Ben A. Bergmann

Nutrient recovery from swine lagoon water by Spirodela punctata.

Spirodela punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments with synthetic swine lagoon water medium was examined for N and P recovery. It has shown a capability to grow in and to remove N and P from synthetic swine lagoon water with high N (240 mg NH4 N/l) and P (31.0 mg PO4 P/l) levels. A lag period of approximately 96 h was observed before the duckweed started to grow. During the lag period, utilization of N and P by the duckweed was very slow. The rates of N and P uptake, and duckweed growth increased with the increase of the initial N and P concentrations in the medium. The highest rates of N and P uptakes, and duckweed growth observed in this study were 0.955. 0.129 mg/l-h, and 1.33 g/m2-h (or 31.92 g/m2-day), respectively. The N:P ratio in swine lagoon water is adequate for growing the duckweed.

In vitro selection of duckweed geographical isolates for potential use in swine lagoon effluent renovation

Plant-based systems for nutrient sequestration into valuable biomass have the potential to help avoid the environmental problems associated with the disposal of large volumes of animal waste. The objective of this study was to select superior duckweed (Lemnaceae) genotypes for the utilization of nutrients in animal wastes. A two-step protocol was used to select promising duckweed geographic isolates to be grown on swine lagoon effluent. Forty-one geographic isolates from the worldwide germplasm collection were used in an in vitro screening test, because they were noted to be fast-growing genotypes during routine collection maintenance. In vitro screening was accomplished by growing geographic isolates on a synthetic medium that approximated swine lagoon effluent in terms of nutrient profile, total ionic strength, pH, and buffering capacity. Large differences among geographic isolates were observed for wet weight gain during the 11-day growing period, percent dry weight, and percent protein in dry biomass. Total protein production per culture jar differed 28-fold between the most disparate of the 41 geographic isolates and was the variable used for selection of superior geographic isolates. The challenge of eight of the 41 geographic isolates with full-strength swine lagoon effluent in the greenhouse led to the selection of three that are promising as genotypes to be grown on lagoon effluent.

Genetic transformation of duckweed Lemna Gibba and Lemna Minor

SummaryWe developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.

NUTRIENT REMOVAL FROM SWINE LAGOON LIQUID BY LEMNA MINOR 8627

Nitrogen and phosphorus removal from swine lagoon liquid by growing Lemna minor 8627, a promising duckweed identified in previous studies, was investigated under in vitro and field conditions. The rates of nitrogen and phosphorus uptake by the duckweed growing in the in vitro system were as high as 3.36 g m–2 day–1 and 0.20 g m–2 day–1, respectively. The highest nitrogen and phosphorus removal rates in the field duckweed system were 2.11 g m–2 day–1 and 0.59 g m–2 day–1, respectively. The highest observed duckweed growth rate was close to 29 g m–2 day–1 in both conditions. Wastewater concentrations and seasonal climate conditions had direct impacts on the duckweed growth and nutrient removal in outdoor tanks. The rate of duckweed production in diluted swine lagoon liquid increased as the dilution rate increased. Duckweed assimilation was the dominant mechanism for nitrogen and phosphorus removal from the swine lagoon liquid when the nutrient concentration in the wastewater was low, but became less important as nutrient concentration increased. Reasonably high light intensity and a longer period of warm temperature could result in a higher growth rate for the duckweed. Pre–acclimation of the duckweed with swine lagoon liquid could accelerate the start–up of a duckweed system to remove nutrients from the wastewater by preventing the lag phase of duckweed growth.

In vitro adventitious shoot production in Paulownia

In vitro adventitious shoot formation was examined in petioles and laminae ofPaulownia elongata, P. fortunei, andP. ‘Henan 1’. Fully expanded, dark green, thick, older leaves were healthier and exhibited greater callus and shoot production than did young leaves. The ten clones used varied greatly in adventitious shoot formation. When single explants from the center of each lamina were cultured,P. ‘Henan 1’ andP. elongata exhibited similar shoot production, and both were more prolific thanP. fortunei. Shoot production per leaf fromP. elongata andP. ‘Henan 1‘ could be greatly increased if petioles were cut in half and leaf laminae were cut into three strips, rather than leaving petiole and laminae explants intact. The growth regulator concentrations required for maximal shoot production differed among clones, but all required 0.2 or 0.5 mg/l naphthaleneacetic acid and 5.0 or 7.0 mg/l benzyladenine. The average adventitious shoot production after 4 weeks in culture for the two most prolific genotypes was 63 shoots per leaf fromP. ‘Henan 1’ and 48 shoots per leaf fromP. elongata.

