Precision emulsification for droplet and capsule production

Emulsions and microcapsules are typical structures in various dispersion formulations for pharmaceutical, food, personal and house care applications. Precise control over size and size distribution of emulsion droplets and microcapsules are important for effective use and delivery of active components and better product quality. Many emulsification technologies have been developed to meet different formulation and processing requirements. Among them, membrane and microfluidic emulsification as emerging technologies have the feature of being able to precisely manufacture droplets in a drop-by-drop manner to give subscribed sizes and size distributions with lower energy consumption. This paper reviews fundamental sciences and engineering aspects of emulsification, membrane and microfluidic emulsification technologies and their use for precision manufacture of emulsions for intensified processing. Generic application examples are given for single and double emulsions and microcapsules with different structure features. © 2013 The Society of Powder Technology Japan. Published by Elsevier B.V.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Cognitive Approach in Castings’ Quality Control

Every year production volume of castings grows, especially grows production volume of non-ferrous metals, thanks to aluminium. As a result, requirements to castings quality also increase. Foundry men from all over the world put all their efforts to manage the problem of casting defects. In this article the authors present an approach based on the use of cognitive models that help to visualize inner cause-and-effect relations leading to casting defects in the foundry process. The cognitive models mentioned comprise a diverse network of factors and their relations, which together thoroughly describe all the details of the foundry process and their influence on the appearance of castings’ defects and other aspects.. Moreover, the article contains an example of a simple die casting model and results of simulation. Implementation of the proposed method will help foundry men reveal the mechanism and the main reasons of casting defects formation.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

Munkaszervezés a lean termelésben – mit magyaráznak a termelésmenedzsment koncepciók = Work organization in lean production – what production concepts explain

A tanulmány a lean termelés munkaszervezését három termelésmenedzsment koncepció segítségével vizsgálja. Az egyes koncepciók a szervezet eltérő metszeteit érintik: (1) a termék-folyamat mátrix (Hayes és Wheelwright, 1979) a termék és a folyamat jellemzőit helyezi középpontba. A lean hatására a szervezet a mátrixban a nagyobb választék és a folyamat alapú működés (nagyobb függőség) irányába mozdul el. Az elmozdulást üzemi szinten a magas elkötelezettségű munkavégzési rendszer gyakorlatainak bevezetése kíséri, mivel azok támogatják a rugalmas működést, a gyors kommunikációt és problémamegoldást. Az elmozdulás „minősége” és így a munkaszervezési gyakorlatok használata (mélyég, száma, munkavállalók bevonása) nagyban függ a termelési stratégiától és a lean érettségtől. (2) A termelési stratégia szakaszai (Wheelwright és Hayes, 1985) a termelés üzleti stratégiában játszott szerepét elemzik. A lean termelés összeegyeztethető a termelési stratégia harmadik szakaszának „command és control” szemléletmódjával. Az ilyen lean termelők költégfókuszúak, a hagyományos munkaerőképben gondolkodnak és körükben kevésbé jellemző az új emberi erőforrás gyakorlatok használata. A lean termelés adaptálása ösztönözheti a vállalatokat a termelési stratégia negyedik szintje felé. A negyedik szint a bevonásra, problémamegoldásra és tanulásra épít, amely megfelel a lean „emberek tisztelete” pillérének. (3) A lean érettségi modell (Hines és társai, 2004) a lean szervezeten belüli elmélyülését és terjedését mutatja be. A lean utazás során a vállalatok az eszköz alapú megközelítéstől a komplex értékrendszerben gondolkodó lean szervezet felé haladnak. A technikai tudásanyag egyre szélesebb körűvé válik, ami rávilágít a tudásátadás (személyek közöttire, de akár struktúrákba, folyamatokba építése is) képességének jelentőségére. Az emberi erőforrás gyakorlatok folyamatosan jelennek meg. De csak a legfejlettebb szakasz, a lean tanuló szervezet megjelenése teszi valóban szükségessé a munkavállalói kép újragondolását is. = This paper examines work organization in lean production with the help of three production concepts. These concepts embrace different dimensions of the organization: (1) the product-process matrix (Hayes and Wheelwright, 1979) is about product and process characteristics. Due to the lean the organization shifts within the matrix – towards higher variability and flow (higher level of interdependencies). On the shop floor the shift is accompanied by the introduction of high commitment work system’s practices, since those support flexible operations, fast communication and problem-solving. The „quality” of the shift and hence the application of these work practices (number of practices, their embeddeness, employee involvement) highly depends on manufacturing strategy and lean maturity. (2) The concept of stages of manufacturing strategy (Wheelwright and Hayes, 1985) analyzes the role of the manufacturing function in the business strategy. Lean production is compatible with the „command and control” approach of the third stage of manufacturing strategy. These lean producers are cost-driven, they have the traditional approach of employees and apply new work organization practices to a less extent. However, the implementation of lean production may drive these companies to the fourth stage. The fourth stage of manufacturing strategy is based on employee involvement, problem-solving and learning. This stage is in full accordance with the „respect for people” pillar of lean production. (3) Lean maturity (Hines et al., 2004) shows the path how lean management deepens and expands within an organization. During the lean journey, companies progress from the tool-based approach to the complex lean value system. The technical knowledge of lean becomes more and more comprehensive and it points out the crucial importance of knowledge conversion capabilities (intrapersonal or even how to build knowledge into structures, processes). Work organization practices constantly appear with the progress, but the review of the traditional approach of employees is only essential at the most advanced stage, when an organization becomes lean learning organization.

