Specific Uptake Rate Research Articles

Response to shock load of titanium dioxide nanoparticles on aerobic granular sludge and algal-bacterial granular sludge processes

Titanium dioxide nanoparticles (TiO2 NPs) are extensively used in various fields and can consequently be detected in wastewater, making it necessary to study their potential impacts on biological wastewater treatment processes. In this study, the shock-load impacts of TiO2 NPs were investigated at concentrations ranging between 1 and 200 mg L−1 on nutrient removal, extracellular polymeric substances (EPSs), microbial activity in aerobic granular sludge (AGS), and algal-bacterial granular sludge (AB-AGS) bioreactors. The results indicated that low concentration (≤10 mg L−1) TiO2 NPs had no effect on microbial activity or the removal of chemical oxygen demand (COD), nitrogen, and phosphorus, due to the increased production of extracellular polymeric substances (EPSs) in the sludge. In contrast, the performance of both AGS and AB-AGS bioreactors gradually deteriorated as the concentration of TiO2 NPs in the influent increased to 50, 100, and 200 mg L−1. Specifically, the ammonia‑nitrogen removal rate in AGS decreased from 99.9 % to 88.6 %, while in AB-AGS it dropped to 91.3 % at 200 mg L−1 TiO2 NPs. Furthermore, the nitrate‑nitrogen levels remained stable in AB-AGS, while NO3-N was detected in the effluent of AGS at 100 and 200 mg L−1. Microbial activities change similarly as smaller decrease in the specific ammonia uptake rate (SAUR) and specific nitrate uptake rate (SNUR) was found in AB-AGS compared to those in AGS. Overall, the algal-bacterial sludge exhibited higher resilience against TiO2 NPs, which was attributed to a) higher EPS volume, b) smaller decrease in LB-EPS, and c) the favorable protein to polysaccharide (PN/PS) ratio. This in turn, along with the symbiotic relationship between the algae and bacteria, mitigates the toxic effects of nanoparticles.

Leveraging the dynamics of microalgal CO2 capture to estimate the maximum inherent photosynthetic potential

AbstractBACKGROUNDNatural photosynthesis, utilizing intelligent molecular machinery to harness sunlight, stands as the benchmark for efficient energy generation. Despite its high quantum efficiency compared to synthetic methods, challenges persist due to dynamic nutritional and light requirements in plant, microalgae, and cyanobacteria‐driven processes. Reported microalgae CO2 uptake rates rely on biomass dry cell weight measurement after certain incubation period and systematic study of required CO2 concentrations for optimal photobioreactor operation remaining unexplored. This research is thus aimed at evaluating the critical dissolved CO2 (Ccrit ∙ CO2) concentration and specific CO2 uptake rates (SCUR) of Chlorella minutissima (CM) culture in the presence of surplus light and other nutrients by online monitoring and measuring the dynamic CO2 uptake rates which will help in maintaining the optimal rate of CO2 delivery for continuous cultivation of microalgae while minimizing the escape of CO2.RESULTSDissolved Ccrit ∙ CO2 was determined to be in the range of 16–20 mg/L, and the maximum SCUR in BBM (Bold Basal Medium) was found to be 0.73 mg CO2 (mg dcw ∙ h)−1. Interestingly, this SCUR value is nearly three times greater than that of the most efficient in vitro artificial photosynthetic novel pathway reported so far.CONCLUSIONFrom the calculated SCUR values, it may be noted that 9.6 g of biomass can be obtained from 1 g of microalgal inoculum in a day, thereby setting a benchmark for the scientists working in the areas of bioprocess engineering, synthetic biology, and metabolic engineering to comply. © 2024 Society of Chemical Industry (SCI).

Development of a defined medium for the heterotrophic cultivation of Metallosphaera sedula

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

Combined Effects of Tetracycline and Copper Ion on Microorganisms During the Biological Phosphorus Removal.

Tetracycline and copper ion are common pollutants in wastewater, and the effects of mixed pollutants on microorganisms in wastewater biological treatment have been less studied. In order to reveal the effects of mixed pollutants of tetracycline and copper ion on the microorganisms during the biological phosphorus removal, three ratios of tetracycline and copper ions were designed by the direct equipartition ray method. The relative abundance and diversity of microbial community were investigated, and the microbial interactions were revealed through microbiological methods. The results demonstrated that, for three different ratios, the inhibitory effect of specific phosphorus uptake rate became more significant with the increase of the tetracycline-copper ions concentration and the reaction time. The microbial community decreased with the increase of the proportion of tetracycline in different ratios. The relative abundance of Acinetobacter decreased with the increase of the proportion of tetracycline, while the relative abundance of Ca.Competibacter was higher under the conditions of low mixtures concentrations. Positive interactions and symbiotic relationships among microorganisms were predominant for three different ratios. However, as the proportion of tetracycline increased, the community structure of microorganisms shifted from phosphate-accumulating organisms to glycogen accumulating organisms and denitrifying bacteria. This study can provide a reference for the effect of mixed pollutants on microorganisms and the mechanism of wastewater treatment.

