The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000 L, and greater, to meet commercial demand. However, the concern of the ‘shear sensitivity’ of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10 g/L are now reported with cell densities greater than 2 × 10⁷ cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of ‘shear sensitivity’ has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, “upper limit” to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.     

Evaluation of the effects and interactions of mixing and oxygen transfer on the production of Fab’ antibody fragments in Escherichia coli fermentation with gas blending

Fermentations carried out at 450-L and 20-L scale to produce Fab’ antibody fragments indicated a serious problem to control levels of dissolved oxygen in the broth due to the large oxygen demand at high cell densities. Dissolved oxygen tension (DOT) dropped to zero during the induction phase and it was hypothesised that this could limit product formation due to inadequate oxygen supply. A gas blending system at 20-L scale was employed to address this problem and a factorial 2² experimental design was executed to evaluate independently the effects and interaction of two main engineering factors: agitation rate and DOT level (both related to mixing and oxygen transfer in the broth) on Fab’ yields. By comparison to the non-gas blending system, results in the gas blending system at same scale showed an increase in the production of Fab’ by 77% independent of the DOT level when using an agitation rate of 500 rpm level and by 50% at an agitation rate of 1,000 rpm with 30% DOT. Product localisation in the cell periplasm of >90% was obtained in all fermentations. Results obtained encourage further studies at 450-L scale initially, to evaluate the potential of gas blending for the industrial production of Fab’ antibody fragments.     

Use of the multi-allelic self-incompatibility gene in apple to assess homozygocity in shoots obtained through haploid induction

 To obtain homozygous genotypes of apple, we have induced haploid development of either the female or the male gametes by parthenogenesis in situ and anther culture, respectively. Of the shoots obtained, which were mainly of a non-haploid nature, some could be derived from fertilised egg cells or from sporophytic anther tissue. In order to select the shoots having a true haploid origin, and thus homozygotes, we decided to use the single multi-allelic self-incompatibility gene as a molecular marker to discriminate homozygous from heterozygous individuals. The rationale behind this approach was that diploid apple cultivars contain 2 different alleles of the S-gene and therefore the haploid induced shoots obtained from them should have only one of the alleles of the single parent. The parental cultivars used were ‘Idared’ (parthenogenesis in situ) and ‘Braeburn’ (androgenesis), and their S-genotypes were known, except for 1 of the ‘Braeburn’S-alleles. To stimulate parthenogenetic development ‘Idared’ styles were pollinated with irradiated ‘Baskatong’ pollen, the S-alleles of the latter (2n) cultivar were also unknown. The cloning and sequence analysis of these 3 unidentified S-alleles, 1 from ‘Braeburn’ and 2 from ‘Baskatong’ is described, and we show that they correspond to the S ₂₄ -, S ₂₆ - and S ₂₇ -alleles. We have optimised a method for analysis of the S-alleles of ‘Idared/Baskatong’- or ‘Braeburn’-derived in vitro plant tissues and have shown that this approach can be applied for the screening of the in vitro shoots for their haploid origin. Received: 18 August 1997 / Accepted: 10 September 1997     

Influence of endogenous and exogenous RNases on the variation of pollen cytosolic-free Ca2+ in Pyrus serotina Rehd

The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca²⁺]_i) variation of pre-germinated Pyrus pollen were studied. [Ca²⁺]_i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T₁ and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca²⁺]_i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca²⁺]_i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca²⁺]_i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca²⁺]_i of ‘Kohsui’ pollen. RNase T₁, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca²⁺]_i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca²⁺]_i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca²⁺]_i changes.     

In vitro screening for cold hardiness of raspberry cultivars

Raspberry (Rubus idaeus L.) cultivars ‘Festival’, ‘Titan’ and ‘Willamette’ were cultured in vitro on three different media: (A) MS medium supplemented with 1.0 mg l^-1 BAP and 0.1 mg l^-1 IBA, (B) MS medium without growth regulators, and (C) MS medium with reduced sucrose (10 g l^-1), and exposed to different low temperature acclimation treatments: (1) control, no acclimation, (2) 1 week at +15 °C, 1 week at +2 °C, 24 h at -2 °C and 3 days at +2 °C, and (3) 2 weeks at +15 °C, 2 weeks at +2 °C, 24 h at −2 °C and 3 days at +2 °C. After acclimation, shoot moisture content was measured, and cold hardiness (LT₅₀) was determined by controlled freezing. Shoot moisture content was generally lower on culture medium B compared to the other media, but not affected by acclimation treatment. In non-acclimated plants, medium composition had no effect on cold hardiness and no cultivar differences in hardiness were observed. After acclimation, plants on culture medium B were on average more cold hardy than on the other media. Acclimation treatment 3 on media A and B allowed the best discrimination between the hardy cultivar ‘Festival’ and less cold hardy ‘Titan’ and ‘Willamette’. When acclimation treatments were tested further using 11 raspberry cultivars with different levels of cold hardiness, discrimination between cultivars was satisfactory only after acclimation treatment 3 on culture medium B. This revised version was published online in June 2006 with corrections to the Cover Date.     