A production/recycling model with quality consideration

An integrated production–recycling system is investigated. A constant demand can be satisfied by production and recycling. The used items might be bought back and then recycled. The not recycled products are disposed off. Two types of models are analyzed. The first model examines and minimizes the EOQ related cost. The second model generalizes the first one by introducing additionally linear waste disposal, recycling, production and buyback costs. This basic model was examined by the authors in a previous paper. The main results are that a pure strategy (either production or recycling) is optimal. This paper extends the model for the case of quality consideration: it is asked for the quality of the bought back products. In the former model we have assumed that all returned items are serviceable. One can put the following question: Who should control the quality of the returned items? If the suppliers examine the quality of the reusable products, then the buyback rate is strongly smaller than one, α<1. If the user does it, then not all returned items are recyclable, i.e. the use rate is smaller than one, δ<1. Which one of the control systems are more cost advantageous in this case?

Visszutas logisztika és termeléstervezés (Reverse logistics and production planning)

A dolgozat a visszutas logisztikát, az újrahasznosítást igyekszik beilleszteni a vállalati termeléstervezés keretei közé. A szükséglettervezési rendszerek (material requirements planning, MRP) célja a készletek és beszerzendő anyagok, alkatrészek időben ütemezett gyártásának és beszerzésének megtervezése. A klasszikus MRP rendszereket az utóbbi időben próbálja a tudomány az újrahasznosítással kibővíteni. Mivel ebben az esetben az új, és újrafelhasználható anyagokat külön kell nyilvántartani, ezért az MRP-táblák és készletek növekednek. A rendelési tételnagyságok meghatározása is nehezebb, összetettebb tételnagysághoz vezet. A dolgozatban egy visszutas logisztikai készletmodellt ismertetünk, valamint annak dinamikus kiterjesztését, amely alapja lehet az SAP-ba beépíthető rendelés állomány meghatározó heurisztikának. ____ The aim of the paper is to extend production planning with reverse logistics and reuse. Material requirements planning (MRP) systems plan and control invetory levels and purchasing activities of the firm. In the last decade scientists on this field try to involve reverse logistics activities in MRP systems. Size of MRP-tables is growing in this case because of the alternative use of newly purchased products and reusable old items. Determination of order quantities will be more complex with these two modes of material supplies. An EOQ-type reverse logistics model is presented in the paper with a dynamic lot size generalization. The generalized model can be seen as a basic model to build in production planning and control system like SAP.

Phosphorus Availability and Salinity Control Productivity and Demography of the Seagrass Thalassia testudinum in Florida Bay

Biomass, net primary productivity (NPP), foliar elemental content, and demography of Thalassia testudinum were monitored in populations from five sites across Florida Bay beginning in January 2001. Sites were selected to take advantage of the spatial variability in phosphorus (P) availability and salinity climates across the bay. Aboveground biomass and NPP of T. testudinum were determined five to six times annually. Short-shoot demography, belowground biomass, and belowground NPP were assessed from a single destructive harvest at each site and short-shoot cohorts were estimated from leaf scar counts multiplied by site-specific leaf production rates. Biomass, relative growth rate (RGR), and overall NPP were positively correlated with P availability. Additionally, a positive correlation between P availability and the ratio of photosynthetic to non-photosynthetic biomass suggests that T. testudinum increases allocation to aboveground biomass as P availability increases. Population turnover increased with P availability, evident in positive correlations of recruitment and mortality rates with P availability. Departures from seasonally modeled estimates of RGR were found to be influenced by salinity, which depressed RGR when below 20 psu or above 40 psu. Freshwater management in the headwaters of Florida Bay will alter salinity and nutrient climates. It is becoming clear that such changes will affect T. testudinum, with likely feedbacks on ecosystem structure, function, and habitat quality.

