Laboratory-scale Bioreactor Research Articles

Microbial community evolution in a lab-scale reactor operated to obtain biomass for biochemical methane potential assays

Biochemical methane potential (BMP) test is an important tool to evaluate the methane production biodegradability and toxicity of different wastes or wastewaters. This is a key parameter for assessing design and feasibility issues in the full-scale implementation of anaerobic digestion processes. A standardized and storable inoculum is the key to obtain reproducible results. In Uruguay, a local enterprise dedicated to design and install anaerobic digesters operated a lab-scale bioreactor as a source of biomass for BMP tests, using a protocol previously described. This reactor was controlled and fed with a mixture of varied organic compounds (lipids, cellulolytic wastes, proteins). Biomass was reintroduced into the reactor after BMP assays to maintain a constant volume and biomass concentration. The aim of this work was to evaluate how the microbial community evolved during this operation and the effect of storing biomass in the refrigerator. The composition of the microbial communities was analyzed by 16S rRNA amplicon sequencing using primers for Bacteria and Archaea. The methanogenic activity was determined, and the methanogens were quantified by mcrA qPCR. One sample was stored for a 5-month period in the refrigerator (4 °C); the activity and the microbial community composition were analyzed before and after storage. Results showed that applying the reported methodology, a reliable methanogenic sludge with an acceptable SMA was obtained even though the reactor suffered biomass alterations along the evaluated period. Refrigerating the acclimatized biomass for 5 months did not affect its activity nor its microbial composition according to the 16S rRNA gene sequence analysis, even though changes in the mcrA abundance were observed.Key points• The applied methodology was successful to obtain biomass suitable to perform BMP assays.• The microbial community was resilient to external biomass addition.• Biomass storage at 4 °C for 5 months did not alter the methanogenic activity.Graphical

Simple, reference-independent assessment to empirically guide correction and polishing of hybrid microbial community metagenomic assembly.

Hybrid metagenomic assembly of microbial communities, leveraging both long- and short-read sequencing technologies, is becoming an increasingly accessible approach, yet its widespread application faces several challenges. High-quality references may not be available for assembly accuracy comparisons common for benchmarking, and certain aspects of hybrid assembly may benefit from dataset-dependent, empiric guidance rather than the application of a uniform approach. In this study, several simple, reference-free characteristics-particularly coding gene content and read recruitment profiles-were hypothesized to be reliable indicators of assembly quality improvement during iterative error-fixing processes. These characteristics were compared to reference-dependent genome- and gene-centric analyses common for microbial community metagenomic studies. Two laboratory-scale bioreactors were sequenced with short- and long-read platforms, and assembled with commonly used software packages. Following long read assembly, long read correction and short read polishing were iterated up to ten times to resolve errors. These iterative processes were shown to have a substantial effect on gene- and genome-centric community compositions. Simple, reference-free assembly characteristics, specifically changes in gene fragmentation and short read recruitment, were robustly correlated with advanced analyses common in published comparative studies, and therefore are suitable proxies for hybrid metagenome assembly quality to simplify the identification of the optimal number of correction and polishing iterations. As hybrid metagenomic sequencing approaches will likely remain relevant due to the low added cost of short-read sequencing for differential coverage binning or the ability to access lower abundance community members, it is imperative that users are equipped to estimate assembly quality prior to downstream analyses.

Pilot-scale process development for recombinant adeno-associated virus (rAAV) production based on high-density Sf9 cell culture

BackgroundIn recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production.MethodsIn this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level.ConclusionsThis successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.

Comparative Study of Mesophilic Biomethane Production in Ex Situ Trickling Bed and Bubble Reactors

