Growing duckweed in swine wastewater for nutrient recovery and biomass production

Spirodela oligorrhiza, a promising duckweed identified in previous studies, was examined under different cropping conditions for nutrient recovery from swine wastewater and biomass production. To prevent algae bloom during the start-up of a duckweed system, inoculating 60% of the water surface with duckweed fronds was required. In the growing season, the duckweed system was capable of removing 83.7% and 89.4% of total nitrogen (TN) and total phosphorus (TP) respectively from 6% swine lagoon water in eight weeks at a harvest frequency of twice a week. The total biomass harvested was 5.30 times that of the starting amount. In winter, nutrients could still be substantially removed in spite of the limited duckweed growth, which was probably attributed to the improved protein accumulation of duckweed plants and the nutrient uptake by the attached biofilm (algae and bacteria) on duckweed and walls of the system.     

An investigation of a fertilizer-tap water medium for mass algal production in outdoor plastic-enclosed systems

The feasibility of using two fertilizers (urea plus superphosphate) in tap water as a medium for the mass culture of green algae (Scenedesmus and Ankistrodesmus) in outdoor plastic-enclosed minipond systems was investigated. Experiments in which the basic fertilizer-tap water medium was enriched with micro- and/or macronutrients revealed no nutrient deficiency symptoms in the algal biomass produced. Biomass production was found to be quantitatively related to the concentration of fertilizer added and maximal production (> 15 g/m(2) day) was achieved following the addition of 30 mg N/L (1.89 g N/m(2) day) and 4.5 mg P/L (0.28 g P/m(2)/day).     

Remediation of Ammonium-Contaminated Abandoned Animal Waste Lagoon Soil: Physical Properties and Growth of Barley

Remediation of the soil beneath closed animal waste lagoons is an important issue, particularly for lagoons in environmentally sensitive regions. Few studies address the possibility of using plants to remediate these soils. The objectives of this research were to determine whether barley (Hor-deum vulgare L.), a salt-tolerant crop, would grow in lagoon soil and to determine the effect of plant residue amendments on barley growth and soil physical properties. The lagoon soil was collected from a closed swine lagoon. Oat-straw and corn-cob residues were added at rates of 0 (control), 15, 30, 45, 60, and 75?g/kg lagoon soil. Barley grew in all soils, including the soil with no residues. Average grain yield of plants in pots with oat straw was more than twice that of plants in pots with corn cobs (10.7?g/pot and 4.5?g/pot, respectively). Bulk density and oxygen diffusion rate decreased with increasing amounts of residue in the soil. Infiltration was lower in pots with oat straw than in pots with corn cobs. Irrigation water moved rapidly through macropores visible in the soil with corn cobs. The soil amended with corn cobs retained less water, causing poor growth of the barley. The results showed that corn-cob residue created preferential flow paths in the lagoon soil.     

Improvement of the conversion of polystyrene to polyhydroxyalkanoate through the manipulation of the microbial aspect of the process: a nitrogen feeding strategy for bacterial cells in a stirred tank reactor

Pseudomonas putida CA-3 has been shown to accumulate the biodegradable plastic polyhydroxyalkanoate (PHA) when fed styrene or polystyrene pyrolysis oil as the sole carbon and energy source under nitrogen limiting growth conditions (67 mg nitrogen per litre at time 0). Batch fermentation of P. putida CA-3 grown on styrene or polystyrene pyrolysis oil in a stirred tank reactor yields PHA at 30% of the cell dry weight (CDW). The feeding of nitrogen at a rate of 1mg N/l/h resulted in a 1.1-fold increase in the percentage of CDW accumulated as PHA. An increase in the rate of nitrogen feeding up to 1.5mg N/l/h resulted in further increases in the percentage of the cell dry weight composed of PHA. However, feeding rates of 1.75 and 2mg N/l/h resulted in dramatic decreases in the percentage of cell dry weight composed of PHA. Interestingly nitrogen was not detectable in the growth medium after 16 h, in any of the growth conditions tested. A higher cell density was observed in cells supplied with nitrogen and thus further increases in the overall production of PHA were observed through nitrogen feeding. The highest yield of PHA was 0.28 g PHA per g styrene supplied with a nitrogen feeding rate of 1.5mg/l/h.     

