A modular and multi-functional purification strategy that enables a common framework for manufacturing scale integrated and continuous biomanufacturing.

Biopharmaceutical manufacture is transitioning from batch to integrated and continuous biomanufacturing (ICB). The common framework for most ICB, potentially enables a global biomanufacturing ecosystem utilizing modular and multi-function manufacturing equipment. Integrating unit operation hardware and software from multiple suppliers, complex supply chains enabled by multiple customized single-use flow paths, and large volume buffer production/storage make this ICB vision difficult to achieve with commercially available manufacturing equipment. Thus, we developed SymphonX™, a downstream processing skid with advanced buffer management capabilities, a single disposable generic flow path design that provides plug-and-play flexibility across all downstream unit operations and a single interface to reduce operational risk. Designed for multi-product and multi-process cGMP facilities, SymphonX™ can perform stand-alone batch processing or ICB. This study utilized an Apollo™ X CHO-DG44 mAb-expressing cell line in a steady-state perfusion bioreactor, harvesting product continuously with a cell retention device and connected SymphonX™ purification skids. The downstream process used the same chemistry (resins, buffer composition, membrane composition) as our historical batch processing platform, with SymphonX™ in-line conditioning and buffer concentrates. We used surge vessels between unit operations, single-column chromatography (protein A, cation and anion exchange) and two-tank batch virus inactivation. After the first polishing step (cation exchange), we continuously pooled product for 6 days. These 6 day pools were processed in batch-mode from anion exchange to bulk drug substance. This manufacturing scale proof-of-concept ICB produced 0.54 kg/day of drug substance with consistent product quality attributes and demonstrated successful bioburden control for unit-operations undergoing continuous operation.

Cool-edge populations of the kelp Ecklonia radiata under global ocean change scenarios: strong sensitivity to ocean warming but little effect of ocean acidification.

Kelp forests are threatened by ocean warming, yet effects of co-occurring drivers such as CO2 are rarely considered when predicting their performance in the future. In Australia, the kelp Ecklonia radiata forms extensive forests across seawater temperatures of approximately 7-26°C. Cool-edge populations are typically considered more thermally tolerant than their warm-edge counterparts but this ignores the possibility of local adaptation. Moreover, it is unknown whether elevated CO2 can mitigate negative effects of warming. To identify whether elevated CO2 could improve thermal performance of a cool-edge population of E. radiata, we constructed thermal performance curves for growth and photosynthesis, under both current and elevated CO2 (approx. 400 and 1000 µatm). We then modelled annual performance under warming scenarios to highlight thermal susceptibility. Elevated CO2 had minimal effect on growth but increased photosynthesis around the thermal optimum. Thermal optima were approximately 16°C for growth and approximately 18°C for photosynthesis, and modelled performance indicated cool-edge populations may be vulnerable in the future. Our findings demonstrate that elevated CO2 is unlikely to offset negative effects of ocean warming on the kelp E. radiata and highlight the potential susceptibility of cool-edge populations to ocean warming.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Identity and functional characterisation of the transporter supporting the Na+ -dependent high-affinity NO3 - uptake in Zostera marina L.

Zostera marina is a seagrass, a group of angiosperms that evolved from land to live submerged in seawater, an environment of high salinity, alkaline pH and usually very low NO3 - . In 2000, we reported the first physiological evidence for the Na+ -dependent high-affinity NO3 - uptake in this plant. Now, to determine the molecular identity of this process, we searched for NO3 - transporters common to other vascular plants encoded in Z. marina's genome. We cloned two candidates, ZosmaNPF6.3 and ZosmaNRT2 with its partner protein ZosmaNAR2. ZosmaNAR2 expression levels increase up to 4.5-fold in Z. marina leaves under NO3 - -deficiency, while ZosmaNRT2 and ZosmaNPF6.3 expressions were low and unaffected by NO3 - . NO3 - transport capacity, kinetic properties and H+ or Na+ -dependence were examined by heterologous expression in the Hansenula polymorpha high-affinity NO3 - transporter gene disrupted strain (∆ynt1). ZosmaNPF6.3 functions as a H+ -dependent NO3 - transporter, without functionality at alkaline pH and apparent dual kinetics (KM = 11.1 µM at NO3 - concentrations below 50 µM). ZosmaNRT2 transports NO3 - in a H+ -independent but Na+ -dependent manner (KM = 1 mM Na+ ), with low NO3 - affinity (KM = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co-expressed, a Na+ -dependent high-affinity NO3 - transport occurs (KM = 5.7 µM NO3 - ), mimicking the in vivo value. These results are discussed in the physiological context, providing evidence that ZosmaNRT2 is a Na+ -dependent high-affinity NO3 - transporter, the first of its kind to be functionally characterised in a vascular plant, that requires ZosmaNAR2 to achieve the necessary high-affinity for nitrate uptake from seawater.

