Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Resource limitation during early ontogeny: constraints induced by growth capacity in larval and juvenile fish

The presence of and mechanisms behind density-dependent growth and resource limitation in larval and juvenile stages of organisms with high mortality such as fish are much debated. We compare observed consumption and growth rates with maximum consumption and growth rates to study the extent of resource limitation in young-of-the-year (YOY) roach (Rutilus rutilus) and perch (Perca fluviatilis). Diet, habitat use, consumption rate and growth rate were measured under varying YOY fish densities over 2 years in four lakes. In the first year, YOY roach and perch were studied under allopatric conditions. Experimental addition of perch roe in the second year also allowed study of YOY of the two species under sympatric conditions in two of the lakes. The diet of YOY roach was dominated by cladoceran zooplankton and YOY roach habitat use was restricted to the shore region in both years. This restricted habitat use did not involve any cost in foraging gain in the first year as consumption and growth rates were very close to maximum rates. During the second year, when the two species coexisted, resources were limited in late season, more so in the littoral than in the pelagic habitat in one lake while the reverse was the case in the other lake. The diet of YOY perch was also dominated by zooplankton, and with increasing perch size the proportion of macroinvertebrate prey in the diet increased. After hatching, YOY perch first utilized the pelagic habitat restricting their habitat use to the shore after 1 to several weeks in the pelagic zone. During the larval period, perch were not resource limited whereas juvenile perch were resource limited in both years. The fact that YOY perch were more resource limited than YOY roach was related to the higher handling capacity and lower attack rate of perch relative to roach, rendering perch more prone to resource limitation. Estimates of resource limitation based on consumption rates and growth rates yielded similar results. This supports the adequacy of our approach to measure resource limitation and suggests that this method is useful for studying resource limitation in organisms with indeterminate growth. Our results support the view that density-dependent growth is rare in larval stages. We suggest that density-dependent growth was absent because larval perch and roach were feeding at maximum levels over a wide range of larvae densities. Received: 14 June 1999 / Accepted: 29 October 1999     

Remote prey detection in Oithona similis: hydromechanical versus chemical cues

We quantified prey encounter rates and prey reaction distancesin the ambush-feeding cyclopoid copepod Oithona similis by videorecording freely swimming copepods at different concentrationsof prey, the dinoflagellate Gymnodinium dominans. Prey encounterrate increased with prey concentration, and a maximal clearancerate of 0.42 ± 0.10 ml h^–1 was estimated. The averagedistance (from the antennules) at which O.similis reacts toprey is 0.014 ± 0.007 cm. A simple prey encounter modelwas used to combine observed predator and prey velocities andprey reaction distance, and yielded a clearance rate similarto that estimated directly from prey encounter rates. The observedprey reaction distance was consistent with that estimated froma published model of hydromechanical prey perception. The possibilityof remote chemodetection was examined by modeling the distributionof solutes leaking out of a swimming cell. The cell leaves along slender chemical trail in its wake. However, since theambush-feeding O.similis is essentially stationary when perceivingprey, it is the width rather than the length of the trail thatmatters. Owing to advection, the chemical signal vanishes almostinstantaneously off the sides of the swimming flagellate, andsolute concentrations are below any likely detection thresholdwithin 40–50 µm from the flagellate. Our observationsare thus inconsistent with remote chemodetection in O.similis.The considerations are generalized, and it is concluded thatambush-feeding copepods, unlike cruisers and suspension feeders,cannot utilize chemical signals for the detection of individualprey, but rely on either hydromechanical detection or directinterception of prey.     

A new side to ubiquitin

Mono-ubiquitination is a common mechanism of protein regulation, and more than ten ubiquitin-interacting domains that recognize the hydrophobic region centered on Ile44 of ubiquitin have been characterized. Two recent reports describe the crystal structure of the Rab5 guanine-nucleotide-exchange factor Rabex-5 and show that it contains two novel ubiquitin-binding domains. One of these is an A20 zinc finger that binds to a polar interaction interface of ubiquitin centered on Asp58. The discovery of an alternative interaction face of ubiquitin opens new avenues for understanding how this small protein regulates protein function.     

