Metabolic modeling for predicting VFA production from protein-rich substrates by mixed-culture fermentation

Proteinaceous organic wastes are suitable substrates to produce high added-value products in anaerobic mixed-culture fermentations. In these processes, the stoichiometry of the biotransformation depends highly on operational conditions such as pH or feeding characteristics and there are still no tools that allow the process to be directed toward those products of interest. Indeed, the lack of product selectivity strongly limits the potential industrial development of these bioprocesses. In this work, we developed a mathematical metabolic model for the production of volatile fatty acids from protein-rich wastes. In particular, the effect of pH on the product yields is analyzed and, for the first time, the observed changes are mechanistically explained. The model reproduces experimental results at both neutral and acidic pH and it is also capable of predicting the tendencies in product yields observed with a pH drop. It also offers mechanistic insights into the interaction among the different amino acids (AAs) of a particular protein and how an AA might yield different products depending on the relative abundance of other AAs. Particular emphasis is placed on the utility of this mathematical model as a process design tool and different examples are given on how to use the model for this purpose.     

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.     

Role of seagrass photosynthesis in root aerobic processes

The role of shoot photosynthesis as a means of supporting aerobic respiration in the roots of the seagrass Zostera marina was examined. O₂ was transported rapidly (10-15 minutes) from the shoots to the root-rhizome tissues upon shoot illumination. The highest rates of transport were in shoots possessing the greatest biomass and leaf area. The rates of O₂ transport do not support a simple gas phase diffusion mechanism. O₂ transport to the root-rhizome system supported aerobic root respiration and in many cases exceeded respiratory requirements leading to O₂ release from the subterranean tissue. Release of O₂ can support aerobic processes in reducing sediments typical of Z. marina habitats. Since the root-rhizome respiration is supported primarily under shoot photosynthetic conditions, then the daily period of photosynthesis determines the diurnal period of root aerobiosis.     

Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatoms

The apoprotein of the major light harvesting pigment-protein complex from the diatom Phaeodactylum tricornutum (UTEX 646) is composed of two similar polypeptides of 17.5 and 18.0 kilodaltons (kD). The in vivo synthesis of these polypeptides is inhibited by the 80s protein synthesis inhibitor cycloheximide, but not by the 70s ribosome inhibitor chloramphenicol. When total poly(A)⁺ RNA was used in in vitro protein synthesis, a number of polypeptides were synthesized with a dominant product at 22 kD. When the polypeptides were immunoprecipitated with monospecific antibodies to the 17.5 and 18.0 polypeptides, a single protein zone of 22 kD was detected. Immunoprecipitation with preimmune serum failed to precipitate detectable levels of protein at any relative molecular weight (M_r). These findings indicate that the two apoprotein polypeptides of the diatom light harvesting pigment-protein are translated from polyadenylated message on cytoplasmic ribosomes as either a single or two (or more) similar M_r precursor proteins. These findings also suggest that this protein is encoded in the nucleus.

Photosynthetic light adaptation features of P. tricornutum UTEX 646 indicate that it responds to low light by increasing cell size and numbers of photosystem I and II reaction centers per cell, but does not change photosynthetic rate per cell or photosynthetic unit sizes significantly. When low light cells are exposed to higher photon flux densities, the in vivo incorporation of label into the apoprotein of the light harvesting complex decreases. In contrast, high light grown cells show rapid (<3 hour) increases in apoprotein synthesis when exposed to low light levels. This is the first demonstration of a specific role of photon flux density in regulating the synthesis of a major light harvesting pigment-protein during photosynthetic light adaptation.

     

Light adaptation and the role of autotrophic epiphytes in primary production of the temperate seagrass, Zostera marina L.

Photosynthetic features of Zostera marina L. and its autotrophic epiphyte community were investigated in a population inhabiting a shallow (1.3 m depth) water meadow in Great Harbor, Woods Hole, MA (U.S.A.). Photosynthesis versus irradiance (P-I) relationships were measured with respect to leaf age determined by the leaf position in the shoot bundle and by location of the tissue along the leaf axis. Therefore both age and light intensity gradients along the leaf axis were considered. The maximum photosynthesis (P_max) per dm² typically increased nearly two-fold along the leaf axis from leaf bases to apices. Photosynthetic rate on a chlorophyll (Chl) basis did not increase as dramatically along the leaf axis, and rates were usually lowest in tissues with the highest Chl content. The P-I relationships of leaves of different ages did not reveal photoinhibition even at light intensities > 1400 μE • m⁻² • s ⁻¹. Furthermore, no photoinhibition was observed in tissues from leaf blade bases, which never experienced high light levels (> 500 μE • m ⁻² • s⁻¹) in situ in Great Harbor. The initial slopes of the P-I curves and light compensation and saturation values varied along the leaf axis in relation to in situ light intensity gradients and in relation to leaf or tissue age. It appeared that leaf and/or tissue age was more important than light environment in determining P-I responses. The contribution of the autotrophic epiphyte community on Z. marina leaves to total photosynthesis per dm² was between 27 and 50%, and between 10 and 44% per mg chlorophyll. These levels of epiphyte photosynthesis can double the primary production of Z. marina leaves. No detrimental effects of epiphyte cover were realized in leaf maximal photosynthesis or P-I relationships. Non-epiphytized leaves and leaves from which epiphytes were removed showed essentially identical photosynthetic features. Light intensity and age gradients along the leaf axis control both the photosynthetic performance of the leaves and epiphyte biomass and photosynthesis.     