DNA methylation and gene transcription act cooperatively in driving the adaptation of a marine diatom to global change.

Genetic changes together with epigenetic modifications such as DNA methylation have been demonstrated to regulate many biological processes and thereby govern the response of organisms to environmental changes. However, how DNA methylation might act cooperatively with gene transcription and thereby mediate the long-term adaptive responses of marine microalgae to global change is virtually unknown. Here we performed a transcriptomic analysis, and a whole-genome bisulfite sequencing, along with phenotypic analysis of a model marine diatom Phaeodactylum tricornutum adapted for 2 years to high CO2 and/or warming conditions. Our results show that the methylated islands (peaks of methylation) mCHH were positively correlated with expression of genes in the subregion of the gene body when the populations were grown under high CO2 or its combination with warming for ~2 years. We further identified the differentially expressed genes (DEGs), and hence the metabolic pathways in which they function, at the transcriptomics level in differentially methylated regions (DMRs). Although DEGs in DMRs contributed only 18-24% of the total DEGs, we found that those DEGs acted cooperatively with DNA methylation and then regulated key processes such as central carbon metabolism, amino acid metabolism, ribosome biogenesis, terpenoid backbone biosynthesis, and degradation of misfolded proteins. Taken together, by integrating transcriptomic, epigenetic, and phenotypic analysis, our study provides evidence for DNA methylation acting cooperatively with gene transcription to contribute to the adaptation of microalgae to global changes.

Avoiding and allowing apatite precipitation in oxygenic photolithotrophs.

The essential elements Ca and P, taken up and used metabolically as Ca2+ and H2 PO4 - /HPO4 2- respectively, could precipitate as one or more of the insoluble forms calcium phosphate (mainly apatite) if the free ion concentrations and pH are high enough. In the cytosol, chloroplast stroma, and mitochondrial matrix, the very low free Ca2+ concentration avoids calcium phosphate precipitation, apart from occasionally in the mitochondrial matrix. The low free Ca2+ concentration in these compartments is commonly thought of in terms of the role of Ca2+ in signalling. However, it also helps avoids calcium phosphate precipitation, and this could be its earliest function in evolution. In vacuoles, cell walls, and xylem conduits, there can be relatively high concentrations of Ca2+ and inorganic orthophosphate, but pH and/or other ligands for Ca2+ , suggests that calcium phosphate precipitates are rare. However, apatite is precipitated under metabolic control in shoot trichomes, and by evaporative water loss in hydathodes, in some terrestrial flowering plants. In aquatic macrophytes that deposit CaCO3 on their cell walls or in their environment as a result of pH increase or removal of inhibitors of nucleation or crystal growth, phosphate is sometimes incorporated in the CaCO3 . Calcium phosphate precipitation also occurs in some stromatolites.

Photon costs of shoot and root NO3-, and root NH4+, assimilation in terrestrial vascular plants considering associated pH regulation, osmotic and ontogenetic effects.

