Recombinant collagen and gelatin for drug delivery.

Lagoon-based municipal wastewater treatment plants (WWTPs) are facing difficulties meeting the needs of rapid population growth as well as the more stringent requirements of discharge permits. Three municipal WWTPs were modified using a high surface area media with upgraded fine-bubble aeration systems. Performance data collected showed very promising results in terms of five-day biochemical oxygen demand (BOD5), ammonia (NH3) and total suspended solids (TSS) removal. Two-year average ammonia effluents were 4.1 mg·L−1 for Columbia WWTP, 4 mg·L−1 for Larchmont WWTP and 2.1 mg·L−1 for Laurelville WWTP, respectively. Two-year average BOD5 effluents were 6.8, 4.9 and 2.7 mg·L−1, and TSS effluents were 15.0, 9.6 and 7.5 mg·L−1. The systems also showed low fecal coliform (FC) levels in their effluents.

Upgrade of three municipal wastewater treatment lagoons using a high surface area media

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor

Use of response surface methodology for optimizing process parameters for the production of α-amylase by Aspergillus oryzae

Lemnaceae or duckweed is an aquatic plant that can be used to recover nutrients from wastewaters. The grown duckweed can be a good resource of proteins and starch, and utilized for the production of value-added products such as animal feed and fuel ethanol. In the last eleven years we have been working on growing duckweed on anaerobically treated swine wastewater and utilizing the duckweed for fuel ethanol production. Duckweed strains that grew well on the swine wastewater were screened in laboratory and greenhouse experiments. The selected duckweed strains were then tested for nutrient recovery under laboratory and field conditions. The rates of nitrogen and phosphorus uptake by the duckweed growing in the laboratory and field systems were determined in the study. The mechanisms of nutrient uptake by the duckweed and the growth of duckweed in a nutrient-limited environment have been studied. When there are nutrients (N and P) available in the wastewater, duckweed takes the nutrients from the wastewater to support its growth and to store the nutrients in its tissue. When the N and P are completely removed from the wastewater, duckweed can use its internally stored nutrients to keep its growth for a significant period of time. A modified Monod model has been developed to describe nitrogen transport in a duckweed-covered pond for nutrient recovery from anaerobically treated swine wastewater. Nutrient reserve in the duckweed biomass has been found the key to the kinetics of duckweed growth. Utilization of duckweed for value-added products has a good potential. Using duckweed to feed animals, poultry, and fish has been extensively studied with promising results. Duckweed is also an alternative starch source for fuel ethanol production. Spirodela polyrrhiza grown on anaerobically treated swine wastewater was found to have a starch content of 45.8% (dry weight). Enzymatic hydrolysis of the duckweed biomass with amylases yielded a hydrolysate with a reducing sugar content corresponding to 50.9% of the original dry duckweed biomass. Fermentation of the hydrolysate using yeast gave an ethanol yield of 25.8% of the original dry duckweed biomass. These results indicate that the duckweed biomass can produce significant quantities of starch that can be readily converted into ethanol.

Growing Duckweed to Recover Nutrients from Wastewaters and for Production of Fuel Ethanol and Animal Feed

Nutrient recovery from swine lagoon water by Spirodela punctata

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.

Nutrient removal from swine lagoon liquid by lemna minor 8627

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

We 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.

Genetic transformation of duckweed lemna gibba and lemna minor

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.

Ben A. Bergmann

Papers

Nutrient recovery from swine lagoon water by Spirodela punctata

Nutrient removal from swine lagoon liquid by lemna minor 8627

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

Genetic transformation of duckweed lemna gibba and lemna minor

In vitro adventitious shoot production in Paulownia