Horticulture

In contrast, other plant species appeared to be unaffected by PPCP exposure. For instance, no significant change in growth rate was noticed after Phragmites australis was exposed to 60 μg L−1 ibuprofen . These findings imply that certain plants may have detoxification mechanisms contributing to their resilience to the stress of such xenobiotics. However, although an increasing amount of research has considered plant accumulation of PPCPs , little is known about the detoxification mechanisms of higher plants toward PPCPs. A common pathway in most organisms for detoxifying xenobiotics is through glutathione conjugation catalyzed by glutathione Stransferases . It has long been recognized that GSTs play an important role in normal cellular metabolism in plants . In addition, GST mRNA responds quickly to exposure of xenobiotics , protecting the cell against oxidative stress or chemical toxicity . Previous research on xenobiotics such as herbicides shows that differences in GSH availability and in the portfolio of GST isoenzymes are associated with xenobiotic resistance . Conjugation with various bio-molecules was observed in plant metabolism of different PPCPs, including diclofenac , ibuprofen , triclosan , benzotriazole and acetaminophen . However, GST-mediated conjugation may play a predominant role, but has been relatively understudied so far . In particular, the entire process of GST mediated detoxification,hydroponic nft from GSH conjugation in the cytoplasm to vacuolar accumulation and processing of conjugates has yet to be elucidated in vivo.

Acetaminophen and structural analogs are phenolic compounds that are among ubiquitous environmental contaminants . Acetaminophen is one of the most used pain and fever relief medicines, and many billions of doses of acetaminophen are consumed each year . Acetaminophen was detected in the aquatic environment at 0.01–0.3 mg L−1 in the South Wales region of the UK . Adverse effects of acetaminophen were also observed in fish and plants . Cucumber is a crop plant, widely consumed, with high economic and ecological relevance, and recommended for use in phytotoxicity studies by U.S. Environmental Protection Agency . In this study, we used acetaminophen to elucidate the mechanisms of GST-mediated detoxification in cucumber plants. Findings of this study may provide evidence for PPCP detoxification via GSH conjugation, and will likely prompt further exploratory investigation on the role and value of this pathway for the numerous emerging contaminants.Cucumber seeds were obtained from Fisher and transferred to a 72-hole plate filled with a mixture of vermiculite and perlite . Plants were cultivated under controlled conditions for 1 week and irrigated with half strength modified Hoagland nutrient solution, which is adequate for the seedlings’ energy demands for cucumber growth and the low ionic strength nutrient solution permits a more precise computation of acetaminophen speciation than do more complete nutrient medium. Details on the solution composition are given in Table S2.

Seedlings in the third-leaf stage were removed from the pots, rinsed with tap water, and placed in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution. Each treatment contained three replicates, and each replicate was prepared from a single plant by separating into roots and shoots. The jars were covered with aluminum foil to prevent evaporation and exposure of roots to light. After 7 days of adaption, plants were exposed to acetaminophen by spiking 25 μL stock solution to yield an initial concentration of 5.0 mg L−1 . The use of such a relatively high concentration was to facilitate metabolite identification. Simultaneously, treatment blanks and plant blanks were included in duplicate to determine abiotic losses of acetaminophen under the experimental conditions. All experimental materials were auto claved, 121 °C for 30 min, before use. A parallel group trial enrolled with acetaminophen at 60 μg L−1 was included to simulate more realistic levels and to validate the high level treatment. The cultivation lasted for 20 days, with the nutrient solution renewed every other day . Additionally, in order to determine if plants can detoxify acetaminophen via GSH conjugation in realistic field environment, cucumber seedlings were cultivated for 20 days in soil amended with 5% bio-solids containing acetaminophen at an initial concentration of 10 mg kg−1 . Cucumber roots were collected and freeze-dried for acetaminophen-GSH analysis. A root exudate control was included to evaluate the effect of plant exudates on microbial degradation and GSH conjugation of acetaminophen in the hydroponic solution. Cucumber seedlings were cultivated in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution, and the plants were removed after 7 days. The solution was brought back to 500 mL with half strength modified Hoagland nutrient solution, and then 25 μL of acetaminophen stock solution was added to yield an initial concentration of 5.0 mg L−1 .

The nutrient solution samples were exposed to the same conditions, and were analyzed for acetaminophen and its conjugates. To investigate whether GSH-acetaminophen can be transported from the root to the shoot, two-week-old cucumber plants were exposed to 1 mg L−1 GSH-acetaminophen for 2 days. Leaves were collected and extracts from the aerial parts were analyzed as described below. To test whether cytochrome P450 was involved in GSH-conjugates formation, two P450 inhibitors, 1-aminobenzotriazole and piperonyl butoxide, were used to pretreat the plant on the basis of previous studies demonstrated specific inhibition effects . Cucumber seedlings were pretreated with 1- aminobenzotriazole or piperonyl butoxide for 12 h in 500 mL glass jars with aerated, half strength modified Hoagland nutrient solution, followed by spiking 25 μL of acetaminophen stock solution to yield an initial concentration of 5.0 mg L−1 . After 96 h incubation, tissue samples were collected from both the treated and untreated plants. Extracts from the tissues were analyzed for GSH-conjugates. The specificity of these P450 inhibitors was evaluated by monitoring cytochrome P450 activity, GSH content and GST activity in cucumber tissues after the treatment. For better clarity, all the experiments performed was schematically represented Table S3. Simultaneously, commercially available common bean , tomato , alfalfa and wheat seeds were germinated and transplanted under the same conditions as above. After 7 days of adaption, plants were exposed to acetaminophen at 5.0 mg L−1 . After 96 h, plant roots were collected and analyzed for acetaminophen and its glutathione, cysteine and N-acetylcysteine conjugates. Sampling,hydroponic channel sample preparation and analysis Cucumber seedlings treated with or without 5 mg L−1 acetaminophen were sacrificed at 0, 12, 24, 48, 72, 96, 120 and 144 h. Before sample preparation, roots were rinsed thoroughly with tap water. Harvested tissues were separated into roots and shoots and then frozen in liquid nitrogen. Enzyme activities of GST, glutathione reductase , γ-glutamylcysteine synthetase , and cytochrome P450, as well as contents of reduced glutathione in both roots and shoots, and lipid peroxidation were immediately analyzed after sampling, as described below. The remaining plant samples were freeze-dried and stored at −80 °C until chemical analysis.