Assessing the Sensitivity of Seabed Species and Biotopes – The Marine Life Information Network (MarLIN)

Science-based approaches to support the conservation of marine biodiversity have been developed in recent years. They include measures of ‘rarity’, ‘diversity’, ‘importance’, biological indicators of water ‘quality’ and measures of ‘sensitivity’. Identifying the sensitivity of species and biotopes, the main topic of this contribution, relies on accessing and interpreting available scientific data in a structured way and then making use of information technology to disseminate suitably presented information to decision makers. The Marine Life Information Network (MarLIN) has achieved that research for a range of environmentally critical species and biotopes over the past four years and has published the reviews on the MarLIN Web site (www.marlin.ac.uk). Now, by linking the sensitivity database and databases of survey information, sensitivity mapping approaches using GIS are being developed. The methods used to assess sensitivity are described and the approach is advocated for wider application in Europe.     

Improving bioreactor cultivation conditions for sensitive cell lines by dynamic membrane aeration

Although the importance of animal cell culture for the industrial (large scale) production of pharmaceutical products is continuously increasing, the sensibility of the cells towards their cultivation environment is still a challenging issue. In comparison to microbial cultures, cell cultures which are not protected by a cell wall are much more sensitive to shear stress and foam formation. Reactor design as well as the selection of ‘robust’ cell lines is particularly important for these circumstances. Nevertheless, even ‘sensitive’ cell lines are selected for certain pharmaceutical processes due to various reasons. These sensitive cell lines have even higher requirements regarding their cultivation environment. Important characteristics for the corresponding reactor design are a high (volumetric) gas mass transfer coefficient, low volumetric power input, low shear stress, low susceptibility to bio-fouling, the ability to cultivate sticky cells and sufficient mixing properties. Membrane aeration has been a long-known possibility to meet some of these requirements, but has not often been applied in recent years. The reasons lie mainly in low gas mass transfer rates, a limited installable volume-specific membrane surface area, restrictions in scalability and problems with membrane fouling. The dynamic membrane aeration bioreactor aeration is a simple concept for bubble-free oxygen supply of such sensitive cultures. It overcomes limitations and draw-backs of previous systems. Consisting of an oscillating, centrally arranged rotor (stirrer) that is wrapped with silicone membrane tubing, it enables doubling the gas mass transfer at the same shear stress in the investigated cultivation scales of 12, 20, 100, and 200 L. Continuous cultivation at these scales allows the same product output as fed-batch cultivation does at tremendously larger reactor volumes. Apart from introducing this novel technology, the presentation comprises selected cultivation results obtained for blood coagulation factor VIII in continuous mode and a therapeutic monoclonal antibody in fed-batch mode in comparison to reference trials.     

Genetic dissection of root growth in rice (Oryza sativa L.) I: a hydrophonic screen

 Root growth is an important component of the adaptation of rice to drought-prone environments. A hydroponic screen was used to study root growth of 28 rice varieties. Both maximum root length and adventitious root thickness varied widely between varieties. In general, japonica varieties had larger root systems than indica varieties. Two F₂ populations involving the thick- and long-rooted upland japonica variety ‘Azucena’ and two poor-rooting varieties, namely the upland indica‘Bala’ and the Italian japonica‘Maratelli’, were made and screened in hydroponics. Generation means analysis revealed significant additive and dominance main effects for the root length traits with a prevalence of dominance gene effects in both crosses. The dominance×dominance type of non-allelic interactions were important for maximum root length from day 7 to day 28, root volume, root thickness and root cell length in the cross ‘Bala’בAzucena’. The heritability (broad-sense) estimates varied from low to high for the traits and displayed differences between populations. This suggested that recombinant lines with improved root traits can be developed from the two crosses with selection methods that involve some form of progeny evaluation. In a companion paper, we report the mapping of quantitative trait loci (QTLs) for root growth traits in the ‘Bala’בAzucena’ population using restriction fragment length polymorphisms (RFLPs). Received: 5 May 1996 / Accepted: 14 February 1997     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Expression of a unique plastid-localized heat-shock protein is genetically linked to acquired thermotolerance in wheat

