High-throughput protein production – Lessons from scaling up from 10 to 288 recombinant proteins per week

The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for handling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.     

Inhibition of methane production from whey by heavy metals – protective effect of sulfide

A whey solution was used as a substrate for methane production in an anaerobic fixed-bed reactor. At a hydraulic retention time of 10 days, equivalent to a space loading of 3.3 kg (m³day)⁻¹, 90% of the chemical oxygen demand was converted to biogas. Only a little propionate remained in the effluent. Toxicity tests with either copper chloride, zinc chloride or nickel chloride were performed on effluent from the reactor. Fifty per cent inhibition of methanogenesis was observed in the presence of ≥10 mg CuCl₂ l⁻¹≥40 mg ZnCl₂ l⁻¹ and ≥60 mg NiCl₂ l⁻¹, respectively. After exposure to Cu²⁺, Zn²⁺ or Ni²⁺ ions for 12 days, complete recovery of methanogenesis by equimolar sulfide addition was possible upon prolonged incubation. Recovery failed, however, for copper chloride concentrations ≥40 mg l⁻¹. If the sulfide was added simultaneously with the three heavy metal salts, methanogenesis was only slightly retarded and the same amount of methane as in non-inhibited controls was reached either 1 day (40 mg ZnCl₂ l⁻¹) or 2 days later (10 mg CuCl₂ l⁻¹). Up to 60 mg NiCl₂ l⁻¹ had no effect if sulfide was present. Sulfide presumably precipitated the heavy metals as metal sulfides and by this means prevented heavy metal toxicity. Received: 8 October 1999 / Received revision: 3 January 2000 / Accepted: 4 January 2000     

Dry anaerobic ammonia–methane production from chicken manure

The effect of temperature on production of ammonia during dry anaerobic fermentation of chicken manure (CM), inoculated with thermophilic methanogenic sludge, was investigated in a batch condition for 8 days. Incubation temperature did not have a significant effect on the production of ammonia. Almost complete inhibition of production of methane occurred at 55 and 65°C while quite low yields of 8.45 and 6.34 ml g⁻¹ VS (volatile solids) were observed at 35 and 45°C due to a higher accumulation of ammonia. In order to improve the production of methane during dry anaerobic digestion of CM, stripping of ammonia was performed firstly on the CM previously fermented at 65°C for 8 days: the stripping for 1 day at 85°C and pH 10 removed 85.5% of ammonia. The first-batch fermentation of methane for 75 days was conducted next, using the ammonia-stripped CM inoculated with methanogenic sludge at different ratios, (CM: thermophilic sludge) of 1:2, 1:1, and 2:1 on volume per volume basis at both 35 and 55°C. Production of methane improved and was higher than that of the control (without stripping of ammonia) but the yield of 20.4 ml g⁻¹ VS was still low, so second stripping of ammonia was conducted, which resulted in 74.7% removal of ammonia. A great improvement in the production of methane of 103.5 ml g⁻¹ VS was achieved during the second batch for 55 days.     

Bioenergy from permanent grassland – A review: 1. Biogas

Grassland biomass is suitable in numerous ways for producing energy. It is well established as feedstock for biogas production. The aim of this review is to summarize current knowledge on suitability and sustainability of grassland biomass for anaerobic digestion. In the first section grassland management for biogas feedstock as well as specifics of harvest, postharvest and digestion technology are described. Methane yields from grass are influenced by many factors. While the effects of some parameters such as grass species, cutting period and management intensity can be regarded as well known, other parameters such as preservation and processing still need investigation. In the second section economic aspects and environmental impacts are discussed. Profitability can be achieved depending on grass silage supply costs and the concept of anaerobic digestion and energy use. Grassland biomass for biogas production competes with other feedstock and other forms of grassland use, in particular animal husbandry. In developed countries a growing production of milk and meat is achieved with decreasing ruminant numbers, resulting in an increasing amount of surplus grassland with a remarkable bioenergy potential. In emerging and developing countries a rapidly rising demand for and production of milk and meat induce growing pressure on grasslands, so that their use for animal feed presumably will take priority over use for bioenergy. Grasslands provide a variety of essential environmental benefits such as carbon storage, habitat function, preservation of ground and surface water quality. When producing biogas from grassland these benefits will remain or even grow, providing appropriate grassland management is implemented. In particular, greenhouse gas emissions can be considerably reduced.     

Evaluating biochemical methane production from brewer’s spent yeast

Anaerobic digestion treatment of brewer’s spent yeast (SY) is a viable option for bioenergy capture. The biochemical methane potential (BMP) assay was performed with three different samples (SY1, SY2, and SY3) and SY1 dilutions (75, 50, and 25 % on a v/v basis). Gompertz-equation parameters denoted slow degradability of SY1 with methane production rates of 14.59–4.63 mL/day and lag phases of 10.72–19.7 days. Performance and kinetic parameters were obtained with the Gompertz equation and the first-order hydrolysis model with SY2 and SY3 diluted 25 % and SY1 50 %. A SY2 25 % gave a 17 % of TCOD conversion to methane as well as shorter lag phase (<1 day). Average estimated hydrolysis constant for SY was 0.0141 (±0.003) day^?1, and SY2 25 % was more appropriate for faster methane production. Methane capture and biogas composition were dependent upon the SY source, and co-digestion (or dilution) can be advantageous.     