Biomethane production via biogas upgrading is regarded as a future renewable gas, further boosting the biogas economy. Moreover, when upgrading is realized by the biogas CO2 conversion to CH4 using surplus renewable energy, the process of upgrading becomes a renewable energy storage method. This conversion can be carried out via microorganisms, and has attracted scientific attention, especially under thermophilic conditions. In this study, mesophilic conditions were imposed using a previously developed enriched culture. The enriched culture consisted of the hydrogenotrophic Methanobrevibacter (97% of the Archaea species and 60% of the overall population). Biogas upgrading took place in three lab-scale bioreactors: (a) a 1.2 L bubble reactor (BR), (b) a 2 L trickling bed reactor (TBR) filled with plastic supporting material (TBR-P), and (c) a 1.2 L TBR filled with sintered glass balls (TBR-S). The gas fed into the reactors was a mixture of synthetic biogas and hydrogen, with the H2 to biogas CO2 ratio being 3.7:1, lower than the stoichiometric ratio (4:1). Therefore, the feeding gas mixture did not make it possible for the CH4 content in the biomethane to be more than 97%. The results showed that the BR produced biomethane with a CH4 content of 91.15 ± 1.01% under a gas retention time (GRT) of 12.7 h, while the TBR-P operation resulted in a CH4 content of 90.92 ± 2.15% under a GRT of 6 h. The TBR-S operated at a lower GRT (4 h), yielding an effluent gas richer in CH4 (93.08 ± 0.39%). Lowering the GRT further deteriorated the efficiency but did not influence the metabolic pathway, since no trace of volatile fatty acids was detected. These findings are essential indicators of the process stability under mesophilic conditions.

A lab-scale frugal solar bioreactor design for cleaner production of bioproducts from thermophilic bacteria in outdoor conditions

Our study introduces an economical solar-powered bioreactor design for producing a model bioproduct, elucidated as a variant of poly-gamma-glutamic acid, using a thermophilic Bacillus licheniformis in a sterile-compromised growth environment. This frugal solar bioreactor (SB) setup repurposes an out-of-service jacketed stirred tank reactor with a working volume of 5 L and incorporates solar photovoltaic (PV) modules and a solar thermal unit to supply the necessary electrical load (1680 Wh) and heat for carrying out 24-hour batch fermentation at 50 °C. By repurposing the condemned equipments, the cost of SB fabrication was brought down by 59 %. In a batch mode of operation, the solar bioreactor yielded a maximum biopolymer concentration of 19.8 ± 0.9 g/L, which was 14.5 % less than the biopolymer titre obtained from a commercial bioreactor under similar production conditions. The molecular weight (MW) of the biopolymer was found to be dependent on the temperature of fermentation, with a maximum MW of 865.7 kDa achieved at 50 °C. It is envisaged that the proposed solar bioreactor design can be used for the effective production of thermophilic bacterial products suited for various low-cost applications.

Valorization of Strawberry Juice Production Wastewater: Possibilities for Polyphenols Recovery and Plant Biostimulant Production.

Fruit juice production is one of the most important branches of the food and beverage industry, considering both the market size and demand. It is also one of the largest generators of industrial wastewater, considering the large consumption of fresh water during fruit processing. Hence, the appropriate treatment strategies are of the utmost importance to minimize the environmental footprint of food industry effluents. This study aimed to investigate the valorization routes for strawberry juice production wastewater (SJPW), both in terms of nutrient recovery and a circular approach to its utilization as a medium for plant biostimulant production. The results show a low antioxidant capacity and low content of polyphenols in SJPW; however, promising results were obtained for the in vitro seed germination and tomato growth promotion when investigating a biostimulant based on Bacillus sp. BioSol021, which was cultivated using SJPW in a lab-scale bioreactor, with root and shoot length improvements of approximately 30% and 25%, respectively, compared to the control samples. The plant growth promotion (PGP) traits indicated the ability of IAA production, in a concentration of 8.55 ± 0.05 mg/L, and the enzymatic activity was evaluated as through the enzymatic activity index (EAI), achieving the following: 2.26 ± 0.04 for cellulolytic activity, 2.49 ± 0.08 for hemicellulolytic activity, 2.91 ± 0.16 for pectinolytic activity, and 1.05 ± 0.00 for proteolytic activity. This study opens a new chapter of possibilities for the development of techno-economically viable circular bioprocess solutions aimed at obtaining value-added microbial products for sustainable agriculture based on the valorization of food industry effluents thus contributing to more sustainable food production at both the agricultural and industrial levels.