 We have used a combination of molecular and classical genetic approaches to delineate the relationship between a specific HSP member and cell viability under heat stress. Using recombinant inbred lines (RILs) of wheat, derived from a cross of the thermotolerant cultivar ‘Mustang’ and the thermosusceptible cultivar ‘Sturdy,’ we have identified a unique HSP and a differentially expressed cDNA sequence, both related to the plastid-localized HSP26 gene family, that are closely associated with acquired thermotolerance in wheat. An isoform of HSP26 was synthesized under heat stress in all examined thermotolerant RILs and ‘Mustang’, and was absent in all examined thermosusceptible RILs and ‘Sturdy.’ Using a modified differential-display method, we have also identified a gene-specific cDNA sequence that is similar to other known members of the wheat HSP26 gene family and is selectively expressed in ‘Mustang’ and most of the examined thermotolerant RILs, but not expressed in ‘Sturdy’ and all the thermosusceptible RILs. These results suggest a genetic linkage between the acquired thermotolerance trait and the differential expression of a unique member of the HSP26 gene family. Received: 21 April 1997 / Accepted: 2 May 1997     

Somatic embryogenesis and plantlet regeneration in mango (Mangifera indica L.)

Summary The ‘Carabao’ or ‘Manila Super’ mango (Mangifera indica L.), a virtually neglected fruit before the advent of KNO₃ flower induction in the early 1970s, is now the third leading Philippine export fruit after banana and pineapple. To apply biotechnology for improvement, a reliable embryogenesis and regeneration protocol is required. We have developed a protocol for somatic embryogenesis and plantlet regeneration in mango: eight strains of ‘Carabao’ and two unidentified varieties, PHL 12384 and PHL 12378. Over 40 batches of nucellar explants from immature fruis (0.75–5.0 cm long) were cultured in vitro from April 1999 to April 2000. Two media were used, MMSE. Mango Medium for Somatic Embryo Induction, Proliferation and Germination and MMPR, Mango Medium for Plantlet Regeneration. These are now routinely used. The protocol is reproducible in 14 other varieties of mango. Shifting the base medium from Gamborg's B5 medium to our own formulation. BP medium (Barba and Pate?a's formulation) effectively controlled browning. Browning has limited the successful in vitro culture of many woody species including the mango. Crop Science Society of the Philippines (CSSP) 2001 Best Paper Award, Asian Agriculture Congress, Westin Philippine Plaza, Manila, Philippines, April 24–27, 2001 and Philippine Fruit Association 2000 Best Poster Award, 8th National Symposium. PCARRD, Los Ba?os, Laguna, Philippines, November 14–16, 2000.     

Genetic variability and structure of natural and domesticated populations of Caribbean pine (Pinus caribaea Morelet)

 Isozyme analysis of seed samples derived from natural and managed populations of the tropical pine Pinus caribaea vars ‘bahamensis’ and ‘caribaea’ was used to assess population genetic structure in its native range and to detect changes occurring during early domestication of the species. Baseline data from natural populations of the two varieties showed that populations sampled as seed are characterized by high gene diversity (mean H_e=0.26) and a low level of inbreeding ( mean F_is=0.15). A UPGMA tree of genetic relatedness among populations indicates that the two varieties represent distinct evolutionary units. Within each variety there is significant differentiation among populations, and this is greater for the more fragmented populations of var ‘bahamensis’ (F_st=0.08) than for var ‘caribaea’ (F_st=0.02). Seed from a seed orchard population of var ‘caribaea’ established within its natural range showed no change in genetic diversity but did show a reduced inbreeding coefficient (F_is=0.09) compared with its progenitor populations, suggesting a decrease in selfing and/or biparental inbreeding. A bulked seed sample from an exotic plantation of var ‘bahamensis’ in Australia displayed a large increase in the inbreeding coefficient (F_is=0.324) compared with that found in natural populations, possibly due to elevated self-fertilization. Finally, a bulked seed sample from an exotic plantation population of var ‘caribaea’ from China showed enhanced genetic diversity, an increase in the inbreeding coefficient and more linkage disequilibrium than its presumed progenitor populations. It was also genetically divergent from them. RFLP analysis of chloroplast DNA variation in the Chinese sample suggested that seeds of the related taxa P. elliottii and P. taeda, or seeds derived from hybridization with these taxa growing in the seed production area, had been included in the seed crop during harvesting. We conclude that monitoring of appropriate genetic markers may be an effective means of identifying potentially deleterious genetic changes occurring during forest tree domestication. Received: 10 August 1998 / Accepted: 8 September 1998     

Molecular mapping of a resistance-specific PCR-based marker linked to a gall midge resistance gene (Gm4t) in rice