Desiccation tolerant ‘resurrection’ grasses from Kenya and West Africa

Summary Only a very small proportion of angiospermae have foliage which can recover (within 24 h) from full airdryness. The number of such plants was extended by five grass species, in the genera Eragrostis, Sporobolus and Tripagon, and by two sedges, Kyllinga spp. The basal leaf meristematic zones in most species survived equilibration to air of 0 to 5% RH, whereas mature leaf tissue in most survived 5 to 30% RH.     

Mechanism of methane monooxygenase – a structural and quantum chemical perspective

 The diiron site of methane monooxygenase (MMO) has the unique ability to activate methane. Structural studies of the MMO diiron site have revealed a limited number of coordination sites for dioxygen and dioxygen derived species. Using quantum mechanical studies of the MMO reaction, several possible reaction paths have been investigated. Energetically feasible geometries have been obtained for the different reaction steps, where the substrate activation is best described by an almost pure hydrogen abstraction step, followed by the formation of a metal-carbon bond. Received: 14 October 1997 / Accepted: 20 January 1998     

Daylight and absenteeism – Evidence from Norway

Based on administrative register data from Norway, we examine the impact of hours of daylight on sick-leave absences among workers. Our preferred estimates imply that an additional hour of daylight increases the daily entry rate to absenteeism by 0.5 percent and the corresponding recovery rate by 0.8 percent, ceteris paribus. The overall relationship between absenteeism and daylight hours is negative. Absenteeism is also sensitive to weather conditions. Heavy snowfall raises the incidence of absence during the winter, while warm weather reduces the probability of returning to work during the summer.     

Biogas production from crop residues on a farm-scale level: is it economically feasible under conditions in Sweden?

Anaerobic digestion would enable the energy potential of agricultural crop residues such as sugar beet tops and straw to be harnessed. Sweden is so spread out that full utilisation of this potential by centralised slurry-based technology is difficult. It appears that simple but effective high-solids reactor systems have a better chance of being economically viable on a farm-scale level (50–500 kW). In the present study, the financial prospects of high-solids digestion, using either single-stage fed-batch or two-stage batch reactor systems, are compared on a farm-scale level (50 kW) with those of conventional slurry digestion, on the basis of experimental results and observations on a laboratory- and pilot-scale. The gas produced can be used for heat, combined heat and power or as vehicle fuel. The results indicate high-solids single-stage fed-batch operations to stand the best chances of being competitive, particularly in connection with organic farming. The methane yield, degree of gas utilisation, and operational costs were found to have the strongest impact on the financial success of the process.     

Rhamnose lipids – biosynthesis, microbial production and application potential

Biosurfactants containing rhamnose and β-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical properties and potential applications. With Pseudomonas species, more than 100 g l⁻¹ rhamnolipids were produced from 160 g l⁻¹ soybean oil at a volumetric productivity of 0.4 g l⁻¹h⁻¹. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m⁻¹ to 25–30 mN m⁻¹ at concentrations of 10–200 mg l⁻¹. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of l-rhamnose, which is already used for the industrial production of high-quality flavor components. Received: 1 July 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

Biogas (natural gas?) production by anaerobic digestion of oil cake by a mixed culture isolated from cow dung

Starting with cow dung, a mixed culture capable of producing biogas by the anaerobic digestion of castor cake (oil expelled) has been isolated and stabilized. The biogas so produced contains small quantities of ethane, propane and butane in addition to methane and carbon dioxide which are the major constituents. This suggests that the mixed culture contains organisms hitherto unisolated and unidentified which are capable of synthesizing these hydrocarbons through the mediation of the alkyl derivatives of coenzyme M.     

Micropylar exudates in Douglas fir – timing and volume of production

 In Pseudotsuga menziesii, a secretion fills the micropylar canal about 7 weeks postpollination until fertilization. Micropylar volumes were measured and found to show variation. Dissection of the ovuliferous scales caused excess fluid to be exuded from the micropylar canal, forming a drop at the tip of the micropyle. This drop was collected, and its production quantified in three trees. Volume and percentage of ovules with drops were greatest when the archegonia in the female gametophyte were at central cell and/or egg cell stage. The volume of exuded drops far exceeded that of the micropyle. Production of subsequent drops by the ovules further confirms that the fluid is actively produced upon dissection Received: 19 September 1998 / Revision accepted: 26 October 1998     

Specific interaction of capsid protein and importin-α/β influences West Nile virus production

West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-α. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-α/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-α/C protein interaction in the context of flavivirus life-cycle.     