Design, experimental optimization, and flow analysis of a novel bioreactor for dynamic mammalian cell culture at laboratory scale using Box-Behnken design

Improving the quality of dynamic cell culture in laboratories is an important field in bioengineering. In this study, a novel lab-scale bioreactor using a vibrating agitator and a modified flask has been introduced to create a strong mixing at low shear stress. This bioreactor has been optimized using Box-Behnken design based on three dimensionless important structural factors including disc diameter, vibration amplitude, and the height of the disc placement. Three growth indicators including the specific growth rate, the natural logarithm of the maximum cell density, and productivity have been considered as biological responses. The results show that the disc diameter has the most important role in these indicators. If the disc diameter, vibration amplitude, and the height of disc placement are set to 0.24, 0.02, and 0.4 of the flask diameter, respectively, the values of the specific growth rate, the maximum cell density, and productivity at this optimum settings are 0.033 (h−1), 13.11, and 5133 (cells/(mL.h)), respectively. These values of the indicators are high and indicate the better performance of this bioreactor than other lab-scale bioreactors. In addition, investigating Reynolds number in the fluid flow indicates that in the range of 780 up to 1150, growth indices are high.

Quantitative investigation of the effect of mechanical and geometrical factors of a laboratory-scale bioreactor using a vibrating agitator on mammalian cell culture indices.

Mammalian cell cultures in laboratories are performed in static and dynamic methods, and cell growth indices are higher in dynamic mode. In this study, a lab-scale stirred bioreactor using a vibrating disc and a suitable setup has been introduced for dynamic cell culture, which creates proper mixing at low shear stress. 15 experiments have been done by Raji cell in batch mode using Box-Behnken design to quantitatively investigate the effect of mechanical and geometrical factors of this bioreactor on cell culture indices. Three structural factors, including disc diameter, vibration amplitude, and the height of the disc placement have been selected as the main factors. Three cell growth indices including the specific growth rate, the maximum cell concentration, and productivity have been considered as biological responses. Resulting models predict the value of each index under different settings of the factors with good accuracy. Results show that the disc diameter has the greatest effect among the investigated factors. Also, the specific growth rate, the natural logarithm of the maximum cell concentration, and productivity are about 0.033(1/h), 13.2, and 5133(cells/hmL), respectively by using a 25 (mm) disc with a vibration amplitude of 2.5 up to 3 (mm), and a placement height of 40 up to 60 (mm).

Characteristics and fouling behaviors of soluble microbial products and sub-visible particles in an anaerobic membrane bioreactor treating sewage at room temperature

A lab-scale anaerobic membrane bioreactor (AnMBR) was employed to investigate the characteristics and fouling behaviors of soluble microbial products (SMP) and sub-visible particles (SVP) in sewage treatment. The study revealed a noteworthy trend, where SMP and SVP concentrations initially surged, subsequently declined, and ultimately stabilized at comparable levels. Compositions and fluorescent property analyses indicated that protein-like substances with tryptophan are the primary fluorescent composition in both SMP and SVP. Notably, SVP exhibited a superior protein concentration and a higher protein-to-polysaccharide ratio, thereby resulting in a higher filtration resistance than SMP. Moreover, microbial species involved in biofouling (Rhodocycales and Desulfovibrionales) also have a higher abundance in SVP, meaning SVP has a higher biofouling potential than SMP. Achievements in this study expanded the knowledge of the characteristics of SMP and SVP and revealed the significant role of SVP in membrane fouling of AnMBR.

Treatment of Synthetic Wastewater Containing Polystyrene (PS) Nanoplastics by Membrane Bioreactor (MBR): Study of the Effects on Microbial Community and Membrane Fouling.