 A PCR-based marker (E20₅₇₀) linked to the gene Gm4t, which confers resistance to a dipteran pest gall midge (Orseolia oryzae), has been mapped using the restriction fragment length polymorphism (RFLP) technique in rice. Gm4t is a dominant resistance gene. We initially failed to detect useful polymorphism for this marker in a F₃ mapping population derived from a cross between two indica parents, ‘Abhaya’בShyamala’, with as many as 35 restriction enzymes. ‘Abhaya’ carries the resistance gene Gm4t and ‘Shyamala’ is susceptible to gall midge. Subsequently, E20₅₇₀ was mapped using another mapping population represented by a F₂ progeny from a cross between ‘Nipponbare’, a japonica variety, and ‘Kasalath’, an indica variety, in which the gene Gm4t was not known to be present. Gm4t mapped onto chromosome 8 between markers R1813 and S1633B. Our method, thus, presents an alternative way of mapping genes which otherwise would be difficult to map because of a lack of polymorphism between closely related parents differing in desired agronomic traits. Received: 1 April 1997 / Accepted: 13 May 1997     

Genetic control theory of developmental events

An earlier theory of cell differentiation and morphogenesis (Wassermann, 1972, 1973, 1978) is combined with the genetic control model of Davidson and Britten (e.g. 1979). The resulting new theory suggests how, bysystematic process algorithms, specifically enumerated combinations of batteries of structural genes can become switched on in particularly enumerated cells, via battery-specific enumerable regulator genes. The systematization is idealized. Up to a certain stage of development in each mitotically arising cell a unique cell-specific combination of structural genes called ‘marker genes’ is active. Marker genes are assumed to code for cell-specifying marker proteins (CSMPs) which permit cells carrying related markers to recognize each other, thus permitting specific cell sorting.Batteries of marker genes could ensure great developmental precision and can safeguard—via redundancies of CSMP types—against accidental loss or detrimental mutational modification of CSMPs or marker genes, respectively. This paper is much concerned with cell lineage in relation to ‘microdifferentiation’, where ‘microdifferentiation’ of a cell refers to a cell's active marker genes and its syntheses of CSMPs. A drastic distinction is made between ‘microdifferentiation’ and ‘gross’ differentiation of a cell, where the same ‘gross’ differentiation may be shared by a large number of cells that could each be uniquely ‘microdifferentiated’. Typical ‘gross’ differentiation could manifest itself in tissue specificity, whereas, up to certain stages of development, all cells of the same gross differentiation type (say tissue specificity) could each be uniquely ‘microdifferentiated’. The theory also assumes that at certain stages of the developmental process some (or in some organisms all) of the previously uniquely specified cells could give rise to small (or occasionally large) clones of equispecified cells, some of which might form clusters that represent complete ‘morphogenetic fields’ Tentative implementation mechanisms are proposed which suggest how the theory could operate in molecular biological terms. In particular, CSMPs could endow cell surface membranes with a highly specific protein network, and an associated equally specific cell surface coat. It is suggested how these highly specified cell surface coats and other systems could provide an ‘extracellular guidance network’ which could help to direct cells to attain energetically optimal locations relative to each other based on the matching of their surface specificities. In numerous experimental situations, where normally present optimal matching of cells is excluded, ‘alternative matching’ based on experiment-specific suboptimal matching could explain many data, notably in experimental development neurobiology (Wassermann, 1978).     

Analysis of hybrids of durum wheat with Thinopyrum junceiforme using RAPD markers

