Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC

Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.     

Sedimentation and Resuspension in Earthen Fish Ponds

Resuspension of particles from pond sediment into the water column may be an important nutrient transfer mechanism in aquaculture ponds. However, the magnitude of sediment re-suspension cannot be determined directly because sediment traps collect particles settling from the water column as well as those re-suspended from the pond bottom. We developed a dilution analysis method to differentiate the magnitude of the two particle source fluxes based upon the concentration of soil-derived elements (Si, Al, and Fe) and water-derived elements (C, N) in material collected by sediment traps placed in earthen ponds. Estimated organic C sedimentation from feed residues and algae was compared with trapped organic C as an independent and approximate measure of resuspension. Resuspension fluxes based independently on analyses of three soil-derived elements and on the estimation of expected C sedimentation were similar and accounted for 60–90% of the total solids flux (121–2,676 g/m² per d) in most ponds sampled. The proportion of total flux that was derived from resuspension in ponds stocked with common carp Cyprinus carpio and tilapia Oreochromis spp . was modeled as a hyperbolic function of fish size and density, with a threshold fish size of 200–300 g. Resuspension flux was conservatively estimated to be equivalent to the daily suspension of a few mm of the pond bottom. These results indicate that sediment resuspension is a major process in carp and tilapia ponds, suggesting that the exchange of nutrients between the sediment and overlying water is intensive.     

Bio-flocs technology application in over-wintering of tilapia

A 50-day experiment was conducted to investigate the effectiveness of the bio-flocs technology for maintaining good water quality in over-wintering ponds for tilapia hybrid fingerlings (Oreochromis niloticus × Oreochromis aureus). To preserve adequate water temperatures in the ponds, they were covered with polyethylene sheets and the water exchange rate was minimized to increase pond water temperature. To avoid water quality deterioration, starch was added to the ponds to stimulate the formation of bio-flocs. Temperature in the covered ponds could easily be controlled and was 0.4–4.9 °C higher than the influent water. Adjusting the C/N ratio in the ponds by adding starch or increasing the amount of carbohydrates added through the feed limited the presence of inorganic nitrogen species when the C/N was about 20, even at high stocking densities of 20 kg/m³ at harvest. Fish survival levels were excellent, being 97 ± 6% for 100 g fish and 80 ± 4% for 50 g fish. Moreover, at harvest the condition of the fish was good in all ponds with a fish condition factor of 2.1–2.3. Overall, these findings can help to overcome over-wintering problems, particularly mass mortality of fish due to low temperatures in the ponds.     

Molecular biology of male gametogenesis

Pollen development has been studied at a molecular level in several systems that are amenable to genetic or transgenic analysis. We have characterized several tomato genes that are expressed late in pollen development. Our goals in this research were 1) to determine the cis- and tran-acting factors that mediate pollen expression, and 2) to determine the functions of the proteins encoded by these genes. We currently favor the hypothesis that pollen-specific gene expression is mediated in a combinatorial manner. Antisense experiments have indicated an important role for the LAT52 protein during pollen hydration.     

Adsorption of systemic fungicides and a herbicide by some components of plant tissues,in relation to some physicochemical properties of the pesticides

Lignin, an important component of plant tissues, adsorbed five systemic fungicides and one herbicide (carbendazim, triadimefon, nuarimol, triarimol, fenarimol and fluometuron) more strongly than bovine serum albumin, cellulose, ethylcellulose or sodium polygalacturonate. Significant correlations were found between the extent of the adsorption of the pesticides on the lignins extracted from three different plant species, and the log Poet (Poet is the octan-1-ol/water partition coefficient) of the compounds. The more lipophilic fungicides triarimol and fenarimol (log Poet about 2.6) were adsorbed to the greatest extent. Fluometuron, triadimefon and nuarimol (log Poet about 2.0) were moderately adsorbed, whereas carbendazim with the lowest log Poet (1.34 at pH 5.0), was adsorbed more than expected from its Poet value. The anomaly of carbendazim is discussed; it is ascribed to it's partial protonation at pH 5.0, whereas the other pesticides were non-ionised.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Shrimp and fish pond soils: processes and management

The pond bottom soil and the accumulated sediments are integral parts of ponds. Concentrations of nutrients, organic matter and microorganism density in the pond bottom are several orders of magnitude greater than in the water. The accumulation of organic sediments may limit pond intensification. The intensive organic matter degradation at the pond bottom and high sediment oxygen demand exceeds the oxygen renewal rate. This leads to the development of anoxic conditions in the sediments and at the sediment–water interface. A series of anaerobic processes, affected by the redox potential of the system, are taking place. A large number of potentially toxic materials are generated. Among those are organic acids, reduced organic sulfur compounds, reduced manganese and sulfides.

Shrimp, as animals that normally live on or near the bottom, are exposed to conditions on the pond bottom. Exposure to toxic materials endanger the well being of the cultured shrimp. Reduced feeding, slower growth, mortality and possibly higher sensitivity to disease are reported.

The rational use of aerators to minimize the area of sludge accumulation, construction of ponds to trap sludge, stirring sediments, chemical poising of the redox system and environmentally accepted treatment and reuse of drained sediments are means to control the conditions at the pond bottom.     

Phosphorus removal in a marine prototype, recirculating aquaculture system

Phosphorus dynamics were examined in a prototype, zero-discharge, marine-recirculating system. Operation of the system without discharge of water and sludge was enabled by recirculation of effluent water through two separate treatment loops. Surface water from the fish basin was pumped over a trickling filter in one loop, while bottom-water was recirculated through a sedimentation basin followed by a fluidized bed reactor in the other treatment loop. Ammonia oxidation to nitrate in the trickling filter and organic matter digestion together with nitrate reduction in the sedimentation basin and fluidized bed reactor were the main biological features of this treatment system. Orthophosphate concentrations did not exceed 15 mg PO₄–P/l in the culture water during more than 1 year of system operation. Much of the phosphorus was retained within the sedimentation basin and fluidized bed reactor. In these treatment stages, the phosphorus content of organic matter was as high as 17.5% and 19%, respectively. High concentrations of total phosphorus and low concentrations of soluble orthophosphate were measured in the initial stages of sedimentation under oxic and anoxic conditions, suggesting that most of the phosphorus was associated with organic matter. Depletion of oxygen and nitrate in the sludge layers of the sedimentation basin coincided with sulfate reduction to sulfide and a release of soluble orthophosphate. The observed phosphorus dynamics in this marine system supported findings from previous studies in which it was demonstrated that denitrifiers underlie phosphorus immobilization under these conditions.     

Nitrogen removal techniques in aquaculture for a sustainable production

As the aquaculture industry intensively develops, its environmental impact increases. Discharges from aquaculture deteriorate the receiving environment and the need for fishmeal and fish oil for fish feed production increases. Rotating biological contactors, trickling filters, bead filters and fluidized sand biofilters are conventionally used in intensive aquaculture systems to remove nitrogen from culture water. Besides these conventional water treatment systems, there are other possible modi operandi to recycle aquaculture water and simultaneously produce fish feed. These double-purpose techniques are the periphyton treatment technique, which is applicable to extensive systems, and the proteinaceous bio-flocs technology, which can be used in extensive as well as in intensive systems. In addition to maintenance of good water quality, both techniques provide an inexpensive feed source and a higher efficiency of nutrient conversion of feed. The bio-flocs technology has the advantage over the other techniques that it is relatively inexpensive; this makes it an economically viable approach for sustainable aquaculture.