Nitrogen Fixation at the Mid‐Atlantic Bight Shelfbreak and Transport of Newly Fixed Nitrogen to the Slope Sea

AbstractContinental shelves contribute a large fraction of the ocean's new nitrogen (N) via N2 fixation; yet, we know little about how physical processes at the ocean's margins shape diazotroph biogeography and activity. Here, we test the hypothesis that frontal mixing favors N2 fixation at the Mid‐Atlantic Bight shelfbreak. Using the 15N2 bubble release method, we measured N2 fixation rates on repeat cross‐frontal transects in July 2019. N2 fixation rates in shelf waters (median = 5.42 nmol N L−1 d−1) were higher than offshore (2.48 nmol N L−1 d−1) but did not significantly differ front frontal waters (8.42 nmol N L−1 d−1). However, specific N2 uptake rates, indicative of the relative contribution of diazotroph‐derived N to particulate N turnover, were significantly higher in frontal waters, suggesting that diazotroph‐derived N is of greater importance in supporting productivity there. This study furthered captured an ephemeral shelf‐water streamer, which resulted from the impingement of a warm core ring on the shelf. The streamer transported shelf‐water diazotrophs (including UCYN‐A and Richelia spp., as assessed by qPCR) offshore with sustained high N2 fixation rates. This feature injected >50 metric tons d−1 of newly fixed N to the Slope Sea—a rate equivalent to ∼4% of the total N flux estimated for the entire Mid‐Atlantic Bight. As intrusions of Gulf Stream meanders and eddies onto the shelf are increasing in frequency due to climate change, episodic lateral fluxes of new N into the Slope Sea may become increasingly important to regional budgets and ecosystem productivity.

Production of poly‐gamma‐glutamic acid and spore formation in Bacillus velezensis 83 are affected by acidic growing conditions and glucose uptake rate

AbstractBACKGROUNDBacillus velezensis 83 is a biocontrol agent and plant growth promoting bacteria that produces antifungal compounds (surfactin and bacilomycin D), plant growth stimulation metabolites such as acetoin and butanediol and the polymer poly‐gamma‐glutamic acid (γ‐PGA), which is essential for bacterial plant colonization. Bacillus sporulation is an important trait in order to obtain long shelf‐life formulations for biocontrol purposes. γ‐PGA production and sporulation are closed‐related processes controlled by glucose availability and modulated by quorum sensing. High spore production (and its recovery) in B. velezensis 83 cultures is limited by the high viscosity oxygen‐limited broths resulting from γ‐PGA accumulation. This work studied the influence of acidic growing conditions and glucose specific uptake rate on growth, quorum sensing‐related metabolites production and sporulation of B. velezensis 83.RESULTSIn batch cultures developed at pH = 5, no bacterial sporulation was detected and quorum sensing related metabolites as γ‐PGA (not detected) and surfactin and bacillomycin were significantly decreased. Instead, acetoin and butanediol were the main products. In contrast, in continuous cultures developed at pH = 5, spores were detected at D = 0.06 h−1. The results indicate that sporulation at pH = 5 is not inhibited by affectations on quorum sensing signals but could be due to a low availability/transport of energy sources (i.e., glucose or acetoin/butanediol) or an affectation on Rap‐Phr system.CONCLUSIONThe findings in this study allow to establish a two‐stage process for Bacillus velezensis 83 spore production: bacterial growth at pH = 5 and sporulation at pH = 6.8. © 2023 Society of Chemical Industry (SCI).