The persistent presence of micro- and nanoplastics (MNPs) in aquatic environments, particularly via effluents from wastewater treatment plants (WWTPs), poses significant ecological risks. This study investigated the removal efficiency of polystyrene nanoplastics (PS-NPs) using a lab-scale aerobic membrane bioreactor (aMBR) equipped with different membrane types: microfiltration (MF), commercial ultrafiltration (c-UF), and recycled ultrafiltration (r-UF) membranes. Performance was assessed using synthetic urban wastewater spiked with PS-NPs, focusing on membrane efficiency, fouling behavior, and microbial community shifts. All aMBR systems achieved high organic matter removal, exceeding a 97% COD reduction in both the control and PS-exposed reactors. While low concentrations of PS-NPs did not significantly impact the sludge settleability or soluble microbial products initially, a higher accumulation increased the carbohydrate concentrations, indicating a protective bacterial response. The microbial community composition also adapted over time under polystyrene stress. All membrane types exhibited substantial NP removal; however, the presence of nano-sized PS particles negatively affected the membrane performance, enhancing the fouling phenomena and increasing transmembrane pressure. Despite this, the r-UF membrane demonstrated comparable efficiency to c-UF, suggesting its potential for sustainable applications. Advanced characterization techniques including pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) were employed for NP detection and quantification.

Effective removal of toxic mixed azo dyes and Cr (VI) ions from wastewater using an integrated approach

The textile industry generates a significant amount of wastewater, including hazardous dyes, pigments, and heavy metals. The integrated approach of isolated bacterial strain and the bacterially synthesized ZnO nanoparticles was used in the removal of mixed azo dyes and Cr(VI) in a lab-scale bioreactor. It resulted in 77.19 % bromocresol purple (4th day), 50.11 % on bromocresol blue (3rd day), and 50.59 % bromocresol green (4th day) removal. Mixed azo dyes and Cr(VI) were removed using bacterial strain, which showed the maximum removal of Cr(VI) was 73.6 % at 25 ppm (3rd day) and mixed azo dyes removal was a maximum of 49.01 % at 75 ppm. A lab-scale reactor study using bacterial strain resulted in 45.89 % (5th day) of mixed azo dye and Cr(VI) removal. However, when the reactor was employed with synthetic wastewater and bacterially synthesized ZnO nanoparticles, achieved a maximum removal of 53.84 % at 120 min. Also, a phytotoxicity study on the Cicer arietinum plant with different concentrations of mixed azo dyes and chromium concentrations showed a maximum length of 5.5 cm at 100 ppm (3rd day). The bacterial strain and synthesized ZnO nanoparticles integrated technique providing an economically viable and scale-up solution for wastewater treatment techniques.

A coupled metabolic flux/compartmental hydrodynamic model for large-scale aerated bioreactors

Spatial variation of microorganism populations in stirred-tank bioreactors, caused by the interactions of complex multiphase flows and microorganism metabolism, results in undesirable performance characteristics and significant challenges in scale-up activities. In this work, a novel modelling approach is developed to investigate these coupled processes in bioreactors, which involves the integration of a computational fluid dynamics-informed compartmental hydrodynamic and a dynamic flux balance (DFB) model. This coupling poses significant computational challenges especially when dealing with transient scenarios. To tackle these challenges we developed a fast point-location algorithm to solve the DFB model at different bioreactor locations. To validate the presented modelling approach, a binary search tree-based metabolic model for E. coli was developed and integrated with a flow-informed compartmental model of a large-scale four-impeller aerated bioreactor in fed-batch operation. The model results exhibit favorable agreement with the concentration profiles reported in literature for both lab-scale and industrial scale bioreactors. Furthermore, the ability of the approach to deal with transient scenarios permitted to study the effect of oxygen closed loop control responses and the occurrence of oscillatory behaviour which were crucial to explain part of the data.

Performances of full-scale biological nitrogen pre-treatment process for drinking water source: Seasonal variations and microbial community

This study investigated the performance of the full-scale unit over a two-year period to enhance nitrification efficiency and provide operational strategies. Results indicated that raw water quality from Donggan River was notably influenced by seasonal variations, particularly during dry and wet seasons, impacting the nitrification efficiency of the biological pretreatment process. Factors such as influent concentrations of ammonia and total Kjeldahl nitrogen were found to have significant effects on nitrification, with temperature and conductivity also showing correlations. The specific rate of ammonia removal was calculated to be approximately 0.1 kg-N/m3/d under the existing operational setup. Moreover, elevating dissolved oxygen levels above 4 mg/L was proposed to potentially boost ammonia oxidation based on findings from experiments conducted in lab-scale bioreactors. In times of increased influent ammonia levels, the elimination of about 1–3 mg-N/L of total nitrogen signified the activation of denitrification processes. This observation was corroborated by results from next-generation sequencing techniques, verifying the existence of denitrifying microorganisms. The real-time PCR analysis results indicated that the abundance of comammox amoA gene was comparable with the abundance of the AOB amoA gene, indicating the presence of comammox Nitrospira and their potential role on nitrification in the system.

