Bioprocess Operation Research Articles

Holistic process control framework for smart biomanufacturing: A deep learning driven approach

This article introduces a cyber-physical system comprising four interactive modules for enabling smart manufacturing in biotherapeutic production. These modules deal with process control, process prognosis, process diagnosis, and act as an external communication platform. An acoustic wave separator, a novel CHO cell clarification device, has been used as a case study with the objective of optimizing and regulating cell clarification process. A deep neural network (DNN)-based model has been proposed for implementing rapid control of bioprocess operations in case of detected anomalies or deviations in real-time. The use of the proposed holistic controller resulted in a 5% mean deviation from the setpoint in the first chamber, with more than 30% mean deviation resulting in the absence of the controller. The intricate layers of the automation framework facilitate real-time monitoring of the critical process parameters at 10-second intervals and ensure robust control action for deviations/anomalies, thus enabling smart manufacturing for biopharmaceutical production.

A Review of Manganese-Oxidizing Bacteria (MnOB): Applications, Future Concerns, and Challenges.

Groundwater serving as a drinking water resource usually contains manganese ions (Mn2+) that exceed drinking standards. Based on the Mn biogeochemical cycle at the hydrosphere scale, bioprocesses consisting of aeration, biofiltration, and disinfection are well known as a cost-effective and environmentally friendly ecotechnology for removing Mn2+. The design of aeration and biofiltration units, which are critical components, is significantly influenced by coexisting iron and ammonia in groundwater; however, there is no unified standard for optimizing bioprocess operation. In addition to the groundwater purification, it was also found that manganese-oxidizing bacteria (MnOB)-derived biogenic Mn oxides (bioMnOx), a by-product, have a low crystallinity and a relatively high specific surface area; the MnOB supplied with Mn2+ can be developed for contaminated water remediation. As a result, according to previous studies, this paper summarized and provided operational suggestions for the removal of Mn2+ from groundwater. This review also anticipated challenges and future concerns, as well as opportunities for bioMnOx applications. These could improve our understanding of the MnOB group and its practical applications.

Quorum sensing responses of r-/K-strategists Nitrospira in continuous flow and sequencing batch nitrifying biofilm reactors.

A better understanding of r-/K-strategists nitrifiers will help to balance the design and operation of bioprocesses for efficient pollution removal from wastewater. The objectives of study were to investigate the nitrite oxidation biokinetics, biofilm property, microbial community and quorum sensing (QS) of nitrifying biofilm in a continuously flow reactor (CFR) and a sequencing batch reactor (SBR). Results showed that nitrite-oxidizing bacteria were estimated to have a nitrite half saturation constant of 76.23 and 224.73 μM in CFR and SBR, respectively. High-throughput and metagenomic sequencing results showed that Nitrospira and Candidatus Nitrospira defluvii were the dominated nitrite-oxidizing taxa performing nitrite oxidation in both reactors. Nitrifying biofilm developed in CFR and SBR showed obviously different properties. Biofilm in SBR had an obviously higher ratio of polysaccharide and protein in extracellular polymeric substances, and higher thickness than in CFR. Metagenomics and chemical analysis revealed various types of acyl-homoserine lactone (AHL) circuit genes (e.g., luxI, lasI, hdtS) and four types of AHL signaling substances (e.g., C6-HSL, C8-HSL, C10-HSL and 3-oxo-C10-HSL) in nitrifying biofilm. The concentrations of these AHLs in biomass and water phases were obviously higher in SBR than that in CFR. Together, AHLs-based QS might affect the formation of nitrifying biofilm and thus contribute to the different biokinetics of Nitrospira in CFR and SBR. Our insights may reveal the molecular mechanism of Nitrospira for different biokinetics, and indicate the AHL association with Nitrospira adaptation to various conditions.

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control.

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%-17%), main (18%-23%), and basic (59%-68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.

Scale-Up of Capsular Polysaccharide Production Process by Haemophilus influenzae Type b Using kLa Criterion.

Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h−1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h−1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.

Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particlewere all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

Cowpea NAC Transcription Factors Positively Regulate Cellular Stress Response and Balance Energy Metabolism in Yeast via Reprogramming of Biosynthetic Pathways.