 The objective of this study was to detect the presence of alien chromatin in intergeneric hybrids of durum wheat (Triticum turgidum, 2n=4x=28; AABB genomes) with the perennial grass Thinopyrum junceiforme (2n=4x=28; J₁J₁J₂J₂) using RAPD markers. The first step was to identify amplification of species-specific DNA markers in the parental grass species and durum wheat cultivars. Initially, the genomic DNA of five grass species (Thinopyrum junceiforme, Th. bessarabicum, Lophopyrum elongatum, Leymus karataviensis and Elytrigia pycnantha) and selected durum cultivars (‘Langdon’, ‘Durox’, ‘Lloyd’, ‘Monroe’, and ‘Medora’) was screened with 40 oligonucleotide primers (nano-mers). Three oligonucleotides that amplified DNA fragments specific to a grass species or to a durum cultivar were identified. Primer PR21 amplified DNA fragments specific to each of the five durum cultivars, and primers PR22 and PR23 amplified fragments specific to each of the grass species. Intergeneric hybrids between the durum cultivars ‘Langdon’, ‘Lloyd’ and ‘Durox’ and Th. junceiforme, and their backcross (BC) progeny were screened with all 40 primers. Six primers amplified parent-specific DNA fragments in the F₁ hybrids and their BC₁ progeny. Three primers, PR22, PR23 and PR41, that amplified Th. junceiforme DNA fragments in both F₁ and BC₁ were further analyzed. The presence of an amplified 1.7-kb Th. junceiforme DNA fragment in the F₁ hybrids and BC₁ progeny was confirmed using Southern analysis by hybridization with both Th. junceiforme genomic DNA and Th. junceiforme DNA amplified with primer PR41. With the exception of line BC₁F₂ no. 5, five selfed progeny of BC₁ and a BC₂ of line 3 (BC₁F₂ no. 3בLloyd’) from a cross of ‘Lloyd’×Th. junceiforme showed the presence of the 1.7-kb DNA fragment. All selfed BC₁ and BC₂ lines retained the 600-bp fragment that was confirmed after hybridization with Th. junceiforme DNA amplified with primer PR22. Other experiments using RFLP markers also showed the presence of up to seven Th. junceiforme DNA fragments in the F₁ hybrids and their BC progeny after hybridization with Th. junceiforme DNA amplified with primer PR41. These studies show the usefulness of molecular markers in detecting alien chromatin/DNA fragments in intergeneric hybrids with durum wheat. Received: 21 November 1996 / Accepted: 21 March 1997     

The establishment of cell suspension cultures ofGladiolus that regenerate plants

Summary Inflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured on solid medium.     

‘Chillum’ jar,a better technique for screening of rhizobia under summer conditions

Summary In the present investigations ‘Chillum’ jar assembly was found to provide more favourable environmental conditions for rhizobia to nodulate leguminous plants particularly under summer conditions than the usual Leonard jar assembly. When thirty pigeon pea rhizobia isolates were tested for their nodulation efficiency in both Leonard jars as well as ‘Chillum’ jars, it was noticed that there was no nodulation in any of the isolates under Leonard jars whereas all isolates were nodulating well under ‘Chillum’ jars conditions. This was probably due to lowering of temperature in ‘Chillum’ jar caused by rapid evaporation from the outer surface of ‘Chillum’ jar assembly. The maximum temperature recorded in ‘Chillum’ jar was 34°C whereas in Leonard jars it was 46.5°C.     

Elevated [CO2], temperature increase and N supply effects on the turnover of below-ground carbon in a temperate grassland ecosystem

The effects of elevated [CO₂] (700 μl l^-1 CO₂) and temperature increase (+3 °C) on carbon turnover in grassland soils were studied during 2.5 years at two N fertiliser supplies (160 and 530 kg N ha^-1 y^-1) in an experiment with well-established ryegrass swards (Lolium perenne) supplied with the same amounts of irrigation water. During the growing season, swards from the control climate (350 μl l^-1 [CO₂] at outdoor air temperature) were pulse labelled by the addition of ¹³CO₂. The elevated [CO₂] treatments were continuously labelled by the addition of fossil-fuel derived CO₂ (^{13 C} of -40 to -50 ‰). Prior to the start of the experimental treatments, the carbon accumulated in the plant parts and in the soil macro-organic matter (‘old’ C) was at −32‰. During the experiment, the carbon fixed in the plant material (‘new’ C) was at −14 and −54‰ in the ambient and elevated [CO₂] treatments, respectively. During the experiment, the ¹³C isotopic mass balance method was used to calculate, for the top soil (0–15 cm), the carbon turnover in the stubble and roots and in the soil macro-organic matter above 200 μ (MOM). Elevated [CO₂] stimulated the turnover of organic carbon in the roots and stubble and in the MOM at N+, but not at N−. At the high N supply, the mean replacement time of ‘old’ C by ‘new’ C declined in elevated, compared to ambient [CO₂], from 18 to 7 months for the roots and stubble and from 25 to 17 months for the MOM. This resulted from increased rates of ‘new’ C accumulation and of ‘old’ C decay. By contrast, at the low N supply, despite an increase in the rate of accumulation of ‘new’ C, the soil C pools did not turnover faster in elevated [CO₂], as the rate of ‘old’ C decomposition was reduced. A 3 °C temperature increase in elevated [CO₂] decreased the input of fresh C to the roots and stubble and enhanced significantly the exponential rate for the ‘old’ C decomposition in the roots and stubble. An increased fertiliser N supply reduced the carbon turnover in the roots and stubble and in the MOM, in ambient but not in elevated [CO₂]. The respective roles for carbon turnover in the coarse soil OM fractions, of the C:N ratio of the litter, of the inorganic N availability and of a possible priming effect between C-substrates are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.