The photon costs of photoreduction/assimilation of nitrate (NO3-) into organic nitrogen in shoots and respiratory driven NO3- and NH4+ assimilation in roots are compared for terrestrial vascular plants, considering associated pH regulation, osmotic and ontogenetic effects. Different mechanisms of neutralisation of the hydroxyl (OH-) ion necessarily generated in shoot NO3- assimilation are considered. Photoreduction/assimilation of NO3- in shoots with malic acid synthesis and either accumulation of malate in leaf vacuoles or transport of malate to roots and catabolism there have a similar cost which is around 35% less than that for root NO3- assimilation and around 20% less than that for photoreduction/assimilation of NO3-, oxalate production and storage of Ca oxalate in leaf vacuoles. The photon cost of root NH4+ assimilation with H+ efflux to the root medium is around 70% less than that of root NO3- assimilation. These differences in photon cost must be considered in the context of the use of a combination of locations of NO3- assimilation and mechanisms of acid-base regulation, and a maximum of 4.9-9.1% of total photon absorption needed for growth and maintenance that is devoted to NO3- assimilation and acid-base regulation.

Why do plants silicify?

Despite seminal papers that stress the significance of silicon (Si) in plant biology and ecology, most studies focus on manipulations of Si supply and mitigation of stresses. The ecological significance of Si varies with different levels of biological organization, and remains hard to capture. We show that the costs of Si accumulation are greater than is currently acknowledged, and discuss potential links between Si and fitness components (growth, survival, reproduction), environment, and ecosystem functioning. We suggest that Si is more important in trait-based ecology than is currently recognized. Si potentially plays a significant role in many aspects of plant ecology, but knowledge gaps prevent us from understanding its possible contribution to the success of some clades and the expansion of specific biomes.

A mechanistic study of the influence of nitrogen and energy availability on the NH4+ sensitivity of nitrogen assimilation in Synechococcus.

In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a ß-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

Potential negative effects of ocean afforestation on offshore ecosystems.

Our scientific understanding of climate change makes clear the necessity for both emission reduction and carbon dioxide removal (CDR). The ocean with its large surface area, great depths and long coastlines is central to developing CDR approaches commensurate with the scale needed to limit warming to below 2 °C. Many proposed marine CDR approaches rely on spatial upscaling along with enhancement and/or acceleration of the rates of naturally occurring processes. One such approach is 'ocean afforestation', which involves offshore transport and concurrent growth of nearshore macroalgae (seaweed), followed by their export into the deep ocean. The purposeful occupation for months of open ocean waters by macroalgae, which do not naturally occur there, will probably affect offshore ecosystems through a range of biological threats, including altered ocean chemistry and changed microbial physiology and ecology. Here, we present model simulations of ocean afforestation and link these to lessons from other examples of offshore dispersal, including rafting plastic debris, and discuss the ramifications for offshore ecosystems. We explore what additional metrics are required to assess the ecological implications of this proposed CDR. In our opinion, these ecological metrics must have equal weight to CDR capacity in the development of initial trials, pilot studies and potential licensing.

Forensic carbon accounting: Assessing the role of seaweeds for carbon sequestration.

Carbon sequestration is defined as the secure storage of carbon-containing molecules for >100 years, and in the context of carbon dioxide removal for climate mitigation, the origin of this CO2 is from the atmosphere. On land, trees globally sequester substantial amounts of carbon in woody biomass, and an analogous role for seaweeds in ocean carbon sequestration has been suggested. The purposeful expansion of natural seaweed beds and aquaculture systems, including into the open ocean (ocean afforestation), has been proposed as a method of increasing carbon sequestration and use in carbon trading and offset schemes. However, to verify whether CO2 fixed by seaweeds through photosynthesis leads to carbon sequestration is extremely complex in the marine environment compared to terrestrial systems, because of the need to jointly consider: the comparatively rapid turnover of seaweed biomass, tracing the fate of carbon via particulate and dissolved organic carbon pathways in dynamic coastal waters, and the key role of atmosphere-ocean CO2 exchange. We propose a Forensic Carbon Accounting approach, in which a thorough analysis of carbon flows between the atmosphere and ocean, and into and out of seaweeds would be undertaken, for assessing the magnitude of CO2 removal and robust attribution of carbon sequestration to seaweeds.

Testing the climate intervention potential of ocean afforestation using the Great Atlantic Sargassum Belt.