Yeast is a dominant host for recombinant production of heterologous proteins, high-value biochemical compounds, and microbial fermentation. During bioprocess operations, pH fluctuations, organic solvents, drying, starvation, osmotic pressure, and often a combination of these stresses cause growth inhibition or death, markedly limiting its industrial use. Thus, stress-tolerant yeast strains with balanced energy-bioenergetics are highly desirous for sustainable improvement of quality biotechnological production. We isolated two NAC transcription factors (TFs), VuNAC1 and VuNAC2, from a wild cowpea genotype, improving both stress tolerance and growth when expressed in yeast. The GFP-fused proteins were localized to the nucleus. Y2H and reporter assay demonstrated the dimerization and transactivation abilities of the VuNAC proteins having structural folds similar to rice SNAC1. The gel-shift assay indicated that the TFs recognize an "ATGCGTG" motif for DNA-binding shared by several native TFs in yeast. The heterologous expression of VuNAC1/2 in yeast improved growth, biomass, lifespan, fermentation efficiency, and altered cellular composition of biomolecules. The transgenic strains conferred tolerance to multiple stresses such as high salinity, osmotic stress, freezing, and aluminum toxicity. Analysis of the metabolome revealed reprogramming of major pathways synthesizing nucleotides, vitamin B complex, amino acids, antioxidants, flavonoids, and other energy currencies and cofactors. Consequently, the transcriptional tuning of stress signaling and biomolecule metabolism improved the survival of the transgenic strains during starvation and stress recovery. VuNAC1/2-based synthetic gene expression control may contribute to designing robust industrial yeast strains with value-added productivity.

Enhancement of co-production of lutein and protein in Chlorella sorokiniana FZU60 using different bioprocess operation strategies

Co-production of multiple compounds is an efficient approach to enhance the economic feasibility of microalgae-based metabolites production. In this study, Chlorella sorokiniana FZU60 was cultivated under different bioprocess strategies to enhance the co-production of lutein and protein. Results showed that both lutein and protein content (7.72 and 538.06 mg/g, respectively) were highest at the onset of nitrogen deficiency under batch cultivation. Semi-batch III strategy, with 75% microalgal culture replacement by fresh medium, obtained similar content, productivity, and yield of lutein and protein as batch cultivation, demonstrating that it can be used for stable and continuous production. Fed-batch II strategy, feeding with 1/3 modified BG11 medium, achieved super-high lutein and protein yield (28.81 and 1592.77 mg/L, respectively), thus can be used for high-output production. Besides, two-stage strategy, combining light intensity shift and semi-batch cultivation, gained extremely high lutein and protein productivity (15.31 and 1080.41 mg/L/day, respectively), thereby is a good option for high-efficiency production. Moreover, the fed-batch II and two-stage strategy achieved high-quality lutein and protein, thus are promising for the co-production of lutein and protein in C. sorokiniana FZU60 for commercial application.

Lentiviral Vector Bioprocessing.

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

Noninvasive online monitoring of Corynebacterium glutamicum fed-batch bioprocesses subject to spent sulfite liquor raw material uncertainty

In this study the use of a particle filter algorithm to monitor Corynebacterium glutamicum fed-batch bioprocesses with uncertain raw material input composition is shown. The designed monitoring system consists of a dynamic model describing biomass growth on spent sulfite liquor. Based on particle filtering, model simulations are aligned with continuously and noninvasively measured carbon evolution and oxygen uptake rates, giving an estimate of the most probable culture state. Applied on two validation experiments, culture states were accurately estimated during batch and fed-batch operations with root mean square errors below 1.1 g L−1 for biomass, 0.6 g L−1 for multiple substrate concentrations and 0.01 g g−1 h−1 for biomass specific substrate uptake rates. Additionally, upon fed-batch start uncertain feedstock concentrations were corrected by the estimator without the need of any additional measurements. This provides a solid basis towards a more robust operation of bioprocesses utilizing lignocellulosic side streams.

Application of two anaerobic membrane bioreactors with different pore size membranes for municipal wastewater treatment

