Marie Just Mikkelsen

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.

Production of 1,3-PDO and butanol by a mutant strain of Clostridium pasteurianum with increased tolerance towards crude glycerol

The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO2. Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping.

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses, Waste Biomasses, Molasses and Syrup Using the Anaerobic, Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.

Partition enrichment of nucleotide sequences (PINS)--a generally applicable, sequence based method for enrichment of complex DNA samples.

The dwindling cost of DNA sequencing is driving transformative changes in various biological disciplines including medicine, thus resulting in an increased need for routine sequencing. Preparation of samples suitable for sequencing is the starting point of any practical application, but enrichment of the target sequence over background DNA is often laborious and of limited sensitivity thereby limiting the usefulness of sequencing. The present paper describes a new method, Probability directed Isolation of Nucleic acid Sequences (PINS), for enrichment of DNA, enabling the sequencing of a large DNA region surrounding a small known sequence. A 275,000 fold enrichment of a target DNA sample containing integrated human papilloma virus is demonstrated. Specifically, a sample containing 0.0028 copies of target sequence per ng of total DNA was enriched to 786 copies per ng. The starting concentration of 0.0028 target copies per ng corresponds to one copy of target in a background of 100,000 complete human genomes. The enriched sample was subsequently amplified using rapid genome walking and the resulting DNA sequence revealed not only the sequence of a the truncated virus, but also 1026 base pairs 5′ and 50 base pairs 3′ to the integration site in chromosome 8. The demonstrated enrichment method is extremely sensitive and selective and requires only minimal knowledge of the sequence to be enriched and will therefore enable sequencing where the target concentration relative to background is too low to allow the use of other sample preparation methods or where significant parts of the target sequence is unknown.

Xdrop: targeted sequencing of long DNA molecules from low input samples using droplet sorting

Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore such technologies are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of genomic DNA. Xdrop is based on isolation of long DNA fragments in millions of double emulsion (DE) droplets, where the DE droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus (HPV) 18 integrated in the genome of human HeLa cells. The enriched DNA was sequenced both on long-read (PacBio and Oxford Nanopore) and short-read (Illumina) platforms. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex regions of the genome where long-range information is required.

Rapid and reliable method for identification of associated endonuclease cleavage and recognition sites

One barrier to cross during genetic engineering is the restriction‐modification system found in many bacteria. In this study, we developed a fast and reliable method for mapping the recognition and cleavage site of the restriction endonucleases. Clostridium pasteurianum, a model organism for the study of nitrogen fixation, has been found to harbour at least two restriction‐modification systems including the restriction endonucleases CpaPI, which is an isoschizomer of MboI and CpaAI. Dam‐methylated DNA was used to isolate the activity of CpaAI. Exposing freshly prepared cell lysate to known nucleotide fragments and directly sequencing the pool of digested nucleotide fragments enabled identification of the cleavage sites in the fragments. By aligning the sequences adjacent to the cleavage site, it was possible to identify the recognition sequence. Using this method, we successfully located all CpaAI recognition and cleavage sites within the template sequence. By modifying DNA with both Dam and CpG methylases (M.SssI) and thereby preventing digestion by CpaPI and CpaAI, no further endonuclease activity was detected.