), while the low upflow velocity reactor had a more diverse PPB community. Methanogens were seen only in the low upflow granules and only in small amounts (≤8%). Granule crude protein content was ~0.60gCP gVS-1 (~0.45gCP gTS-1), similar to that from other PPB production technologies. The growth of a rapid settling and discrete PPB granular biomass on synthetic wastewater suggests methods for resource recovery using PPB can be diversified to also include granular biomass.

Autotrophic sulfide removal by mixed culture purple phototrophic bacteria.

Current H2S treatment methods for sour gases require considerable amounts of chemicals and energy, or in case of biological treatment, unwanted diluents such as oxygen or nitrogen may be introduced. In order to reduce those requirements, the viability of an anaerobic biological H2S removal process using purple phototrophic bacteria (PPB) was investigated in this study. PPB can use sunlight, and centrate as nutrient source, thus potentially reducing energy and chemical requirements. An added benefit is the production of biomass with potential uses, such as single cell protein. An inoculum of PPB enriched from domestic wastewater was grown photoautotrophically with sulfide as the electron donor and inorganic carbon in a mixed culture. Additionally, synthetic medium and centrate as well as high (56 ±â€¯11 Wm-2) and low (27 ±â€¯3 Wm-2) IR irradiation were trialled. Finally, a process model was developed to study biomass specific removal rates and yield. The results showed that a mixed culture of PPB removed sulfide completely in synthetic media (121 ±â€¯9 mg-S.L-1) at a maximum rate of 1.79 ±â€¯0.16 mg-S(Lh)-1 (low irradiance) and 2.9 mg-S(Lh)-1 (high irradiance). The pH increased in both experiments from about 8.5 to 9. Sulfide removal rates using centrate and low irradiance were similar. However Fe and Mn were found to be limiting growth and sulfide removal. In all experiments, Chromatiaceae (purple sulfur bacteria) were most abundant at the end of the experiment, while at the start purple non-sulfur bacteria were most abundant (from the inoculum). Process modelling and experimental work identified the sulfide oxidation to be a multi-step process with accumulation of intermediates. Specific rates were directly dependent on light input, doubling at high irradiance. Sulfide oxidation was estimated at 0.100 ±â€¯0.014 h-1 (0.085 ±â€¯0.012 g-S(g-VS.h)-1) at low irradiance, and the biomass yield at 0.86 ±â€¯0.05 mg-COD.mg-COD-1. This process model enables the virtual evaluation of autotrophic sulfide removal by PPB in a continuous scaled-up process. Overall, the photoautotrophic removal of sulfide seems to be a viable option, especially because of the possibility of using sunlight as an energy source and centrate as a nutrient source.