Bifidobacterium animalis subsp. lactis HN019 live probiotics and postbiotics: production strategies and bioactivity evaluation for potential therapeutic properties.

Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer's needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 1010 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions.

Unravelling the impact of oxic-settling-anaerobic cycle implementation and solid retention time on sludge generation, membrane operation, and contaminant removal in membrane bioreactors

Sewage sludge production represents a critical issue nowadays. The oxic-settling-anaerobic (OSA) system is a promising strategy for reducing sludge production during biological wastewater treatment. However, the distinct effect of OSA process and solid retention time (SRT) on sludge production has not been clarified yet. Therefore, during this study, the impacts of OSA system implementation at different SRT on sludge generation, membrane operation, and contaminant removal were elucidated in two lab-scale membrane bioreactors working at 20 d (MBR20) and 60 d (MBR60). After OSA implementation, the MBR-OSA20 and MBR-OSA60 were operated at a hydraulic retention time in the sludge holding tank (HRTSHT) of 6–12 h and of 6–24 h, respectively. The estimated observed sludge yield (Yobs) showed a reduction of 38–48 % for the MBR-OSA20, but no beneficial effect was observed for MBR-OSA60 at 6 h of HRTSHT. At the HRTSHT of 24 h, Yobs decreased by 17 % due to hydrolysis and acidogenic biomass fermentation. OSA implementation and long SRT had no negative effects on organic carbon and ammonium removal (96(±3) % and 100 %, respectively) and improved total nitrogen (TN) reduction by 19–41 %. Membrane fouling and sludge filterability were not significantly affected, whereas sludge settleability worsened after the implementation of the OSA cycle. This activity provided additional insights about the distinct impact of the OSA process and SRT on sludge production and characteristics, confirming the OSA process as a valid solution for reducing sludge production under a moderate SRT, but revealing a negligible effect on Yobs under a prolonged SRT and a short HRTSHT.

A low-cost, open-source gas flow meter for laboratory-scale bioreactors

Biogas production measurement is a fundamental aspect of anaerobic digestion research. Lab-scale experiments usually require devices capable of measuring low flowrates and low relative pressures. Despite the existence of multiple commercially available alternatives for such demands, each with particular advantages and limitations, researchers have often resorted to in-house developed systems. This inclination is motivated by cost savings and the potential for of customization to suit specific research needs. Those systems, however, are seldom reproduced beyond laboratory walls due to overly complex designs, limited component availability or unpublished software. In this work, we developed a fully open-source lab-scale biogas flow meter equipped with datalogging capabilities. Our approach relied on inexpensive, widely available electronic modules. The flow meter was calibrated, tested and validated in batch biogas production experiments, alongside a comparative assessment with a proprietary device widely used in the field. The semi-continuous gas meter presented a resolution of 7.45 ± 0.13 mL and maintained a stable pulse volume (under 2 % relative standard deviation) for flowrates ranging from 60 mL h−1 to 1120 mL h−1. The device was not sensitive to the evaporative loss of up to 7 mL of packing liquid, demonstrating its applicability to long-term experiments. The developed system was shown to be robust and reliable and can be easily reproduced, revised and enhanced in laboratories everywhere.