Trends in greenhouse gas emissions from consumption and production of animal food products – implications for long-term climate targets

To analyse trends in greenhouse gas (GHG) emissions from production and consumption of animal products in Sweden, life cycle emissions were calculated for the average production of pork, chicken meat, beef, dairy and eggs in 1990 and 2005. The calculated average emissions were used together with food consumption statistics and literature data on imported products to estimate trends in per capita emissions from animal food consumption. Total life cycle emissions from the Swedish livestock production were around 8.5 Mt carbon dioxide equivalents (CO₂e) in 1990 and emissions decreased to 7.3 Mt CO₂e in 2005 (14% reduction). Around two-thirds of the emission cut was explained by more efficient production (less GHG emission per product unit) and one-third was due to a reduced animal production. The average GHG emissions per product unit until the farm-gate were reduced by 20% for dairy, 15% for pork and 23% for chicken meat, unchanged for eggs and increased by 10% for beef. A larger share of the average beef was produced from suckler cows in cow–calf systems in 2005 due to the decreasing dairy cow herd, which explains the increased emissions for the average beef in 2005. The overall emission cuts from the livestock sector were a result of several measures taken in farm production, for example increased milk yield per cow, lowered use of synthetic nitrogen fertilisers in grasslands, reduced losses of ammonia from manure and a switch to biofuels for heating in chicken houses. In contrast to production, total GHG emissions from the Swedish consumption of animal products increased by around 22% between 1990 and 2005. This was explained by strong growth in meat consumption based mainly on imports, where growth in beef consumption especially was responsible for most emission increase over the 15-year period. Swedish GHG emissions caused by consumption of animal products reached around 1.1 t CO₂e per capita in 2005. The emission cuts necessary for meeting a global temperature-increase target of 2° might imply a severe constraint on the long-term global consumption of animal food. Due to the relatively limited potential for reducing food-related emissions by higher productivity and technological means, structural changes in food consumption towards less emission-intensive food might be required for meeting the 2° target.     

Carbon dioxide and methane fluxes in boreal peatland microcosms with different vegetation cover—effects of ozone or ultraviolet-B exposure

O₃ concentrations in the troposphere are rising and those in the stratosphere decreasing, the latter resulting in higher fluxes of solar ultraviolet-B (UV-B) radiation to the earth's surface. We assessed whether the fluxes of CO₂ and CH₄ are altered by enhanced UV-B radiation or elevated tropospheric O₃ concentrations in boreal peatland microcosms (core depth 40 cm, diameter 10.5 cm) with different vegetation cover. At the end of the UV-B experiment which lasted for a growing season, net CO₂ exchange (NEE) and dark ecosystem respiration (R _TOT) were sevenfold higher, and CH₄ efflux 12-fold higher, in microcosms with intact vegetation dominated by Eriophorum vaginatum L. and Sphagnum spp., compared to microcosms from which we removed E. vaginatum. Vegetation treatment had minor effects on CH₄ production and consumption potentials in the peat, suggesting that the large difference in CH₄ efflux is mainly due to efficient CH₄ transport via the aerenchyma of E. vaginatum. Ambient UV-B supplemented with 30% and elevated O₃ concentrations (100 and 200 ppb, for 7 weeks) significantly increased R _TOT in both vegetation treatments. Elevated O₃ concentrations reduced NEE over time, while UV-B had no clear effects on the fluxes of CO₂ or CH₄ in the cloudy summer of the study. Field experiments are needed to assess the significance of increasing UV-B radiation and elevated tropospheric O₃ concentration on peatland gas exchange in the long-term.     

Future consequences and challenges for dairy cow production systems arising from climate change in Central Europe – a review

It is well documented that global warming is unequivocal. Dairy production systems are considered as important sources of greenhouse gas emissions; however, little is known about the sensitivity and vulnerability of these production systems themselves to climate warming. This review brings different aspects of dairy cow production in Central Europe into focus, with a holistic approach to emphasize potential future consequences and challenges arising from climate change. With the current understanding of the effects of climate change, it is expected that yield of forage per hectare will be influenced positively, whereas quality will mainly depend on water availability and soil characteristics. Thus, the botanical composition of future grassland should include species that are able to withstand the changing conditions (e.g. lucerne and bird's foot trefoil). Changes in nutrient concentration of forage plants, elevated heat loads and altered feeding patterns of animals may influence rumen physiology. Several promising nutritional strategies are available to lower potential negative impacts of climate change on dairy cow nutrition and performance. Adjustment of feeding and drinking regimes, diet composition and additive supplementation can contribute to the maintenance of adequate dairy cow nutrition and performance. Provision of adequate shade and cooling will reduce the direct effects of heat stress. As estimated genetic parameters are promising, heat stress tolerance as a functional trait may be included into breeding programmes. Indirect effects of global warming on the health and welfare of animals seem to be more complicated and thus are less predictable. As the epidemiology of certain gastrointestinal nematodes and liver fluke is favourably influenced by increased temperature and humidity, relations between climate change and disease dynamics should be followed closely. Under current conditions, climate change associated economic impacts are estimated to be neutral if some form of adaptation is integrated. Therefore, it is essential to establish and adopt mitigation strategies covering available tools from management, nutrition, health and plant and animal breeding to cope with the future consequences of climate change on dairy farming.     

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved β-glucocerebrosidase enzymes have no effect on their function or distribution.