Carbon monoxide concentrations in syngas are often high, but tolerance toward CO varies a lot between homoacetogenic bacteria. Analysis of the autotrophic potential revealed that the first isolated ...acetogenic bacterium Clostridium aceticum was able to use CO as sole carbon and energy source for chemolithoautotrophic carbon fixation but simultaneously showed little tolerance to high CO concentrations. Not yet reported, autotrophic ethanol production by
C. aceticum was discovered with CO as a substrate in batch processes. Growth rates estimated in batch processes at varying CO partial pressures were used to identify the CO inhibition kinetics of
C. aceticum, using a substrate inhibition model.
C. aceticum shows a strong CO inhibition with an optimum CO partial pressure of only 5.4 mbar in the gas phase at cell dry weight concentrations of up to 0.5 g·L
−1. At optimum conditions, growth and acetate formation rates were estimated to be 0.24 hr
−1 and 0.52 g·g
−1·hr
−1, respectively. Syngas fermentation at high partial pressures of up to 280 mbar CO in the inlet gas phase was enabled by applying a continuously operated stirred‐tank bioreactor with submerged membranes with total cell retention. Around 70% CO conversion was achieved continuously in the membrane bioreactor with strongly CO inhibited
C. aceticum resulting in space‐time yields of up to 0.85 g·L
−1·hr
−1 acetate.
First isolated acetogenic microorganism Clostridium aceticum was shown to be strongly inhibited by the gaseous substrate CO at high partial pressures. Substrate inhibition kinetics was identified by growth rate determination at varying CO partial pressures in independent batch processes. Application of a continuously operated stirred‐tank bioreactor with submerged membranes for total cell retention enabled continuous syngas fermentation even at high CO partial pressures in the inlet gas phase, resulting in distinctly increased product space‐time yields.
l‐tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a ...variety of economic and metabolic advantages. Process performance of the recombinant
l‐tryptophan producer
Escherichia coli NT367 was studied in controlled fed‐batch processes. The chromosome of the recombinant
l‐tryptophan producer was equipped with additional genes coding for enzymes of the aromatic amino acids biosynthetic pathway and
l‐serine biosynthesis, including genes for feedback‐resistant enzyme variants (
trpE
fbr,
aroFBL, and
serA
fbr
), deletions of enzymatic steps for the degradation of precursors or the product
l‐tryptophan (
sdaB and
tnaA), and alterations in the regulation of
l‐tryptophan metabolism (deletion of
trpL and
trpR). The impact of glycerol supply rates as well as the application of a multicopy plasmid (pF112‐
aroFBL
‐kan) were investigated in fully controlled stirred‐tank bioreactors on a 15 L scale. The combination of
E. coli NT367 carrying pF112‐
aroFBL
‐kan and an appropriate biomass‐specific glycerol supply‐rate resulted in the highest final product concentration of 12.5 g L
−1
l‐tryptophan with the lowest concentrations of other aromatic amino acids. Fed‐batch production of
l‐tryptophan from glycerol was shown for the first time with recombinant
E. coli.
L‐tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of variants of the recombinant L‐tryptophan producer Escherichia coli NT367 was studied in controlled fed‐batch processes.
Summary
The reduction of CO2 emissions is a global effort which is not only supported by the society and politicians but also by the industry. Chemical producers worldwide follow the strategic goal ...to reduce CO2 emissions by replacing existing fossil‐based production routes with sustainable alternatives. The smart use of CO and CO2/H2 mixtures even allows to produce important chemical building blocks consuming the said gases as substrates in carboxydotrophic fermentations with acetogenic bacteria. However, existing industrial infrastructure and market demands impose constraints on microbes, bioprocesses and products that require careful consideration to ensure technical and economic success. The mini review provides scientific and industrial facets finally to enable the successful implementation of gas fermentation technologies in the industrial scale.
Today, chemical industry follows the strategic goal to reduce the manufacturing CO2 footprint. The use of CO‐enriched gas as a substrate for fermentation processes is an attractive alternative to the use of fossile ressources. This review provides an overview of metabolic and biochemical engineering backgrounds and outlines the particular needs to integrate gas fermentation in existing infrastructure of value added chains in chemical industry.
•Gas fermentation (CO2/H2) in controlled stirred-tank bioreactors with continuous gas supply.•Standardized comparative gas fermentation of 8 different acetogenic bacteria out of 6 genera.•Acetate was ...the main product of all studied strains with concentrations of up to 32.2gL−1.•Ethanol was produced with up to 0.42gL−1 (Sporomusa ovata) by 4 strains.•Butyrate was produced with up to 0.14gL−1 (Eubacterium aggregans) by 3 strains.