Pore size is one of the most important properties in the successful operation of membrane-based bioprocesses for the treatment of municipal wastewater. The characteristics of two anaerobic membrane bioreactors (AnMBRs), one with a hollow fiber membrane of 0.4 μm pore size (AnMBR1), and the other with a membrane of 0.05 μm pore size (AnMBR2) were investigated for the treatment of real municipal wastewater at room temperature (25 °C) under varied hydraulic retention times (HRTs). Process performance was evaluated in terms of organic removal efficiency, biogas production and membrane filtration behaviours during a long-term continuous operation. Both AnMBRs showed good organic removal performance with COD and BOD removal efficiencies of around 89% and 93%, respectively. High energy recovery potential was achieved, with the biogas yield ranging between 0.20 and 0.26 L-gas/g-CODrem and a methane content of approximately 75%. The differences in the membrane filtration behaviours in the two AnMBRs included different permeate flux and total filtration resistance (Rt). In the AnMBR with a 0.4 μm pore size membrane, an average Rt of 1.08 × 10^12 m−1 was obtained even when the permeate flux was a high 0.274 m/day, while a higher average Rt of 1.51 × 10^12 m−1 was observed in the AnMBR with 0.05 μm pore size membrane even when the flux was a low 0.148 m/day. The off-line membrane cleaning strategy used for AnMBR1 indicated that the membrane restoration efficiency was 90.2%.

Experimental and Model-Based Analysis to Optimize Microalgal Biomass Productivity in a Pilot-Scale Tubular Photobioreactor.

A dynamic coarse-grained model of microalgal growth considering light availability and temperature under discontinuous bioprocess operation was parameterized using experimental data from 15 batch cultivations of Nannochloropsis granulata in a pilot-scale tubular photobioreactor. The methodology applied consists of a consecutive two-step model parameter estimation using pooled, clustered and reorganized data to obtain initial estimates and multi-experiment fitting to obtain the final estimates, which are: maximum specific growth rate μmax = 1.56 d−1, specific photon half-saturation constant KS,ph = 1.89 , specific photon maintenance coefficient mph = 0.346 and the cardinal temperatures Tmin = 2.3°C, Topt = 27.93°C and Tmax = 32.59°C. Biomass productivity prediction proved highly accurate, expressed by the mean absolute percent error MAPE = 7.2%. Model-based numerical optimization of biomass productivity for repeated discontinuous operation with respect to the process parameters cultivation cycle time, inoculation biomass concentration and temperature yielded productivity gains of up to 35%. This optimization points to best performance under continuous operation. The approach successfully applied here to small pilot-scale confirms an earlier one to lab-scale, indicating its transferability to larger scale tubular photobioreactors.

Biodiversity, isolation and genome analysis of sulfamethazine-degrading bacteria using high-throughput analysis.

Sulfamethazine (SM2) is one of the sulfonamide antibiotics that is frequently detected in aquatic environment. Given the complex structure of SM2 and its potential threat to the environment, it is necessary to determine the degradation behavior of high-concentration SM2. The mechanisms of community structure and diversity of activated sludge were analyzed. A novel SM2-degrading strain YL1 was isolated which can degrade SM2 with high concentration of 100mgL-1. Strain YL1 was identified as Paenarthrobacter ureafaciens and there was also a significant increase in the genus during acclimation. Additional SM2 metabolic mechanisms and genomic information of YL1 were analyzed for further research. The succession of the community structure also investigated the effect of SM2 on the activated sludge. This result not only advances the current understanding of microbial ecology in activated sludge, but also has practical implications for the design and operation of the environmental bioprocesses for treatment of antimicrobial-bearing waste streams.

Using benchtop NMR spectroscopy as an online non-invasive in vivo lipid sensor for microalgae cultivated in photobioreactors

The production of lipids by microalgae is widely studied, especially to find the best bioprocess operating conditions and optimize the productivity of the targeted product. In this context, being able to monitor online the evolution of the lipid concentration is a great advantage regarding the control and/or the optimization of the production. Yet, most non-invasive analyses hit a brick wall on the interference of the lipid signal with the ubiquitous water of the culture medium. This article shows how a compact NMR spectrometer connected to a photobioreactor can circumvent this drawback and measure, in real-time and in a non-invasive manner, the total lipid concentration, and that directly on the entire cells grown in their culture medium. The water signal could be enough-selectively removed using the W5 version of the WATERGATE pulse sequence. The NMR signal nicely correlates (R² > 0.99) with the offline FAME (Fatty Acid Methyl Ester) total lipid analysis as performed by GC-FID (Gas Chromatography coupled to Flame Ionization Detector) within limits of detection and quantification of respectively 9 and 30 mg.L−1. The lipid specific signal appears also quite robust regarding the dissolved dioxygen, making the benchtop NMR spectroscopy an appropriate universal device for the online monitoring of lipids produced in bioprocesses.

The hydraulic retention time influences the abundance of Enterobacter, Clostridium and Lactobacillus during the hydrogen production from food waste.