Energy supply of the okapi in captivity: fermentation characteristics of feedstuffs

A variety of feeds are used in the nutrition of browsing ruminants. During digestion trials on okapis, feedstuffs of different facilities were sampled and the Hohenheim gas test was used as in vitro fermentation method to quantify their fermentative behavior. Forty‐six feeds were analyzed (7, fruit and vegetable; 11, energy concentrates and pelleted compounds; 13, forage; 9, browse leaf; 6, small and large twig samples). Gas production of these samples was recorded after 2, 4, 6, 8, 10, 12, and 24 hr of fermentation. Browse leaf samples were additionally analyzed with a tannin‐binding agent (polyethylene‐glycol) to assess limiting effects of condensed tannins. Metabolizable energy (ME) was estimated from 24 hr gas production according to standard regressions. Vegetables and particularly fruits were found to yield very high gas productions during the first 2 hr of fermentation, whereas unmolassed beet pulp was found to have a more even distribution of gas production/energy release over total fermentation time. Feeds like rolled oats or bread were evaluated to yield very high energy contents of >14 MJ ME/kg dry matter (DM). Alfalfa (Medicago sativa) hay had a comparable fermentation pattern to fresh browse samples, characterized by a high fermentation rate. In conclusion, energy‐rich constituents for captive ruminant diets should not include larger amounts of vegetables and especially fruits, due to their very fast fermentation during the initial phase of fermentation and the connected risk of rumen acidosis. Energy‐concentrates like beet pulp (unmolassed) showed moderate fermentation characteristics and energy content and are well suited as a component of zoo ruminant diets. Energy‐concentrates with very high energy densities (>13 MJ ME/kg DM) like bread or rolled oats are not suitable for a diet that is intended to promote long feeding times. Various aspects are involved in the decision for appropriate forage for browsing ruminants; based on fermentation pattern, alfalfa hay seems to be a reasonable substitute for browse leaves. Zoo Biol 0:1–16, 2006. © 2006 Wiley‐Liss, Inc.     

The impact of different water gas levels on cataract formation, muscle and lens free amino acids, and lens antioxidant enzymes and heat shock protein mRNA abundance in smolting Atlantic salmon, Salmo salar L

The present experiment was conducted to examine if freshwater (FW) oxygen and carbon dioxide regimes cause physiological responses that lead to cataract formation in Atlantic salmon (Salmo salar L.) smolt. Duplicate groups of 50 g Atlantic salmon smolts were exposed to three freshwater oxygen saturation regimes (95, 112 or 125% saturation), with or without addition of carbon dioxide (measured 17–18 and 2–3 mg L^− 1, respectively), for six weeks before transfer to seawater (SW). The FW exposure groups were followed up for another six weeks under a common SW regime. Fish were screened for cataract and sampled accordingly, at start, after 6 weeks in FW and after 6 weeks in SW. Increased growth related cataract incidences and severities were recorded in SW, mainly in the groups previously exposed to normoxic and hyperoxic conditions in FW, as compared to the respective groups added carbon dioxide. The concentration of histidine compounds (imidazoles) in muscle and lens tissue, used as quantitative risk markers of cataract, were lower than observed in earlier studies, however, neither were affected by the present water gas regimes in FW nor after follow up in SW. Independently of water oxygenation in FW, muscle free amino acid profiles in salmon groups concomitantly exposed to elevated carbon dioxide indicated use of selected free amino acids for energy purposes. Significantly lower abundance of heat shock protein 70 mRNA and trends towards stepwise reduction of antioxidant enzymes mRNA in the lens from fish exposed to increased water oxygenation were recorded, probably linked to increased growth and/or external stress during smoltification. This represents a first communication on using early molecular markers to express reduced protection of the fish lens against external stress to explain cataract development.     

Seasonal development of secondary xylem and phloem in <Emphasis Type="Italic">Schizolobium parahyba</Emphasis> (Vell.) Blake (Leguminosae: Caesalpinioideae)

The cambial activity and periodicity of secondary xylem and phloem formation have been less studied in tropical tree species than in temperate ones. This paper describes the relationship between seasonal cambial activity, xylem and phloem development, and phenology in Schizolobium parahyba, a fast growing semideciduous seasonal forest tree from southeastern Brazil. From 2002 to 2003, wood samples were collected periodically and phenology and climate were recorded monthly in the same period. S. parahyba forms annual growth increments in wood, delimited by narrow initial parenchyma bands. The reduction of the cambial activity to a minimum correlates to the dry season and leaf fall. The higher cambial activity correlates to the wet season and the presence of mature leaves. In phloem, a larger conductive region was observed in the wet season, when the trees were in full foliage. The secondary phloem did not exhibit any incremental zone marker; however, we found that the axial parenchyma tends to form irregular bands.     

A claim in search of evidence: reply to Manger’s thermogenesis hypothesis of cetacean brain structure

In a recent publication in Biological Reviews, Manger (2006) made the controversial claim that the large brains of cetaceans evolved to generate heat during oceanic cooling in the Oligocene epoch and not, as is the currently accepted view, as a basis for an increase in cognitive or information‐processing capabilities in response to ecological or social pressures. Manger further argued that dolphins and other cetaceans are considerably less intelligent than generally thought. In this review we challenge Manger’s arguments and provide abundant evidence that modern cetacean brains are large in order to support complex cognitive abilities driven by social and ecological forces.