Ensuring that global warming remains <2 °C requires rapid CO2 emissions reduction. Additionally, 100-900 gigatons CO2 must be removed from the atmosphere by 2100 using a portfolio of CO2 removal (CDR) methods. Ocean afforestation, CDR through basin-scale seaweed farming in the open ocean, is seen as a key component of the marine portfolio. Here, we analyse the CDR potential of recent re-occurring trans-basin belts of the floating seaweed Sargassum in the (sub)tropical North Atlantic as a natural analogue for ocean afforestation. We show that two biogeochemical feedbacks, nutrient reallocation and calcification by encrusting marine life, reduce the CDR efficacy of Sargassum by 20-100%. Atmospheric CO2 influx into the surface seawater, after CO2-fixation by Sargassum, takes 2.5-18 times longer than the CO2-deficient seawater remains in contact with the atmosphere, potentially hindering CDR verification. Furthermore, we estimate that increased ocean albedo, due to floating Sargassum, could influence climate radiative forcing more than Sargassum-CDR. Our analysis shows that multifaceted Earth-system feedbacks determine the efficacy of ocean afforestation.

The maximum growth rate hypothesis is correct for eukaryotic photosynthetic organisms, but not cyanobacteria.

The (maximum) growth rate (µmax ) hypothesis predicts that cellular and tissue phosphorus (P) concentrations should increase with increasing growth rate, and RNA should also increase as most of the P is required to make ribosomes. Using published data, we show that though there is a strong positive relationship between the µmax of all photosynthetic organisms and their P content (% dry weight), leading to a relatively constant P productivity, the relationship with RNA content is more complex. In eukaryotes there is a strong positive relationship between µmax and RNA content expressed as % dry weight, and RNA constitutes a relatively constant 25% of total P. In prokaryotes the rRNA operon copy number is the important determinant of the amount of RNA present in the cell. The amount of phospholipid expressed as % dry weight increases with increasing µmax in microalgae. The relative proportions of each of the five major P-containing constituents is remarkably constant, except that the proportion of RNA is greater and phospholipids smaller in prokaryotic than eukaryotic photosynthetic organisms. The effect of temperature differences between studies was minor. The evidence for and against P-containing constituents other than RNA being involved with ribosome synthesis and functioning is discussed.

Determinants, and implications, of the shape and size of thylakoids and cristae.

Thylakoids are flattened sacs isolated from other membranes; cristae are attached to the rest of the inner mitochondrial membrane by the crista junction, but the crista lumen is separated from the intermembrane space. The shape of thylakoids and cristae involves membranes with small (5-30 nm) radii of curvature. While the mechanism of curvature is not entirely clear, it seems to be largely a function of Curt proteins in thylakoids and Mitochondrial Organising Site and Crista Organising Centre proteins and oligomeric FOF1 ATP synthase in cristae. A subordinate, or minimal, role is attributable to lipids with areas of their head group area greater (convex leaflet) or smaller (concave leaflet) than the area of the lipid tail; examples of the latter group are monogalactosyldiglyceride in thylakoids and cardiolipin in cristae. The volume per unit area on the lumen side of the membrane is less than that of the chloroplast stroma or cyanobacterial cytosol for thylakoids, and mitochondrial matrix for cristae. A low volume per unit area of thylakoids and cristae means a small lumen width that is the average of wider spaces between lipid parts of the membranes and the narrower gaps dominated by extra-membrane components of transmembrane proteins. These structural constraints have important implications for the movement of the electron carriers plastocyanin and cytochrome c6 (thylakoids) and cytochrome c (cristae) and hence the separation of the membrane-associated electron donors to, and electron acceptors from, these water-soluble electron carriers. The donor/acceptor pairs, are the cytochrome fb6Fenh complex and P700+ in thylakoids, and Complex III and Complex IV of cristae. The other energy flux parallel to the membranes is that of the proton motive force generated by redox-powered H+ pumps into the lumen to the proton motive force use in ATP synthesis by H+ flux from the lumen through the ATP synthase. For both the electron transport and proton motive force movement, concentration differences of reduced and oxidised electron carriers and protonated and deprotonated pH buffers are involved. The need for diffusion along a congested route of these energy transfer agents may limit the separation of sources and sinks parallel to the membranes of thylakoids and cristae.