The influence of hydraulic retention time (HRT) on the microbial communities was evaluated in an anaerobic sequencing batch reactor (AnSBR) using organic waste from a restaurant as the substrate. The relationship among Lactobacillus, Clostridium and Bacillus as key micro-organisms on hydrogen production from organic solid waste was studied. The effect of the HRT (8-48h) on the hydrogen production and the microbial community was evaluated. Quantitative PCR was applied to determine the abundance of bacteria (in particular, Enterobacter, Clostridium and Lactobacillus genera). An AnSBR fermentative reactor was operated for 111 cycles, with carbohydrate and organic matter removal efficiencies of 80±15·42% and 22·1±4·49% respectively. The highest percentage of hydrogen in the biogas (23·2±11·1 %), and the specific production rate (0·42±0·16mmol H2 gVSadded -1 d-1 ) were obtained at an HRT of 48h. The decrease in the HRT generated an increase in the hydrogen production rate but decreasing the content of the hydrogen in the gas. HRT significantly influence the abundance of Enterobacter, Clostridium and Lactobacillus during the hydrogen production from food waste leading the hydrogen production as well as the metabolic pathways. The microbial analysis revealed a direct relationship between the HRT and the presence of fermentative bacteria (Enterobacter, Clostridium and Lactobacillus genera). Clostridium sp. predominated at an HRT of 48h, while Enterobacter and Lactobacillus predominated at HRTs between 8 and 24h. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: It was demonstrated that hydrogen production using food waste was influenced by the hydraulic retention time (HRT), and closely related to changes in microbial communities together with differences in metabolic patterns (e.g. volatile fatty acids, lactate, etc.). The decrease in the HRT led to the dominance of lactic acid bacteria within the microbial community whereas the increase in HRT favoured the emergence of Clostridium bacteria and the increase in acetic and butyric acids. Statistical data analysis revealed a direct relationship existing between the HRT and the microbial community composition in fermentative bacteria. This study provides new insight into the relationship between the bioprocess operation and the microbial community to understand better and control the biohydrogen production.

Online monitoring of the redox potential in microaerobic and anaerobic Scheffersomyces stipitis fermentations.

A correlation among different volumetric oxygen transfer coefficients (kLa) and the oxireduction potential (ORP) in batch fermentations using Scheffersomyces stipitis was evaluated. Experiments were performed using a mixture of xylose and glucose as the substrates. Microaerophilic condition (kLa = 4.9h-1) have shown to be suitable when compared to complete anaerobiosis (kLa = 0), providing an ethanol yield and a productivity after 48h of 64.3% and 0.45g ethanol L-1 h-1, respectively; the maximum ethanol titer obtained was 21.50g ethanol L-1. Values of ORP varying from - 270 to - 330mV resulted in high ethanol production from xylose by S. stipitis. Different ORP values were found in anaerobiosis and kLa 4.9h-1, suggesting that for ethanol production by S. stipitis, values from - 270 to - 330mV are favorable under the studied circumstances. In this ORP range, the greatest rates of xylose consumption and ethanol production were registered. ORP monitoring was demonstrated to be a suitable option for online control throughout the fermentation processes, which may provide a more efficient bioprocess operation with a very low O2 concentration.

Optimizing turbidostatic microalgal biomass productivity: A combined experimental and coarse-grained modelling approach

A combined, iterative experimental and model-based approach towards the optimization of microalgal biomass productivity has been conducted based on five long-term turbidostatic experiments using a strain of the unicellular green algae Nannochloropsis granulata. A coarse-grained model has been developed to describe and predict light-limited steady-state biomass productivity and dilution rate under turbidostatic bioprocess operation. It consists of three main components: Description of the specific growth rate (Monod-based), description of the average internal light intensity (Lambert-Beer-based) and calculation of the biomass-specific photon availability rate. The model employs just three parameters: The mean specific decadic extinction coefficient(ϵ∅ = 0.749 m2 gX-1) as obtained from direct measurements during the experiments as well as the maximum specific growth rate (μmax = 0.90 d−1) and the photon half saturation constant (KS,ph = 0.058 molph gX−1 d−1) as estimated by parameter identification from the experimental data. Following the initial parameter identification based on three experiments, two further separate identifications have been carried out iteratively using the data of the respective next consecutive experiment. Before carrying out the next experiment, the so far established model was used to predict biomass productivity and dilution rate for the respective set operating point (i.e. the controlled biomass concentration). Each resulting model was validated using the predicted subsequent productivity. The achieved model prediction accuracy, expressed as mean absolute percent error (MAPE), stayed below 2% in all three cases.

