Fertilization of SRC Willow,I: Biomass Production Response

Short rotation coppice (SRC) willow is often regarded as one of the most promising crops to increase biomass production and thereby meet the growing demand for renewable energy. This study is based on the hypotheses that biomass production of SRC willow responds positively to increasing doses of nitrogen, and that similar biomass production response can be achieved by use of mineral fertilizer, sewage sludge and animal manure. A 2-year experiment was established with the clone Tordis grown on a sandy soil in northern Jutland, Denmark. The experiment included mineral fertilizer, sludge and manure, and treatments of different doses up to 360 kg nitrogen ha^?1. The fertilization led to a modest but significant increase in biomass production. The largest production of 11.9 oven dried tons/ha/year was obtained for the application of 60 kg nitrogen ha^?1 annually. Higher doses did not lead to increased biomass production; in fact, production seemed to decline with increasing fertilization application (not significant). We found no difference in production between different types of fertilizers. The limited response of the fertilization may be caused by a high fertility of the soil due to former agricultural fertilization. The number of sagging shoots increased significantly with increasing nitrogen dose.     

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut⁺ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014     

Metabolic Engineering of Saccharomyces cerevisiae

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.     

Myotubes from Severely Obese Type 2 Diabetic Subjects Accumulate Less Lipids and Show Higher Lipolytic Rate than Myotubes from Severely Obese Non-Diabetic Subjects

About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several severely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to fundamental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexibility were examined in human myotubes derived from severely obese subjects without (BMI 44±7 kg/m²) and with type 2 diabetes (BMI 43±6 kg/m²). Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to severely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also similar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher lipolysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes.     

Cyanogenic glycosides and their metabolic enzymes in barley, in relation to nitrogen levels

The possible role for cyanogenic glycosides as nitrogen storage compounds was studied in barley, Hordeum vulgare (cv. Golf), cultivated under different nitrogen regimes. Cyanogenic glycosides were absent in seeds and roots but were synthesized in seedlings where they accumulated at a level of about 150 nmol shoot⁻¹ in control plants and 110 nmol shoot⁻¹ in nitrogen-starved plants. An enzyme involved in the breakdown of cyanogenic glycosides, β-glucosidase (EC 3.2.1.-) exhibited high activity in seeds and was also detected in roots and shoots. The activity of β-cyanoalanine synthase (EC 4.4.1.9), which is involved in the metabolism of HCN, was low in seeds but very high in roots and shoots. There was no correlation between the activities of the two enzymes and the content of cyanogenic glycosides or nitrogen. The relative content of nitrogen in cyanogenic glycosides never exceeded 0.3% of total nitrogen, and the amount of cyanogenic glycosides decreased at a low rate even at a stage when nitrogen limitation inhibited growth.     

Why do leaves and leaf cells of N-limited barley elongate at reduced rates?

The objective of the present study was to assess whether, in barley, nitrogen supply limits the rate of leaf elongation through a reduction in (relative) cell elongation rate and whether this is attributable to a reduced turgor, a reduced availability of osmolytes or, by implication, changed wall properties. Plants were grown on full-strength Hoagland solution (“Hoagland”-plants), or on N-deficient Hoagland solution while receiving N at a relative addition rate of 16 or 8% N · plant-N⁻¹ · d⁻¹ (“16%-” and “8%-plants”). Hoagland-plants were demand-limited, whereas 16%- and 8%-plants were supply-limited in N. Third leaves were analysed for leaf elongation rate and final epidermal cell length, and, within the basal growing region, for the spatial distribution of relative segmental elongation rates (RSER, pin-pricking method), epidermal cell turgor (cell-pressure probe), osmotic pressure (OP, picolitre osmometry) and water potential (Ψ). During the development of the third leaf, plants grew at relative growth rates (relative increase in fresh weight ) of 18.2, 15.6 and 8.1% · d⁻¹ (Hoagland-, 16%- and 8%-plants, respectively). Final leaf length and leaf elongation rate were highest in Hoagland plants (ca. 34.1 cm and 2.33–2.60 mm · h⁻¹, respectively), intermediate in 16%- plants (31.0 cm and 1.89–1.96 mm · h⁻¹) and lowest in 8%-plants (29.4 cm and 1.41–1.58 mm · h⁻¹). These differences were accompanied by only small differences in final cell length, but large differences in cell-flux rates (146, 187 and 201 cells · cell-file⁻¹ · d⁻¹ in 8%-, 16%- and Hoagland-plants, respectively). The length of the growth zone (32–38 mm) was not much affected by N-levels (and nutrient technique). A decrease in RSER in the growth zone distal to 10 mm produced the significant effect of N-levels on leaf elongation rate. In all treatments, cell turgor was almost constant throughout the growing region, as were cell OP and Ψ in 16%- and 8%-plants. In Hoagland-plants, however, cell OP increased by ca. 0.1 MPa within the zone of highest elongation rates and, as a consequence, cell Ψ decreased simultaneously by 0.1 MPa. Cell Ψ increased considerably where elongation ceased. Within the zone where differences in RSERs were highest between treatments (10–34 mm from base) average turgor was lowest, OP highest and Ψ most negative in Hoagland- compared to 8%- and 16%-plants (P < 0.001), but not significantly different between 8%- and 16%-plants. Received: 9 January 1997 / Accepted: 6 March 1997     

Gastric bypass surgery reveals independency of obesity and diabetes melitus type 2

Background

Roux-en-Y gastric bypass surgery is widely applied to ameliorate morbid obesity, including diabetes in people with type 2 diabetes. The latter vanish a few days after surgery for many, but not in all patients before any weight reduction has occurred. The explanation for this change in metabolic status is poorly understood, but the observation may suggest that the fate obesity and diabetes is only partly linked after surgery.

Methods

The trajectories of weight reduction measured as reduced body mass index (BMI) in 741obese subjects with and without diabetes were evaluated. Evaluation was performed on three groups: 1) subjects that were non-diabetic before and after surgery; 2) subjects that were diabetics before surgery but non-diabetics after surgery; and 3) subjects that were diabetics before surgery and remained diabetics after surgery. The diabetic state was established at HbA1c above 48 mmol/mol.

Results

The trajectories differ significantly between groups and any sub-populations of groups, the latter identified by the distance between individual trajectories using a k-means procedure. The results suggest that different domains in the enormous genetic network governing basic metabolism are perturbed in obesity and diabetes, and in fact some of the patients are affected by two distinct diseases: obesity and diabetes mellitus type 2.

Conclusion

Although RYGB “normalized” many glycaemic parameters in some of the diabetic subjects apparently converting to a non-diabetics state, other diabetic subjects stay diabetic in the context of the new gut anatomy after surgery. Thus, the obesity part of the glycaemic derangement may have been ameliorated, but some defects of the diabetic state had not.