Prevalence and transmission of antimicrobial resistance among <Emphasis Type="Italic">Aeromonas</Emphasis> populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh

In order to investigate the influence of a duckweed aquaculture based hospital sewage water recycling plant on the prevalence and dissemination of antibiotic resistance, we made use of an existing collection of 1,315 Aeromonas isolates that were previously typed by the biochemical fingerprinting PhP-AE system. In these treatment plant, hospital raw sewage water is first collected in a settlement pond (referred to as sewage water in this study) and is then transferred to a lagoon, where the duckweed (Lemnaceae) is grown (referred to as lagoon). The duckweed is harvested and used as feed for the fish in a separate pond (referred to as fish pond). From this collection, representatives of 288 PhP types were subjected to antibiotic susceptibility testing for eight antimicrobials by broth microdilution method. The overall resistance rates among Aeromonas isolates from the treatment plant were highest for ampicillin (87%) and erythromycin (79%) followed by cephalothin (58%), nalidixic acid (52%), streptomycin (51%), tetracycline (31%), chloramphenicol (13%) and gentamicin (8%). A significantly lower prevalence of antibiotic resistance was found in Aeromonas from environmental control water, patient stool samples, duckweed and fish compared to sewage water isolates. The prevalence of resistance in the sewage water was not significantly reduced compared to the lagoon water and fish pond. Throughout the treatment system, the frequencies of resistant strains were found to diminish during the sewage water purification process, i.e. in the lagoon where sewage water is used to grow the duckweed. However, the frequency of resistant strains again increased in the fish pond where sewage grown duckweed is used for aquaculture. Among the selected isolates, two multiresistant clonal groups of Aeromonas caviae HG4 were identified that exhibited indistinguishable PhP and amplified fragment length polymorphism fingerprints and shared a common plasmid of approximately 5 kb. Representatives of both groups were recovered from almost every part of the sewage treatment plant but not in the control ponds nor in human samples, which suggests that specific multiresistant Aeromonas clones are able to persist and spread throughout the entire purification process.     

Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.)

Summary Suspension cultures which maintained embryogenic potency for more than 18 months were established from excised immature embryos of rice (Oryza sativa L. cv. Konansou). The cultures were subcultured every three days in N6 medium supplemented with proline (10 mM), casein hydrolysate (300 mg/l), sucrose (30 g/l) and 2,4-D (1 mg/l). The frequency of embryogenesis from the embryogenetic suspension cultures reached about 90% when cell clusters (about 1 mm in diameter) were transferred to a solid medium which consisted of N6 medium, NAA (1 mg/l), kinetin (5 mg/l), sucrose (30 g/l) and Gelrite (2 g/l). When smaller clusters of cells (approximately 200–400 m in diameter) were transferred to a liquid medium which consisted of salts of N6 medium diluted with an equal volume of water plus sucrose (45 g/l), NAA (0.01 mg/l) and 4-PU (0.1 mg/l) at a cell density of 13 clusters/ml in 2 ml of medium, somatic embryogenesis was initated at high frequency (about 50%). Morphological evidence is provided to demonstrate that the regeneration occurred via embryogenesis. This is the first report of high-frequency embryogenesis in suspension cultures of rice cells.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

Initial crop growth in soil collected from a closed animal waste lagoon

In the 21st century, remediation of the soil beneath animal waste lagoons will become an important issue, as they are closed due to environmental regulations or to abandonment. The possibility of growing crops in the soil, which has high concentrations of ammonium-N, has not been studied. The objective of this experiment was to determine if crop species would germinate and grow in lagoon soil. Soil was gathered from a lagoon that had received wastes from swine (Sus scrofa) and beef (Bos taurus) since 1968. Eight crops were grown in greenhouse pots containing the lagoon soil: winter barley (Hordeum vulgare L. 'Weskan'); field corn (Zea mays L., Cargill's hybrid 7997); 'Plainsman' winter rapeseed [Brassica napus L. spp. oleifera (Metzg.) Sinsk. f. biennis]; soybean [Glycine max (L.) Merr. 'KS 4694'); forage sorghum [Sorghum bicolor (L.) Moench 'Norkan']; sunflower (Helianthus annuus L. 'Hysun 354'); and winter wheat (Triticum aestivum L.)--two cultivars: '2137' and 'Turkey.' Plants were grown for 35 days in lagoon soil or an agricultural soil (Haynie very fine sandy loam; coarse-silty, mixed, superactive, calcareous, mesic Mollic Udifluvent) obtained from a field near the closed lagoon. Ammonium-N (average value of 692 mg/kg) was about 70-85 times greater than the average value of 8-10 mg/kg NH4-N in Kansan soils. The lagoon soil was nonsodic and had a salinity ranking of "medium" with an electrical conductivity averaging 2.29 dS/m. The high ammonium-N concentration in the lagoon soil was not inhibitory to emergence and growth. The eight crops grew taller in the lagoon soil than in the agricultural soil. Except for '2137' wheat, dry weight was higher in the lagoon soil than in the agricultural soil. The results showed that the lagoon soil is not detrimental to early growth of eight crops.     