Potential of enriched phototrophic purple bacteria for H2 bioconversion into single cell protein

Single cell protein (SCP) has emerged as an alternative protein source, potentially based on the recovery of carbon and nutrients from waste-derived resources as part of the circular economy. From those resources, gaseous substrates have the advantage of an easy sterilization, allowing the production of pathogen-free SCP. Sterile gaseous substrates allow producing pathogen-free SCP. This study evaluated the use of an enriched phototrophic purple bacteria (PPB) consortium for SCP production using H2 and CO2 as electron and C sources. The influence of pH (6.0–8.5), temperature (15–50 °C) and light intensity (0–50 W·m−2) on the growth kinetics and biomass yields was investigated using batch tests. Optimal conditions were found at pH 7, 25 °C and light intensities over 30 W·m−2. High biomass and protein yields were achieved (~ 1 g CODbiomass·g CODH2consumed−1 and 3.9–4.4 g protein·g H2−1) regardless of the environmental conditions, being amongst the highest values reported from gaseous streams. These high yields were obtained thanks to the use of light as a sole energy source by the PPB consortium, allowing a total utilization of H2 for growth. Hydrogen uptake rates varied considerably, with values up to 61 ± 5 mg COD·d−1 for the overall H2 consumption rates and 2.00 ± 0.14 g COD·g COD−1·d−1 for the maximum specific uptake rates under optimal growth conditions. The latter value was estimated using a mechanistic model able to represent PPB growth on H2. The biomass exhibited high protein contents (>50 % w/w) and adequate amino acid profiles, showing its suitability as SCP for feed. PPB were the dominant bacteria during the experiments (relative abundance over 80 % in most tests), with a stable population dominated by Rhodobacter sp. and Rhodopseudomonas sp. This study demonstrates the potential of enriched PPB cultures for H2 bioconversion into SCP.

Modeling of simultaneous carbon and nitrogen removal (SCNR) in an anaerobic/anoxic reactor treating salmon fishery wastewater

Mathematical modeling can help to clarify some aspects of the process that promote the simultaneous removal of organic matter and nitrogen (SCNR) from the fish processing industry wastewater that still require elucidation, such as the competition between methanogenic and denitrifying microorganisms for the substrate, for example. Therefore, the aim of this paper is to present the application of the Anaerobic Digestion Model No. 1 (ADM1) for modeling the SCNR process in a single stage anaerobic/anoxic reactor fed with a mix of a synthetic substrate and a saline protein-rich salmon processing industry. The ADM1 was not specifically developed for the treatment of this wastewater, therefore, initially, a parametric sensitivity analysis was performed to define the most sensitive parameters. The sensitivities were quantified in terms of the variation of measurable process variables under the perturbation of model parameters in their neighborhood domain. After this analysis, the model was calibrated and validated using previously published experimental data. Based on a sensitivity analysis, five parameters of the original ADM1 and of the denitrification processes were estimated using iterative methods, which optimized the parameters with experimental results. Namely: proteins hydrolysis constant (khyd,pr = 0.05 h−1); Monod maximum specific uptake rate for amino acids utilizers (km,aa = 1.87 h−1); half saturation value for amino acids utilizers (KS,aa = 0.23 kg COD m−3); Monod maximum specific uptake rate for acetate utilization rates by the denitrifiers (kno3,ac = 0.09 h−1) and half saturation value for the utilization of the acetate by the denitrifiers (KS,no3,ac = 0.38 kg COD m−3). Simulation with calibrated parameters indicated good agreement with experimental data, with the mean absolute error below 40 %. Considering these optimal values, the complete reduction of the oxidized forms of nitrogen occurred up to 11 h of reaction time. Cross-validation results indicated that the calibrated model was able to predict the experimental results of effluent COD, acetate, propionate, butyrate, nitrate and nitrite flows with good accuracy showing a mean absolute error below 40 % (19 %–36 %).

Autogenerative high-pressure anaerobic digestion modelling of volatile fatty acids: Effect of pressure variation and substrate composition on volumetric mass transfer coefficients, kinetic parameters, and process performance

Pressurised anaerobic digestion is a valuable process that allows the production of biogas with a high methane content, reducing the energy costs for the biogas upgrading and injection into the distribution grid. This technology has attracted scientific research attention in the last decade, and kinetic models have been formulated. In this work, a modified Anaerobic Model Digestion n.1 of an autogenerative high‑pressure anaerobic digestion of volatile fatty acids in batch configuration is proposed by evaluating the effect of increasing the autogenerative pressure in the reactor on the prediction of the dynamic performances and kinetic parameters. Experimental results from the literature were used for developing, calibrating, and validating the model. Results highlighted the dependence of the mass transfer coefficient of the main component of biogas on the increasing autogenerative pressure: the higher the pressure, the higher the volumetric mass transfer coefficient. At atmospheric pressure, the volumetric mass transfer coefficient is almost equal (about 0.065–0.067 1/s). By increasing the pressure, H2 showed the highest value (0.098 1/s at 20 bar), while for CH4 and CO2, comparable values were assessed (0.085 1/s at 20 bar). Results also highlighted the effect of the pressure variation on acetate, butyrate, and propionate Monod maximum specific uptake rate, half saturation value, the first order decay rate, and the Upper and Lower pH levels for acetate degraders. Moreover, hydrogen produced during the process was considered an additional substrate, for which the kinetic parameters were assessed. Finally, simulation findings are consistent with the literature data in terms of volatile fatty acid consumption.

