Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.     

Provenance- and life-history stage-specific responses of the dwarf shrub Calluna vulgaris to elevated vapour pressure deficit

Climate change may alter microscale-effective ecosystem properties such as atmospheric water vapour pressure, but consequences for plant growth are insufficiently understood. Within a northwest German heathland an open-top chamber experiment was established to analyse the effects of elevated vapour pressure deficit (eVPD) on growth responses of Calluna vulgaris considering both plant origin (Atlantic (AP), sub-Atlantic (SAP), sub-Continental (SCP)) and life-history stage (1-year vs. 10-year old plants). We hypothesised that the plants’ sensitivity to eVPD decreases (i) from AP to SCP and (ii) with progressing life-history stage. Elevated VPD caused a provenance-specific decrease of shoot increment whilst aboveground biomass productivity remained unaffected. AP and SAP responded with increasing belowground biomass δ¹³C signatures to eVPD, whereas δ¹³C values decreased for SCP. Moreover, eVPD increased and decreased belowground biomass δ¹³C signatures of 1- and 10-year old plants, respectively. These responses to eVPD were related to differences in morphological-chemical traits and the plants’ trait plasticity in response to eVPD. SCP showed the highest aboveground tissue mass density and significantly increased tissue C:N ratios under eVPD. One-year old plants had a tenfold higher shoot:root ratio than 10-year old plants, making young plants more sensitive to eVPD. Our findings demonstrate that the atmospheric water status affects the morphology and physiology of Calluna independent of the soil water status. The results have implications for the conservation of heathlands under climate change: (i) SCP may constitute an appropriate ecotype for assisted migration-approaches, and (ii) management needs to weigh different options for heathland rejuvenation.

     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

On the reporting and review requirements of ISO 14044

The International Journal of Life Cycle Assessment -     

Habitat matters – Strong genetic and epigenetic differentiation in Linum catharticum from dry and wet grasslands

Plant species differ in their ecological amplitude, with some species occurring in very different habitats under strongly differentiated environmental conditions. We were interested in to what extent the occurrence of Linum catharticum in dry calcareous grasslands (Bromion) and wet litter meadows (Molinion), two habitats on opposing ends concerning, for example, moisture level, is reflected on the genetic and epigenetic level. Using AFLP (amplified fragment length polymorphisms) and MSAP (methylation sensitive amplification polymorphisms) analyses, we studied the genetic and epigenetic variation of L. catharticum from calcareous grasslands and litter meadows. From each habitat, we included five study sites with 16 individuals per sampling location. We observed lower genetic than epigenetic diversity, but considerable differentiation among habitats, which was stronger on the genetic than the epigenetic level. Additionally, we observed a strong correlation of genetic and epigenetic distance, irrespective of geographic distance. The dataset included a large portion of fragments exclusively found in individuals from one or the other habitat. Some epigenetic fragments even occurred in different methylation states depending on the habitat. We conclude that environmental effects act on both the genetic and epigenetic level, producing the clear differentiation among plant individuals from calcareous grasslands and litter meadows. These results may also point into the direction of ecotype formation in this species.     

PpGRAS12 acts as a positive regulator of meristem formation in Physcomitrium patens

Plant Molecular Biology - This study focused on the key regulatory function of Physcomitrium patens GRAS12 gene underlying an increasing plant complexity, an important step in plant...     

8,000 years of climate,vegetation, fire and land-use dynamics in the thermo-mediterranean vegetation belt of northern Sardinia (Italy)

Vegetation History and Archaeobotany - Knowledge about the vegetation history of Sardinia, the second largest island of the Mediterranean, is scanty. Here, we present a new sedimentary record...