Combining constructed wetlands and aquatic and soil filters for reclamation and reuse of water

Abstract Reclamation and reuse of water and nutrients at their source provide the opportunity to use simple, less costly technologies and lessens potentials for catastrophic effects due to centralized treatment system failures. The combination of multiple treatment environments within constructed wetlands can provide water quality suitable for reuse. A current project in rural Chatham County, NC, uses simple, aesthetically pleasing treatment components constructed both outdoors and indoors to reclaim domestic sewage for toilet flushing, landscape irrigation and aesthetic water features. A courtyard containing constructed wetlands and a solarium with modular soil filter components and aquatic chambers are designed to treat sewage from within a small business facility and to provide recreational space for its 60 employees. The combination of vertical flow and horizontal flow constructed wetlands with fill and draw controls provides the necessary environments for nitrification–denitrification, removal of organic materials and phosphorus adsorption reactions. The system is designed to treat and reuse 4500 l day −1 (1200 gal day −1 ) of domestic sewage from the business. Some of the plants used are selectively bred or genetically engineered to maximize their water reclamation potential. Utilization of simple treatment and reuse technology has permitted the business owner to renovate an abandoned and deteriorating school building into a home for two thriving and internationally based businesses and to protect the water quality of a nearby reservoir.

NUTRIENT REMOVAL FROM SWINE LAGOON EFFLUENT BY DUCKWEED

ABSTRACT. Three duckweed geographic isolates were grown on varying concentrations of swine lagoon effluent in a greenhouse to determine their ability to remove nutrients from the effluent. Duckweed biomass was harvested every other day over a 12-day period. Duckweed biomass production, nutrient loss from the swine lagoon effluent, and nutrient content of duckweed biomass were used to identify effluent concentrations/geographic isolate combinations that are effective in terms of nutrient utilization from swine lagoon effluent and production of healthy duckweed biomass. When Lemna minor geographic isolate 8627 was grown on 50% swine lagoon effluent, respective losses of TKN, NH 3 -N, TP, OPO 4 -P, TOC, K, Cu, and Zn were 83, 100, 49, 31, 68, 21, 28, and 67%.

Five years of Paulownia field trials in North Carolina

Performance of Paulownia elongata, Paulownia fortunei, and PaulowniaבHenan 1’ was monitored at three sites in North Carolina between 1996 and 2000, and P. elongata clones were tested at an additional two sites. The primary objective was to define the potential for growing Paulownia in the state. Species and clones within species differed in survival and height and diameter growth whenever genotype was included as a treatment. P. elongata was superior at two and P. fortunei was superior at one of the three sites that included P. elongata, P. fortunei, and P.בHenan 1’. Trees produced through vegetative means were more likely to survive and were taller and of greater DBH than seed produced trees. Mortality in trees produced from seed was greater for P. fortunei and P. בHenan 1’ than for P. elongata. Application of animal waste or different rates of nitrogen as inorganic fertilizer did not have a pronounced effect on tree survival and growth.

In vitro rooting and early greenhouse growth of micropropagated Paulownia elongata shoots

Experiments focused on improving methods for rooting micropropagated Paulownia elongata shoots and enhancing the quality of plants grown from rooted shoots transferred to the greenhouse. Micropropagated shoots rooted on basal Murashige and Skoog medium, but inclusion of 0.2 mg/l naphthaleneacetic acid and 0.4 mg/l indole-3-butyric acid during a 10-day rooting treatment resulted in a shorter rooting time, a greater number of roots per shoot, and shorter roots that were more easily manipulated during transfer to the greenhouse. Shoots with a greater initial height had greater rooting success than short shoots. Shoots from 1.1 to 2.0 cm in height were preferable to taller shoots because: (1) less time is required to produce them, (2) their smaller leaf size makes them easier to handle, (3) they root with about the same frequency, (4) rooted shoots are more easily transferred to the greenhouse, and (5) once in the greenhouse they attain a height that is indistinguishable from shoots that were classified as very tall at the start of the rooting process. Inclusion of sucrose in the rooting medium promoted rooting as compared to shoots rooted without sucrose. Of the fertilizer treatments applied to plants newly transferred to the greenhouse, 17-17-17 (N-P-K) and 0-44-0 each applied at 1 g/l with each watering resulted in the highest portion of plant fresh mass allocated to roots (49%).

Susceptibility of Paulownia elongata to Agrobacterium and production of transgenic calli and hairy roots by in vitro inoculation

Susceptibility of Paulownia elongata S.Y. Hu (princess tree) to Agrobacterium tumefaciens and A. rhizogenes was demonstrated by inoculating in vitro shoots. Shoots had a gall formation frequency of ≥83% when inoculated with any of three A. tumefaciens strains (542, A281, or C58). Timing of gall appearance and type of callus proliferation differed among A. tumefaciens strains. Rapidly proliferating callus was produced from explants that were inoculated with A. tumefaciens. Hairy roots were produced directly from wound sites on 33% of shoots inoculated with A. rhizogenes strain R1601. Rapidly growing detached roots were produced from explants that were inoculated with A. rhizogenes. Opine analyses demonstrated the expression of foreign genes in proliferating galls/hairy roots shortly after emergence from wound sites and in callus and roots after 12 weeks of in vitro culture. Southern analyses demonstrated the presence of tDNA in long-term callus and root cultures.