Robotic workflows for automated long-term adaptive laboratory evolution: improving ethanol utilization by Corynebacterium glutamicum

BackgroundAdaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation.ResultsHere, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW−1 h−1 and a growth rate of 0.15 ± 0.01 h−1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements.ConclusionsThe established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

KLa based scale-up cultivation of the extremophilic archaeon Sulfolobus acidocaldarius: from benchtop to pilot scale.

The two major scale-up criteria in continuously stirred bioreactors are 1) constant aerated power input per volume (Pg/Vl), and 2) the volumetric O2 mass transfer coefficient (kla). However, Pg/Vl is only influenced by the stirrer geometry, stirrer speed, aeration and working volume, while the kla is additionally affected by physiochemical properties of the medium (temperature, pH, salt content, etc.), sparging of gas and also by the bioreactor design. The extremophilic archaeon Sulfolobus acidocaldarius, thriving at 75°C and pH 3.0, has the potential for many biotechnological applications. However, previous studies imply that the family Sulfolobaceae might be affected by higher oxygen concentration in the headspace (>26%). Hence, adequate oxygen supply without being toxic has to be ensured throughout the different scales. In this study, the scale-up criteria Pg/Vl and kla were analyzed and compared in a 2L chemostat cultivation of S. acidocaldarius on a defined growth medium at 75°C and a pH value of 3.0, using two different types of spargers at the same aerated power input. The scale-up criterion kLa, ensuring a high specific growth rate as well as viability, was then used for scaleup to 20L and 200L. By maintaining a constant kla comparable dry cell weight, specific growth rate, specific substrate uptake rates and viability were observed between all investigated scales. This procedure harbors the potential for further scale-up to industrial size bioreactors.

Characterisation of acetogen formatotrophic potential using Eubacterium limosum.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280 ± 56mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO2 (150 ± 11mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. KEY POINTS: First Eubacterium limosum steady-state formatotrophic growth omics dataset High formate specific uptake rate, however carbon dioxide was the major product Formate may be the cause of intracellular stress and biofilm formation.

Characterizing and utilizing oxygen-dependent promoters for efficient dynamic metabolic engineering

Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dissolved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and additionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear particularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F1-subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth-arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction.

Removal of radioactive material (Uranium-VI) from low level wastewaters and subsequent disposal of loaded biomass

The concentration of radioactive material i.e., Uranium, when disposed from nuclear reactor as low-level wastewater is under the limit value of 10 mg/l and concentration from mine tailing wastes is in the order of 10–100 mg/l. Biosorption is very cost-effective adsorption mechanism, discussed in this paper for removal Uranium (VI) from LLW using nonviable fungus called Ganoderma lucidum. Non-viable fungus has its advantage over viable fungus like for viable fungus one has to maintain temperature, pH and nutrient level. In this paper, the fungus called Ganoderma lucidum (GL) was used for sequestering uranium (VI) followed by studying their equilibrium and kinetics. Results show sorption rate is satisfactory, and data fitted a model of second order. Also, GL to be very effective biosorbent with 70 % of the total sequestration took place during the first 5 min for pure uranyl solution the corresponding specific uranium uptake rates being 0.106 mg/g-min at equilibrium concentration of 10 mg/L (Uranium VI) and pH maintained is 5. In this paper effect of competing co-ions were also studied and it is found out that effect of iron on uranium uptake is quite significant whereas the effect of other co-ions is at best marginal. Thus, biosorption by GL fungus is energy efficient and cost-effective method for removal of radioactive material from low level wastewater.