Estimating the availability of nutrients from processed swine lagoon solids through incubation studies

Potential environmental hazards from the excess accumulation of swine (Sus scrofa domesticus) manure in eastern North Carolina and new state guidelines on treatment alternatives have necessitated the reevaluation of best management practices for disposal of swine waste (manure and effluent) as a fertilizer source on local crop land. Creation of a value-added product is one viable means of utilizing and economically redistributing the nutrients in swine manure. Incubation studies using four agricultural soils from eastern North Carolina were conducted with pelletized processed swine lagoon solids (PSLS) (1.7% N, 2.5% P, 0.12% Cu and 0.18% Zn) composed of dewatered swine lagoon sludge plus rock flour. The PSLS was added at three application rates (0, 200, 400 mg N kg⁻¹ soil) and incubated for 16 weeks at 25°C. The soil was sampled at 0, 1, 2, 4, 8, 12, and 16 weeks and analyzed for NO₃–N, NH₄–N and Mehlich III-extractable P, Zn, and Cu. High concentrations of NH₄–N and low concentrations of NO₃–N were present in the soils shortly after addition of PSLS. After the second week, extractable NH₄–N dropped to <4 mg kg⁻¹, while the soil concentration of NO₃–N increased rapidly. The amount of NO₃–N generated reached 90% of its final value after the fourth week. Across the four soils, 24–35% of the added N, 15–50% of the added P, 20–50% of the added Zn and 15–20% of the added Cu was extractable after 8 weeks. The PSLS is an excellent source of P, but may require additional N if used as a fertilizer source for most row crops.     

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant

Because of increased concern about surface water eutrophication from nutrient-enriched agricultural runoff, many swine producers are encouraged to decrease application rates of waste-based P. Precipitation and subsequent removal of magnesium ammonium phosphate (MgNH(4)PO(4) x 6H(2)O), commonly known as struvite, is a promising mechanism for N and P removal from anaerobic swine lagoon effluent. The objectives of this research were to (i) quantify the effects of adjusting pH and Mg:P ratio on struvite precipitation and (ii) determine the rate constant pH effect for struvite precipitation in anaerobic swine lagoon liquid. Concentrations of PO(4)-P in liquid from two anaerobic swine lagoons were determined after 24 h of equilibration for a pH range of 7.5-9.5 and Mg:P ratios between 1:1 and 1.6:1. Struvite formation reduced the PO(4)-P concentration in the effluents to as low as 2 mgl(-1). Minimum concentrations of PO(4)-P occurred between pH 8.9 and 9.25 at all Mg:P ratios. Struvite precipitation decreased PO(4)-P concentrations by 85% within 20 min at pH 9.0 for an initial Mg:P ratio of 1.2:1. The rate of PO(4)-P decrease was described by a first-order kinetic model, with rate constants of 3.7, 7.9, and 12.3 h(-1) at pH 8.4, 8.7 and 9.0 respectively. Our results indicate that induced struvite formation is a technically feasible method to remove N and P from swine lagoon liquid and it may allow swine producers to recover nutrients for off-farm sale.     

Ecological responses and remediation ability of water fern (Azolla japonica) to water pollution

The ability of the water fern Azolla japonica to remediate phosphorus (P), nitrogen (N), and iron (Fe) contamination, and its physiological responses to three common sources of water pollution (landfill leachate, swine lagoon sewage, and fish farm sewage) and standard solution were investigated. The biomass, water content, and chlorophyll content of Azolla japonica in each solution were measured, and the concentrations and accumulation rates of polluting elements in the solutions were determined. A. japonica showed over eight-fold increase in biomass within only 20 d in every solution except in swine lagoon sewage, extremely high in N concentration. Consistent chlorophyll and water contents of the plant in most solutions showed that A. japonica can adapt to highly concentrated solutions. N, P, and Fe concentrations of the solutions decreased significantly within the 20 d. In most treatments, A. japonica showed high N accumulation and also showed total uptake of P and Fe from the solutions. In reference to this result, using this species as a phytoremediator plant would have additional benefits of helping maintaining endangered populations of A. japonica. Therefore, the plant??s fast growth, good element remediation efficiency, and conservation needs makes A. japonica a suitable plant species for pollution remediation.     