Low-intensity subnanosecond fluorescence study of the light-harvesting chlorophyll ab protein

The fluorescence decay characteristics of the isolated light-harvesting chlorophyll $\text{a}\text{b}$ protein have been studied using low-intensity subnanosecond-resolution time-correlated single-photon counting. In the monomeric state in detergent micelles, the chlorophyll $\text{a}\text{b}$ protein exhibits biexponential decay (τ₁ = 1.2 ns, τ₂ = 3.3 ns) with the two components having very similar weights. The decay parameters do not depend on emission wavelength. These results are discussed in relation to the Van Metter-Knox-Shepanski model (Van Metter, R.M. (1977) Biochim. Biophys. Acta 462, 642–657; Shepanski, J.S. and Knox, R.S. (1982) Isr. J. Chem., in the press) of the chlorophyll $\text{a}\text{b}$ protein, and a kinetic analysis of the energy-transfer processes. The influence of detergent composition and concentration on the fluorescence decay of the chlorophyll protein is also described.     

Carbon Partitioning in Eelgrass (Regulation by Photosynthesis and the Response to Daily Light-Dark Cycles)

Diel variations in rates of C export, sucrose-phosphate synthase (SPS) and sucrose synthase (SS) activity, and C reserves were investigated in Zostera marina L. (eelgrass) to elucidate the environmental regulation of sucrose formation and partitioning in this ecologically important species. Rates of C flux and SPS activity increased with leaf age, consistent with the ontogenic transition from sink to source status. Rates of C export and photosynthesis were low but quantitatively consistent with those of many terrestrial plant species. The Vmax activity of SPS approached that of maize, but substrate-limited rates were 20 to 25% of Vmax, indicating a large pool of inactive SPS. SPS was unresponsive to the day/night transition or to a 3-fold increase in photosynthesis generated by high [CO2] and showed little sensitivity to inorganic phosphate. Consequently, regulation of eelgrass SPS appeared similar to starch- rather than to sugar-accumulating species even though eelgrass accumulates sucrose. Leaf [sucrose] was constant and high throughout the diel cycle, which may contribute to the down-regulation of SPS. Root sucrose synthase activity was high but showed no response to nocturnal anoxia. Root [sucrose] also showed no diel cycle. The temporal stability of [sucrose] confers an ability for eelgrass to buffer the effects of prolonged light limitation that may be key to its survival and ecological success in environments subject to periods of extreme light limitation and chaotic daily variation in light availability.     

Antenna structure and excitation dynamics in photosystem I. II. Studies with mutants of Chlamydomonas reinhardtii lacking photosystem II.

Using time-resolved single photon counting, fluorescence decay in photosystem I (PS I) was analyzed in mutant strains of Chlamydomonas reinhardtii that lack photosystem II. Two strains are compared: one with a wild-type PS I core antenna (120 chlorophyll a/P700) and a second showing an apparent reduction in core antenna size (60 chlorophyll a/P700). These data were calculated from the lifetimes of core antenna excited states (75 and 45 ps, respectively) and from pigment stoichiometries. Fluorescence decay in wild type PS I is composed of two components: a fast 75-ps decay that represents the photochemically limited lifetime of excited states in the core antenna, and a minor (less than 10%) 300-800 ps component that has spectral characteristics of both peripheral and core antenna pigments. Temporal and spectral properties of the fast PS I decay indicate that (a) excitations are nearly equilibrated among the range of spectral forms present in the PS I core antenna, (b) an average excitation visits a representative distribution of core antenna spectral forms on all pigment-binding subunits regardless of the origin of the excitation, (c) reduction in core antenna size does not alter the range of antenna spectral forms present, and (d) transfer from peripheral antennae to the PS I core complex is rapid (less than 5 ps).     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

Binding sites of acetylcholine in the aromatic gorge leading to the active site of acetylcholinesterase

Theoretical calculations performed on the interactions of acetylcholine with the aromatic gorge of acetylcholinesterase indicate the existence of a number of local minima for the substrate. These minima are clustered in four regions of increasing interactions from top to bottom of the gorge, culminating in the region of the active site. The results allow the delineation of the role of the different aminoacids lining the walls, emphasizing, in particular, that of Trp 279 and Trp 84 while smaller interactions involve tyrosines 70, 121, 130, 334 and Phe 330. The influence of D72 is stressed, as well as the orientating role of A 201 and the strong driving influence of E199.