Ecological processes underpinning microbial community structure during exposure to subinhibitory level of triclosan

Ecological processes shaping the structure and diversity of microbial communities are of practical importance for managing the function and resilience of engineered biological ecosystems such as activated sludge processes. This study systematically evaluated the ecological processes acting during continuous exposure to a subinhibitory level of antimicrobial triclosan (TCS) as an environmental stressor. 16S rRNA gene-based community profiling revealed significant perturbations on the community structure and dramatic reduction (by 20–30%) in species diversity/richness compared to those under the control conditions. In addition, community profiling determined the prevalence of the deterministic processes overwhelming the ecological stochasticity. Analysis of both community composition and phenotypes in the TCS-exposed communities suggested the detailed deterministic mechanism: selection of TCS degrading (Sphingopyxis) and resistant (Pseudoxanthomonas) bacterial populations. The analysis also revealed a significant reduction of core activated sludge members, Chitinophagaceae (e.g., Ferruginibacter) and Comamonadaceae (e.g., Acidovorax), potentially affecting ecosystem functions (e.g., floc formation and nutrient removal) directly associated with system performance (i.e., wastewater treatment efficiency and effluent quality). Overall, our study provides new findings that inform the mechanisms underlying the community structure and diversity of activated sludge, which not only advances the current understanding of microbial ecology in activated sludge, but also has practical implications for the design and operation of environmental bioprocesses for treatment of antimicrobial-bearing waste streams.

An integrated meta-omics approach reveals substrates involved in synergistic interactions in a bisphenol A (BPA)-degrading microbial community

BackgroundUnderstanding microbial interactions in engineering bioprocesses is important to enhance and optimize performance outcomes and requires dissection of the multi-layer complexities of microbial communities. However, unraveling microbial interactions as well as substrates involved in complex microbial communities is a challenging task. Here, we demonstrate an integrated approach of metagenomics, metatranscriptomics, and targeted metabolite analysis to identify the substrates involved in interspecies interactions from a potential cross-feeding model community—bisphenol A (BPA)-biodegrading community, aiming to establish an identification method of microbial interactions in engineering or environmental bioprocesses.ResultsThe community-level BPA-metabolic pathway was constructed using integrated metagenomics and targeted metabolite analyses. The dynamics of active functions and metabolism of major community members were identified using metagenomic and metatranscriptomic analyses in concert. Correlating the community BPA biodegradation performance to the individual bacterial activities enabled the discovery of substrates involved in a synergistic interaction of cross-feeding between BPA-degrading Sphingonomas species and intermediate users, Pseudomonas sp. and Pusillimonas sp. This proposed synergistic interaction was confirmed by the co-culture of a Sphingonomas sp. and Pseudomonas sp. isolates, which demonstrated enhanced BPA biodegradation compared to the isolate of Sphingonomas sp. alone.ConclusionThe three types of integrated meta-omics analyses effectively revealed the metabolic capability at both community-wide and individual bacterial levels. The correlation between these two levels revealed the hidden connection between apparent overall community performance and the contributions of individual community members and their interactions in a BPA-degrading microbial community. In addition, we demonstrated that using integrated multi-omics in conjunction with culture-based confirmation approach is effective to elucidate the microbial interactions affecting the performance outcome. We foresee this approach would contribute the future application and operation of environmental bioprocesses on a knowledge-based control.

Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies

A lutein-enriched mutant, Chlorella sorokiniana MB-1-M12 was grown mixotrophically for lutein production. The lutein production efficiency of the strain was enhanced via optimizing the operating strategies. The results show that using semi-continuous cultivation with a medium replacement ratio of 75% resulted in a higher lutein productivity and lutein concentration of 6.24 mg/L/d and 50.6 mg/L, respectively, which were markedly higher than those obtained from batch and fed-batch cultivation. Cultivation under simulated outdoor cultivation conditions (i.e., temperature of 35 °C/25 °C for a 12 h/12 h light/dark cycle) could achieve the highest lutein productivity and lutein concentration of 3.34 mg/L/d and 30.8 mg/L, respectively. Lutein production via outdoor cultivation of MB-1-M12 strain with a 60-L tubular photobioreactor was performed using semi-continuous operation. With a medium replacement ratio of 75%, a good lutein productivity (4.46 mg/L/d) and concentration (27.4 mg/L) was obtained, indicating the feasibility of producing lutein under outdoor cultivation of the microalgal strain.