The production of chemicals by syngas fermentation is a promising alternative to heterotrophic fermentation processes. The autotrophic process performances of the so far not well studied acetogens Acetobacterium fimetarium, Acetobacterium wieringae, Blautia hydrogenotrophica, Clostridium magnum, Eubacterium aggregans, Sporomusa acidovorans, Sporomusa ovata and Terrisporobacter mayombei were characterized. Acetobacterium woodii was used as reference strain. Standardized batch experiments with continuous supply of the gaseous substrates CO2 and H2 were performed in fully controlled stirred-tank bioreactors. A. wieringae and S. ovata showed by far the highest growth rates and maximum biomass concentrations among the acetogens under study. Aside from the reference strain A. woodii, highest volumetric (17.96gL−1d−1) as well as cell specific acetate formation rates (21.03gg−1d−1) were observed with S. ovata resulting in a final acetate concentration of 32.2gL−1. Accumulation of formate with up to 4.8gL−1 was observed with all acetogens. Ethanol was produced autotrophically with up to 0.42gL−1 by four of the acetogenic bacteria under study (A. wieringae, C. magnum, S. acidovorans and S. ovata) and also by A. woodii. Butyrate was formed with up to 0.14gL−1 by three of the acetogenic bacteria under study (C. magnum, B. hydrogenotrophica and E. aggregans). Due to its superior process performances S. ovata may be a promising host for redirecting carbon fluxes by applying metabolic engineering and tools of synthetic biology to produce non-natural chemicals from syngas.
Artificial single‐stranded DNA (ssDNA) with user‐defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies ...such as genome editing and DNA origami. However, currently available biotechnological approaches for mass‐producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross‐contaminating manufacturer plants with self‐replicating phages. Here we overcome this problem with an efficient, scalable, and cross‐contamination‐free method for the phage‐free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self‐replication in the absence of the helper plasmid. This enables cross‐contamination‐free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by‐products and increased the maximal product concentration up to 83 mg L−1 of ssDNA in a stirred‐tank bioreactor, thus realizing up to a 40‐fold increase in maximal product concentration over previous scalable phage‐free ssDNA production methods.
“Reverse Infection” with phagemid particles encoding one gene of the M13 phage and a user‐defined single‐stranded DNA (ssDNA)‐sequence leads to phagemid (ssDNA) production by Escherichia coli in presence of a helper plasmid encoding the remaining genes of the M13 phage. E. coli cells without helper plasmid can be infected by the phagemid but do not produce new phagemid particles. This phage‐free process for the production of user‐defined ssDNA supports cross‐contamination‐free large‐scale contract manufacturing.
DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable ...in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn
-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.
The bioprocessing industry relies on packed‐bed column chromatography as its primary separation process to attain the required high product purities and fulfill the strict requirements from ...regulatory bodies. Conventional column packing methods rely on flow packing and/or mechanical compression. In this work, the application of ultrasound and mechanical vibration during packing was studied with respect to packing density and homogeneity. We investigated two widely used biochromatography media, incompressible ceramic hydroxyapatite, and compressible polymethacrylate‐based particles, packed in a laboratory‐scale column with an inner diameter of 50 mm. It was shown that ultrasonic irradiation led to reduced particle segregation during sedimentation of a homogenized slurry of polymethacrylate particles. However, the application of ultrasound did not lead to an improved microstructure of already packed columns due to the low volumetric energy input (~152 W/L) caused by high acoustic reflection losses. In contrast, the application of pneumatic mechanical vibration led to considerable improvements. Flow‐decoupled axial linear vibration was most suitable at a volumetric force output of ~1,190 N/L. In the case of the ceramic hydroxyapatite particles, a 13% further decrease of the packing height was achieved and the reduced height equivalent to a theoretical plate (rHETP) was decreased by 44%. For the polymethacrylate particles, a 18% further packing consolidation was achieved and the rHETP was reduced by 25%. Hence, it was shown that applying mechanical vibration resulted in more efficiently packed columns. The application of vibration furthermore is potentially suitable for in situ elimination of flow channels near the column wall.
Flow cytometry and its technological possibilities have greatly advanced in the past decade as analysis tool for single cell properties and population distributions of different cell types in ...bioreactors. Along the way, some solutions for automated real‐time flow cytometry (ART‐FCM) were developed for monitoring of bioreactor processes without operator interference over extended periods with variable sampling frequency. However, there is still great potential for ART‐FCM to evolve and possibly become a standard application in bioprocess monitoring and process control. This review first addresses different components of an ART‐FCM, including the sampling device, the sample‐processing unit, the unit for sample delivery to the flow cytometer and the settings for measurement of pre‐processed samples. Also, available algorithms are presented for automated data analysis of multi‐parameter fluorescence datasets derived from ART‐FCM experiments. Furthermore, challenges are discussed for integration of fluorescence‐activated cell sorting into an ART‐FCM setup for isolation and separation of interesting subpopulations that can be further characterized by for instance omics‐methods. As the application of ART‐FCM is especially of interest for bioreactor process monitoring, including investigation of population heterogeneity and automated process control, a summary of already existing setups for these purposes is given. Additionally, the general future potential of ART‐FCM is addressed.
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