Dirammox-dominated microbial community for biological nitrogen removal from wastewater

Direct ammonia oxidation (Dirammox) might be of great significance to advance the innovation of biological nitrogen removal process in wastewater treatment systems. However, it remains unknown whether Dirammox bacteria can be selectively enriched in activated sludge. In this study, a lab-scale bioreactor was established and operated for 2 months to treat synthetic wastewater with hydroxylamine as a selection pressure. Three Dirammox strains (Alcaligenes aquatilis SDU_AA1, Alcaligenes aquatilis SDU_AA2, and Alcaligenes sp. SDU_A2) were isolated from the activated sludge, and their capability to perform Dirammox process was confirmed. Although these three Dirammox bacteria were undetectable in the seed sludge (0%), their relative abundances rapidly increased after a month of operation, reaching 12.65%, 0.69%, and 0.69% for SDU_A2, SDU_AA1, and SDU_AA2, respectively. Among them, the most dominant Dirammox (SDU_A2) exhibited higher nitrogen removal rate (32.35%) than the other two strains (13.57% of SDU_AA1 and 14.52% of SDU_AA2). Comparative genomic analysis demonstrated that the most dominant Dirammox bacterium (SDU_A2) possesses fewer complete metabolic modules compared to the other two less abundant Alcaligenes strains. Our findings expanded the understanding of the application of Dirammox bacteria as key functional microorganisms in a novel biological nitrogen and carbon removal process if they could be well stabilized.Key points• Dirammox-dominated microbial community was enriched in activated sludge bioreactor.• The addition of hydroxylamine played a role in Dirammox enrichment.• Three Dirammox bacterial strains, including one novel species, were isolated.Graphical abstract

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered β-fructofuranosidase enzyme in Pichia pastoris

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Laboratory investigation and prediction of permeability of fresh to five-year-old municipal solid wastes of low and high food contents

The permeability of municipal solid wastes (MSWs) is important for the design and operation of landfills. This study presented the experimental investigation of the permeability of low food content- (LF-) and high food content- (HF-) MSWs prepared in laboratory-scale bioreactors for up to 5 years. The permeability of MSWs with diverse degrees of decomposition (DOBs), void ratios, and permeation liquids was measured (288 tests). The measured permeability was compared to that predicted from the (modified) Kozeny-Carman (K-C) equations in four different forms. The results indicated that the permeability of both LF- and HF-MSWs decreased significantly (p < 0.05) with decomposition under a given void ratio. The predicted permeability using the original K-C equation fitted well with that of fresh MSWs. The permeability of decomposed MSWs was closer to the predicted results using the modified K-C equation with the effective void ratio. This can be attributed to the increase in the fine fractions due to degradation. The reduction in the effective voids was more significant with HF-MSWs. The parameters required in the (modified) K-C equations showed a good correlation with DOB and effective particle size (d10). The predicted permeability based on the relationship between DOB (or d10) and equation parameters was within 3 times the difference compared to the measured values. The above results indicated that the modified K-C equation can be adopted to predict the permeability of fresh and degraded MSWs while more field-scale experiments should be conducted to further evaluate its feasibility.

Decipher syntrophies within C2-C4 organic acids-degrading anaerobic microbiomes: A multi-omic exploration

In methanogenic ecosystems, carboxylic acid degradation is a crucial process facilitated by specialized bacteria working in syntrophy with methanogens. This study explores the transcriptomic responses in 178 metagenome-assembled genomes upon changes in feeding composition, specifically lactate, butyrate, propionate, and acetate, in four lab-scale bioreactors. Methanothrix soehngenii and Methanoculleus sp. emerged as key biomethanation contributors. Through metatranscriptomics, machine learning-based functional annotation, and flux balance analysis the underlying microbial interactions and dynamics were deciphered. Syntrophomonadaceae and Mesotoga species maintained mutualistic metabolite exchanges with hydrogenotrophic methanogens, degrading primarily butyrate and lactate, respectively. Acetate was mainly consumed by Smithellaceae sp., in competition with M. soehngenii in all reactors. Furthermore, the acetoclastic archaeon exhibited a previously undocumented capability to metabolize lactate, thereby confirming the prevalence of acetotrophic pathway. Transcriptomic profiles revealed an additional layer of complexity, where propionate dismutation and beta-oxidation pathways are interconnected by the exchanges of butyrate between putative syntrophs, including Syntrophomonadaceae and Smithellaceae species. Integrating multi-omics data with genome-scale metabolic modeling enabled the accurate reconstruction of dynamics within the controlled ecosystem. This composite novel approach was applied to the AD system to unveil the intricate relationships operating within the microbiome to promote thriving. Results elucidated the still poorly explored organization of the so-called microbial dark matter.