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

Intensive culture of grass carp, Ctenopharyngodon idella, in circular tanks

Fingerling grass carp ( Ctenopharyngodon idella ) were cultured in 380 1 circular fiberglass tanks at stocking densities of 0.53, 1.06, 1.59 and 2.11 fish/1. Fish were fed duckweed ( Lemna minima ) to satiation. Fish grew from 2.7 g to a maximum mean weight of 72.7 g in 88 days. Fish stocked at 0.53 fish/1 grow more rapidly than those stocked at higher densities. Mean daily consumption rates were strongly correlated with oxygen levels and when oxygen levels dropped below 4 mg/1 consumption was reduced approximately 40%. High biomass, growth, and survival rates obtained during this study indicate grass carp are well suited for intensive tank culture.     

Arsenic Uptake by Lemna minor in Hydroponic System

Arsenic is hazardous and causes several ill effects on human beings. Phytoremediation is the use of aquatic plants for the removal of toxic pollutants from external media. In the present research work, the removal efficiency as well as the arsenic uptake capacity of duckweed Lemna minor has been studied. Arsenic concentration in water samples and plant biomass were determined by AAS. The relative growth factor of Lemna minor was determined. The duckweed had potential to remove as well as uptake arsenic from the aqueous medium. Maximum removal of more than 70% arsenic was achieved at initial concentration of 0.5 mg/l arsenic on 15th day of experimental period of 22 days. Removal percentage was found to decrease with the increase in initial concentration. From BCF value, Lemna minor was found to be a hyperaccumulator of arsenic at initial concentration of 0.5 mg/L, such that accumulation decreased with increase in initial arsenic concentration.     

Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel

A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.     

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10⁻⁶ g of wet pig feces in 500 ml of phosphate-buffered saline and 10⁻⁴ g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon,nitrogen and metal ions

To investigate the feasibility of using fresh water from Mangueira Lagoon (Rio Grande do Sul, Brazil) for biomass production in open raceway ponds (0.7 m long, 0.18 m wide, 0.075 m deep) we studied the influence of nutrient addition (carbon as sodium bicarbonate, nitrogen as urea, phosphate, sulfate, ferric iron, magnesium and potassium) on the growth rate of the cyanobacteria Spirulina platensis using a 22 factorial design. In unsupplemented lagoon water production of S platensis was 0.78 +/- 0.01 g/l (dry weight basis) while the addition of 2.88 g/l of sodium bicarbonate (without added urea, phosphate, sulfate or metal ions) resulted in 0.82 +/- 0.01 g/l after 400 hours of culture. The further addition of phosphate and metal ions resulted in growth for up to 750 h and a final S. platensis biomass of 1.23 +/- 0.04 to 1.34 +/- 0.03 g/l.     

The use of dialysis culture in a study of chlorophyll budget in a brackish lagoon,Japan

Growth rates of the entire phytoplankton community of a brackish lagoon in northeastern Japan were estimated by measuring increasing chlorophyll a content in dialysis bags during the summer and early autumn of 1986. The chlorophyll a contents of lagoon water fluctuated between 20 and 200 mg m^–3. At lower densities of phytoplankton (20–50 mg chl. a m^–3), growth rates (the rate of increase of chlorophyll a) exceeded 1 turnover per day, while at higher densities (more than 50 mg chl. a m^–3), the growth rate decreased rapidly. Tidal exchanges of chlorophyll a showed net exports of chlorophyll a from the lagoon to adjacent waters. The exchange rate of chlorophyll a was estimated to be 0.65 d^–1. At about 140 mg m^–3 of chlorophyll a concentration, the increase of chlorophyll in the lagoon water compensated for tidal export. Only a small proportion of primary production was consumed by zooplankton in the lagoon. There were also net exports of ammonium and phosphate from the lagoon. Nutrient flux from sediment exceeded the phytoplankton requirement and was the major source of the ammonium and phosphate exports from the lagoon. The low inorganic N/P atom supply ratio in the lagoon suggests that nitrogen is a major nutrient limiting phytoplankton growth.     

Production of beta-carotene by a Rhodotorula glutinis mutant in sea water medium

Mutant 32, derived from Rhodotorula glutinis NCIM 3353 produced 76-fold more beta-carotene than the parent strain. In the growth medium prepared in seawater, the total carotenoid content and dry cell mass was 86 mg/l and 16 g/l, respectively, as compared to 70 mg/l and 12 g/l obtained with a medium prepared in distilled water. A 2-fold increase in beta-carotene with simultaneous 2.3-fold decrease in torulene content was also observed. When grown in seawater medium at pH 6.0, 83 +/- 5% carotenoids could be extracted from the cells without any mechanical disintegration.