Learning-by-doing: experience from 20 years of teaching LCA to future engineers

Purpose

In support of the sustainable development of our societies, future engineers should have elementary knowledge in sustainability assessment and use of life cycle assessment. Publications on pedagogical experience with teaching life cycle assessment (LCA) in high-level education are however scarce. Here, we describe and discuss 20 years of experience in teaching LCA at MSc level in an engineering university with the ambition to share our insights and inspire teaching of LCA as part of a university curriculum.

Methods

We detail the design of an LCA course taught at the Technical University of Denmark since 1997. The course structure relies on (i) a structured combination of theoretical teaching, practical assignments and hands-on practice on LCA case studies, and (ii) the conduct of real-life LCA case studies in collaboration with companies or other organisations. Through the semester-long duration of the course, students from different engineering backgrounds perform full-fledged LCA studies in groups, passing through two iterations—a screening LCA supporting a more targeted LCA.

Results and discussion

The course design, which relies on a learning-by-doing principle, is transparently described to inspire LCA teachers among the readers. Historical evolution and statistics about the course, including its 192 case studies run in collaboration with 105 companies and institutions, are analysed and serve as basis to discuss the benefits and challenges of its different components, such as the theory acquisition, the assignment work, the LCA software learning, the conduct of case studies, the merits of industrial collaborations and grading approaches.

Conclusions

We demonstrate the win-win situation created by the setting of the course, in which the students are actively engaged and learn efficiently how to perform an LCA while the collaborating companies often get useful insights into their analysed case studies. The course can also be an eye opener for companies unfamiliar with LCA, who get introduced to life cycle thinking and the potential benefits of LCA. We have no hesitation in recommending industries and LCA teachers to engage into such collaborations even in the fundamental teaching of LCA techniques.

     

Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.     

Bacterial and fungal communities in bulk soil and rhizospheres of aluminum-tolerant and aluminum-sensitive maize (Zea mays L.) lines cultivated in unlimed and limed Cerrado soil

Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.     

Wild boar (Sus scrofa) harvesting using the espera hunting method: side effects and management implications

Harvesting of wildlife by man has been linked to demographic and evolutionary impacts in many populations. We investigated the sex ratio and age class structure in hunting bags of wild boar harvested by espera—nocturnal single hunt at bait—during four hunting seasons in Alentejo (Portugal). In addition, we assessed whether the hunting method is a significant predictor of the probability of harvesting an animal of a particular gender, of particular age class or of a particular combination of these two attributes. We found that the espera hunting method allows very selective harvesting regimes, and thus, it seems a highly effective population management tool. Removing a large proportion of adult males, however, may bias the population sex ratio towards females, reduce male life expectancy and raise the degree of polygyny. Our results suggest that recruitment rates are resilient to this skewed sex ratio, and possibly the higher proportion of females in the adult population may even increase productivity.

     

Side-effects of organic and synthetic pesticides on cold-stored diapausing prepupae of <Emphasis Type="Italic">Trichogramma cordubensis</Emphasis>

The side-effects of three insecticides (deltamethrin, lambda-cyhalothrin and Bacillus thuringiensis Berliner) and one fungicide (basic copper sulphate) were tested on cold-stored diapausing prepupae of Trichogramma cordubensis Vargas and Cabello (Hymenoptera: Trichogrammatidae). Pesticides were directly sprayed on parasitized host eggs (being the diapausing parasitoids in the prepupal stage) after cold storage (3°C) for three different periods (60, 120 and 180 days). Regardless of the period of cold storage, both pyrethroids reduced the emergence rates of T. cordubensis (both <25%) compared to the control (emergence varied from 83% to 89%). The most toxic pyrethroid was lambda-cyhalothrin; Trichogramma cordubensis adult emergence varied from 7% to 15%. Lambda-cyhalothrin also negatively affected the longevity and fecundity of parasitoids cold stored for 60 days. Bacillus thuringiensis Berliner subsp. kurstaki and basic copper sulphate had little or no adverse effect on emergence rates (generally >80%), longevity nor fecundity of T. cordubensis, indicating that these pesticides could be successfully integrated into pest management programs using wasps that were cold stored under diapause. Such integration would be valuable to pest management programs by reducing the costs of T. cordubensis mass rearing and by allowing producers to stockpile parasitoids for release in the growing season. However, since the emergence rate, longevity and fecundity of T. cordubensis generally decreased with increasing duration in cold storage, wasps to be used in integrated pest management programs should only be stored at 3°C for 60 days maximum. Handling Editor: Sam Cook.     

Determination of protein concentration in downstream biomanufacturing processes by in-line index of refraction

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.     

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist.We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.     

Imaging plant cells by two-photon excitation

Summary. Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.Correspondence and reprints: Instituto Gulbenkian de Ciência. 2780-156 Oeiras, Portugal.     

Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess

The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate. Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)). No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K. lactis and other known Crabtree-negative yeasts. During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions. When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change. Our results suggest that the low tendency for ethanol formation in K. marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.     

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.