Temperature-light shapes the nutritional strategy of a mixotrophic green alga, Picochlorum sp. GLMF1 (Trebouxiophyceae)

Constitutive mixotrophs (CM), combining phagotrophy with inherent phototrophy, are critical primary producers and consumers in marine pelagic food webs. However, how mixotrophs, especially obligate CM, balance phototrophy and phagotrophy in response to changing environmental conditions remains equivocal. Here, we examined the growth performance, photosynthesis traits, and phagotrophic activities of a mixotrophic green alga, Picochlorum sp. GLMF1, over a wide range of temperature-irradiance combinations. The growth of Picochlorum sp. exhibited a unimodal thermal response under all the experimental light intensities. The optimal growth temperature decreased dramatically when the irradiance exceeded 80 μmol m-2 s-1, whereas the maximum growth rate kept a relatively constant and high value (around 1.1 d-1). This might be attributed to the trade-off between photosynthesis and ingestion under various light and temperature conditions. We found that the photosynthetic electron transport rate (ETR) and chlorophyll a specific inorganic carbon uptake rate increased with increasing irradiance. The maximum ETR decreased from 22.26 ± 1.93 μmol e m-2 s-1 at 22°C to 8.96 ± 0.42 μmol e m-2 s-1 at 31°C, suggesting a significant constraint of photosynthesis at high temperature. By contrast, ingestion increased with increasing temperature in most cases, and was significantly lower under high irradiance conditions. Combining the contributions of phototrophic and phagotrophic processes to total carbon acquisition, Picochlorum sp. showed a flexible nutritional strategy in response to temperature and light. Increasing light favors photosynthesis, whereas warming stimulates phagotrophy. Such complementation allows them to flourish under a wide range of temperature and irradiance. Our study adds knowledge to the nutritional strategy of mixotrophic green algae, with significant implications for their ecological role in marine food webs under projected climate change.

Phosphorus Removal from Aerobic Granular Sludge: Proliferation of Polyphosphate-Accumulating Organisms (PAOs) under Different Feeding Strategies

Aerobic granular sludge (AGS) is known for high phosphorus removal from wastewaters, and phosphorus can be recovered from high phosphorus-containing waste sludge granules. This study aimed at determining the feeding strategy that provides the best performance in terms of the proliferation of polyphosphate-accumulating organisms (PAOs) and phosphorus removal. Using three AGS bioreactors, this study compared phosphorus removal and the proliferation dynamics of PAOs under three different feeding strategies: anaerobic slow feeding (R1), pulse feeding + anaerobic mixing (R2), and pulse feeding (R3). Results indicate that R1 and R2 achieved significantly higher phosphorus removal (97.6 ± 3% for R1 and 98.3 ± 1% for R2) than R3 (55 ± 11%). The anaerobic slow feeding procedure (R1) achieved the highest specific phosphorus release rate (SPRR) and specific phosphorus uptake rate (SPUR) as compared to the other two feeding conditions. 16S ribosomal ribonucleic acid (rRNA) gene sequencing assay of the microbial community for the three feeding strategies indicated that although the feeding strategy impacted reactor performance, it did not significantly alter the microbial community. The bacteria community composition maintained a similar degree of diversity. Proteobacteria, Bacteroidetes, and Verrucomicrobia were the dominant bacterial phyla in the system. Dominant PAOs were from the class Betaproteobacteria and the genera Paracoccus and Thauera. Glycogen-accumulating organisms were significantly inhibited while other less-known bacteria such as Wandonia and Hyphomonas were observed in all three reactors.

Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E. coli on glycerol always required either the addition of electron acceptors for anaerobic respiration (e.g. fumarate) or the supplementation with complex and relatively expensive additives (tryptone or yeast extract). In the present work, driven by model-based calculations, we propose and validate a novel and simple strategy to enable fermentative growth of E. coli on glycerol in defined minimal medium. We show that redox balance could be achieved by uptake of small amounts of acetate with subsequent reduction to ethanol via acetyl-CoA. Using a directed laboratory evolution approach, we were able to confirm this hypothesis and to generate an E. coli strain that shows, under anaerobic conditions with glycerol as the main substrate and acetate as co-substrate, robust growth (μ = 0.06 h−1), a high specific glycerol uptake rate (10.2 mmol/gDW/h) and an ethanol yield close to the theoretical maximum (0.92 mol per mol glycerol). Using further stoichiometric calculations, we also clarify why complex additives such as tryptone used in previous studies enable E. coli to achieve redox balance. Our results provide new biological insights regarding the fermentative metabolism of E. coli and offer new perspectives for sustainable production processes based on glycerol.