Hydrological Conditions Control P Loading and Aquatic Metabolism in an Oligotrophic, Subtropical Estuary

Using high-resolution measures of aquatic ecosystem metabolism and water quality, we investigated the importance of hydrological inputs of phosphorus (P) on ecosystem dynamics in the oligotrophic, P-limited coastal Everglades. Due to low nutrient status and relatively large inputs of terrestrial organic matter, we hypothesized that the ponds in this region would be strongly net heterotrophic and that pond gross primary production (GPP) and respiration (R) would be the greatest during the “dry,” euhaline estuarine season that coincides with increased P availability. Results indicated that metabolism rates were consistently associated with elevated upstream total phosphorus and salinity concentrations. Pulses in aquatic metabolism rates were coupled to the timing of P supply from groundwater upwelling as well as a potential suite of hydrobiogeochemical interactions. We provide evidence that freshwater discharge has observable impacts on aquatic ecosystem function in the oligotrophic estuaries of the Florida Everglades by controlling the availability of P to the ecosystem. Future water management decisions in South Florida must include the impact of changes in water delivery on downstream estuaries.

Quality Control Circles May Answer Industry's Needs

The concept of the quality control circle (QCC) has worked well in Japanese industry in increasing efficiency, production, and profits. The author explores the QCC, its history and advantages, and tells how it could be adapted quite easily and effectively to the hospitality industry

Design and implementation of a web based shop floor control system to maintain schedule feasibility in a dynamic job shop environment

The effective control of production activities in dynamic job shop with predetermined resource allocation for all the jobs entering the system is a unique manufacturing environment, which exists in the manufacturing industry. In this thesis a framework for an Internet based real time shop floor control system for such a dynamic job shop environment is introduced. The system aims to maintain the schedule feasibility of all the jobs entering the manufacturing system under any circumstance. The system is capable of deciding how often the manufacturing activities should be monitored to check for control decisions that need to be taken on the shop floor. The system will provide the decision maker real time notification to enable him to generate feasible alternate solutions in case a disturbance occurs on the shop floor. The control system is also capable of providing the customer with real time access to the status of the jobs on the shop floor. The communication between the controller, the user and the customer is through web based user friendly GUI. The proposed control system architecture and the interface for the communication system have been designed, developed and implemented.

Physiological response measures (respiration, clearance, byssus production, survival) of Perna viridis during a 91 day exposure experiment to microplastics in Bogor, Indonesia, 2014

Perna viridis from the Bay of Jakarta was exposed to different concentrations (0, 21.6, 216 and 2160 mg/l) of PVC microplastic particles for 91 days in a controlled laboratory experiment. Particles were negatively buoyant, but were regularly resuspended from the sediment, mimicking tidal events. The particles were contaminated with the organic pollutant fluoranthene, except for one control group, which was exposed to the highest plastic concentration (2160 mg/l) but with clean particles. Within the 91 days survival was monitored. After 40 - 44 days of the exposure, physiological responses of all mussel individuals were measured. Respiration rates were measured as the decrease of oxygen in a sealed container in 20 minutes. Clearance rates were determined by measuring the depletion of algal cells in the water in 30 minutes. Byssus production was assessed by counting the number of newly formed byssus discs within 24 hours.

KOSMOS Finland 2012 mesocosm study: primary production and respiration

Anthropogenic carbon dioxide (CO2) emissions are reducing the pH in the world's oceans. The plankton community is a key component driving biogeochemical fluxes, and the effect of increased CO2 on plankton is critical for understanding the ramifications of ocean acidification on global carbon fluxes. We determined the plankton community composition and measured primary production, respiration rates and carbon export (defined here as carbon sinking out of a shallow, coastal area) during an ocean acidification experiment. Mesocosms (~ 55 m3) were set up in the Baltic Sea with a gradient of CO2 levels initially ranging from ambient (~ 240 µatm), used as control, to high CO2 (up to ~ 1330 µatm). The phytoplankton community was dominated by dinoflagellates, diatoms, cyanobacteria and chlorophytes, and the zooplankton community by protozoans, heterotrophic dinoflagellates and cladocerans. The plankton community composition was relatively homogenous between treatments. Community respiration rates were lower at high CO2 levels. The carbon-normalized respiration was approximately 40 % lower in the high CO2 environment compared with the controls during the latter phase of the experiment. We did not, however, detect any effect of increased CO2 on primary production. This could be due to measurement uncertainty, as the measured total particular carbon (TPC) and combined results presented in this special issue suggest that the reduced respiration rate translated into higher net carbon fixation. The percent carbon derived from microscopy counts (both phyto- and zooplankton), of the measured total particular carbon (TPC) decreased from ~ 26 % at t0 to ~ 8 % at t31, probably driven by a shift towards smaller plankton (< 4 µm) not enumerated by microscopy. Our results suggest that reduced respiration lead to increased net carbon fixation at high CO2. However, the increased primary production did not translate into increased carbon export, and did consequently not work as a negative feedback mechanism for increasing atmospheric CO2 concentration.