Engineering in Life Sciences最新文献

The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.

Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases (FAS) and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N-terminus or C-terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system.

Shifting the current fossil economy to a carbon neutral supply is an enormous challenge. Current geopolitical issues that severely endanger long-term established fossil supply chains function as catalysts fostering novel solutions. In this context, the goals for establishing a resilient economy meet climate protection demands for reducing the human footprint in atmosphere. Biotechnological processes for producing fine chemicals and commodities by utilizing renewable resources may play an important role to make the transformation to a circular economy happen.

Bioprocesses already successfully documented their outstanding quality not only to compete with established fossil routes but also to complement and even to replace them in industrial practice. Long-term established examples comprise the microbial production of amino acids, organic acids, technical enzymes, food additives, active pharma ingredients … and many more.

Interestingly enough, mono-cultures are used, predominately. This reflects the steady improvement of molecular tools for efficiently manipulating microbes to produce the molecule of interests. However, the question may arise whether mono-cultures should be the first choice for developing novel bioprocesses. Often enough product formation demands for precursors, reduction equivalents, energy demands, etc. that contradict cellular needs for growth and maintenance. Furthermore, the genetic engineering of hosts may well reach technical limits if targeted product formation opposes the lifestyle of the cells. Additionally, many examples for the production of antibiotics are known outlining that antibiotic production only starts in the presence of another interacting strain.

Consequently, the implementation of co-cultures with well-equilibrated interactions is a promising approach for establishing a new generation of bioproduction processes. Accordingly, this topic (cell-to-cell interactions) is highlighted in the current special issue and is also a central theme in the priority program ‘InterZell SPP2170’ of the German Science Foundation (DFG) that co-fuels the special issue.

Once developed in the labs, novel bioprocesses should find their way into large-scale bioreactors to translate innovation into practice. Often enough the so-called scale-up reveals the deterioration of key performance criteria such as titer, rates, and yield (TRY). This basically reflects cellular responses on mixing heterogeneities that inevitably occur in industrial scale. For mitigating related performance losses profound research is necessary. Consequently, the special issue also covers related studies (cell-to-bioreactor interactions) analyzing microbial responses in detail and developing novel scale-down devices.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS-CoV-2 can spread irrespective of a patient's course of disease, these institutions’ continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS-CoV-2 screening facilities with a built-in biosafety level (BSL)-2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build-in laboratory with two similar stations commencing operation until June 2020. During the 15-month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.

Quality by Design (QbD) is one of the most important tools for the implementation of Process Analytical Technology (PAT) in biopharmaceutical production. For optimal characterization of a monoclonal antibody (mAb) upstream process a stepwise approach was implemented. The upstream was divided into three process stages, namely inoculum expansion, production, and primary recovery, which were investigated individually. This approach enables analysis of process parameters and associated intermediate quality attributes as well as systematic knowledge transfer to subsequent process steps. Following previous research, this study focuses on the primary recovery of the mAb and thereby marks the final step toward a holistic characterization of the upstream process. Based on gained knowledge during the production process evaluation, the cell viability and density were determined as critical parameters for the primary recovery. Directed cell viability adjustment was achieved using cytotoxic camptothecin in a novel protocol. Additionally, the cell separation method was added to the Design of Experiments (DoE) as a qualitative factor and varied between filtration and centrifugation. To assess the quality attributes after cell separation, the bioactivity of the mAb was analyzed using a cell-based assay and the purity of the supernatant was evaluated by measurement of process related impurities (host cell protein proportion, residual DNA). Multivariate data analysis of the compiled data confirmed the hypothesis that the upstream process has no significant influence on the bioactivity of the mAb. Therefore, process control must be tuned towards high mAb titers and purity after the primary recovery, enabling optimal downstream processing of the product. To minimize amounts of host cell proteins and residual DNA the cell viability should be maintained above 85% and the cell density should be controlled around 15 × 10⁶ cells/ml during the cell removal. Thereby, this study shows the importance of QbD for the characterization of the primary recovery of mAbs and highlights the useful implementation of the stepwise approach over subsequent process stages.

Bioconversion of CO₂ into liquid fuels or chemicals, preferred medium chain carboxylic acids (caproic and caprylic acid), is an attractive CO₂ utilization technology. The present study aims to investigate the effects of different ratios of H₂/CO₂ on regulating the distribution of C2-C8 carboxylic acid products, while the headspace pressure of 1.5 bar was set to amplify the effect of different ratios. The H₂/CO₂ ratio of 4:1 was more suitable for preparing acetic acid, where the highest acetic acid yield was 17.5 g/L. And the H₂/CO₂ ratio of 2:1 showed excellent chain elongation ability with the highest n-caprylic yield of 2.4 g/L. Additionally, the actual H₂/CO₂ ratios of 4:1 reactors were higher than that in 2:1 may be course chain elongation often accompanied by H₂ production. The 16S rRNA genes analysis shows that the genus Terrisporobacter and Coriobacteriales may be related to acetic acid production enriched in H₂/CO₂ ratio 4:1 reactors, and the genus Clostridium and Paenibacillaceae may associate with the chain elongation pathway were enriched in H₂/CO₂ ratio 2:1 reactors.

An anaerobic granular sludge was enriched to utilize H₂/CO₂ in a continuous gas-fed up-flow anaerobic sludge reactor by applying operating conditions expected to produce acetic acid, butyric acid, and ethanol. Three stages of fermentation were found: Stage I with acetic acid accumulation with the highest concentration of 35 mM along with a pH decrease from initial 6 to 4.5. In Stage II, H₂/CO₂ was replaced by 100% H₂ to induce solventogenesis, whereas butyric acid was produced with the highest concentration of 2.5 mM. At stage III with 10 µM tungsten (W) addition, iso-valeric acid, valeric acid, and caproic acid were produced at pH 4.5–5.0. In the batch tests inoculated with the enriched sludge taken from the bioreactor (day 70), however, methane production occurred at pH 6. Exogenous 15 mM acetate addition enhanced both the H₂ and CO₂ consumption rate compared to exogenous 10, 30, and 45 mM acetate by the enriched sludge. Exogenous acetate was failed to be converted to ethanol using H₂ as electron donor by the enriched acetogens.