Optimized Operating Conditions for a Biological Treatment Process of Industrial Residual Process Brine Using a Halophilic Mixed Culture

Residual process brine is a sustainable raw material for chlor-alkali electrolysis processes. This study investigates the influence of critical process parameters on the performance of a continuous treatment process for residual process brine using halophilic microorganisms. The goal of the bioprocess is an efficient degradation of the organic impurities formate, aniline, phenol, and 4,4′-methylenedianline from this residual stream. It was shown that formate could be degraded with high efficiencies (89–98%) during the treatment process. It was observed that formate degradation was influenced by the co-substrate glycerol. The lowest residual formate concentrations were achieved with specific glycerol uptake rates of 8.0–16.0 × 10−3 g L−1 h−1 OD600−1. Moreover, a triple-nutrient limitation for glycerol, ammonium, and phosphate was successfully applied for continuous cultivations. Furthermore, it was shown that all aromatic impurities were degraded with an efficiency of 100%. Ultimately, this study proposed optimized operating conditions, allowing the efficient degradation of organics in the residual process brine under various process conditions. Future optimization steps will require a strategy to prevent the accumulation of potential intermediate degradation products formed at high aniline feed concentrations and increase the liquid dilution rates of the system to achieve a higher throughput of brines.

Mechanistic modeling of glycerol fermenting and sulfate-reducing processes by granular sludge under sulfidogenic conditions

Glycerol can be converted to ethanol, 1,3-propanediol, formate, acetate, propionate, and inorganic carbon under anaerobic conditions through oxidative and reductive pathways in the absence and presence of sulfate. A structured mathematical model considering multiple pathways of glycerol fermentation combined with sulfate reduction was set up in this work, where three mechanisms were proposed and verified by modeling. Finally a mechanism properly predicting both glycerol fermenting and sulfate-reducing processes was chosen. Concentrations of multiple intermediates measured in batch activity tests were satisfactorily described by the model. The intermediate products of glycerol fermentation included formate, propionate, ethanol, 1,3-propanediol, and 3-hydroxypropionate (3HP). The main pathways of glycerol fermentation were the oxidative pathway to produce ethanol and the reductive pathway to produce 1,3-propanediol. The former accounted for 40.6% of the total glycerol converted whereas the latter accounted for 42.6%. 1,3-propanediol was converted to 3HP coupled to sulfate reduction. 3HP was mainly further oxidized to acetate. The kinetic parameters of maximum specific uptake rates of substrate were calibrated and then, the sulfate reduction process was validated. The confidence intervals of estimated parameters were assessed according to the Fisher information matrix (FIM) method. The low confidence intervals obtained indicated that the experimental behavior was satisfactorily described with the proposed kinetic model.

Modelling of autogenerative high-pressure anaerobic digestion in a batch reactor for the production of pressurised biogas

BackgroundPressurised anaerobic digestion allows the production of biogas with a high content of methane and, at the same time, avoid the energy costs for the biogas upgrading and injection into the distribution grid. The technology carries potential, but the research faces practical constraints by a.o. the capital investment needed in high-pressure reactors and sensors and associated sampling limitations. In this work, the kinetic model of an autogenerative high-pressure anaerobic digestion of acetate, as the representative compound of the aceticlastic methanogenesis route, in batch configuration, is proposed to predict the dynamic performance of pressurised digesters and support future experimental work. The modelling of autogenerative high-pressure anaerobic digestion in batch configuration, which is not extensively studied and simulated in the present literature, was developed, calibrated, and validated by using experimental results available from the literature.ResultsUnder high-pressure conditions, the assessment of the Monod maximum specific uptake rate, the half-saturation constant and the first-order decay rate was carried out, and the values of 5.9 kg COD kg COD−1 d−1, 0.05 kg COD m−3 and 0.02 d−1 were determined, respectively. By using the predicted values, excellent fittings of the final pressure, the CH4 molar fraction and the specific methanogenic yield calculation were obtained. Likewise, the variation in the gas–liquid mass transfer coefficient by several orders of magnitude showed negligible effects on the model predictive values in terms of methane molar fraction of the produced biogas, while the final pressure seemed to be slightly influenced.ConclusionsThe proposed model allowed to estimate the Monod maximum specific uptake rate for acetate, the half-saturation rate for acetate and the first-order decay rate constant, which were comparable with literature values reported for well-studied methanogens under anaerobic digestion at atmospheric pressure. The methane molar fraction and the final pressure predicted by the model showed different responses towards the variation of the gas–liquid mass transfer coefficient since the former seemed not to be affected by the variation of the gas–liquid mass transfer coefficient; in contrast, the final pressure seemed to be slightly influenced. The proposed approach may also allow to potentially identify the methanogens species able to be predominant at high pressure.