At each sampling time, nutrient solution samples and the aqueous root exudate samples were collected and mixed with equal volumes of methanol. The mixture was added with d4-acetaminophen as a surrogate, filtered through a PTFE syringe filter and stored at −20 °C before analysis. To quantify acetaminophen and its glutathione, cysteine and Nacetylcysteine conjugates, the freeze-dried plant tissue samples were ground to a fine powder with a mortar and pestle, and a 0.5-g aliquot was used for solvent extraction. Each sample was spiked with d4- acetaminophen as the recovery surrogate, and extracted, sequentially, with 20 mL methyl tert-butyl ether , acetonitrile, and 0.5 mM HCl solution in an ultrasonic water bath for 20 min for each extraction. The supernatant from MTBE and acetonitrile extraction was combined after centrifugation and reconstituted in 1.0 mL methanol after drying under nitrogen . The extract was pooled with the supernatant from the HCl extraction. The pooled extract was loaded onto an Oasis™ HLB cartridge that was preconditioned with 6 mL methanol and 12 mL deionized water, and eluted with 15 mL methanol under gravity. The eluate was evaporated to dryness under N2, and the residue was recovered in 1.5 mL methanol:water mixture and filtered before analysis. The instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography coupled to a Waters Micromass Triple Quadrupole mass spectrometer equipped with an electrospray ionization interface . Acetaminophen, and its glutathione, cysteine and N-acetylcysteine conjugates were separated using ACQUITY UPLC BEH C18 column with water and methanol as mobile phases. Electrospray ionization was operated in the positive mode. Detailed information on the instrumental analysis is given in Table S4.Fresh cucumber roots were frozen in liquid nitrogen, and homogenized on ice with 5 mL of 50 mM potassium phosphate buffer containing 1 mM EDTA and 1% polyvinylpyrrolidone. After centrifugation at 15,000 g at 4 °C for 20 min, the supernatant was used for the assay of activities of glutathione S-transferase, glutathione reductase, and cytochrome P450 . Measurement of GST activity was carried out spectrophotometrically after the glutathione and 1-chloro-2,4-dinitrobenzene adduct synthesis reaction . Brieflfly, GST activity was measured in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM CDNB, 5 mM GSH and 100 μL enzyme extract. The increase in absorbance as a result of GSH-CDNB synthesis was determined at 340 nm for 5 min on a Cary 50 UV– Visible spectrophotometer . The GST activity was calculated using the extinction coefficient of GSH-CDNB . To determine the activity of GR , a 200 μL aliquot of enzyme extract was added to the reaction mixture of 0.15 mM NADPH and 0.5 mM oxidized glutathione in 50 mM PBS . The absorbance decrease was monitored at 340 nm for 3 min after GSSG-dependent consumption of NADPH . For the assay of γ-ECS , plant tissues were homogenized and suspended in 5 mL of 0.1 M HCl. After centrifugation at 20,000 g for10 min at 4 °C, the supernatant was collected. The γ-ECS activity was measured according to Shan and Liang . An incubation solution was prepared with 800 μL 50 mM TrisHCl containing 0.25 mM glutamate, 10 mM ATP, 1 mM dithioerythritol, 2 mM cysteine, and 400 μL of the γ-ECS sample. The mixed solution was incubated for 1 h at 25 °C, and then 800 μL phosphorus agent was added and mixed. After incubation at 45 °C for 25 min, the absorbance at 660 nm was measured on the UV–Visible spectrophotometer. One unit of γ-ECS activity was defined as 1 mmol cysteine-dependently generated PO4 3− per minute. The levels of proteins in the enzyme extracts were determined using Coomassie Brilliant Blue G-250 . Briefly, 100 μL of enzyme extract was added to 5 mL of 0.01% Coomassie Brilliant Blue G- 250. After 5 min, the absorbance at 595 nm was measured on the UV– Visible spectrophotometer. The protein content of enzyme extracts was calculated by comparison with a standard curve using bovine serum albumin as the standard.The content of GSH was determined spectrophotometrically according to Jiang et al. . Plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice and centrifuged at 12,000 g at 4 °C for 15 min, and the supernatant was analyzed for GSH. To 0.5 mL of supernatant, 0.5 mL PBS and 0.5 mL of 5′5′-dithiobis-2- nitrobenzonic acid were added. After thorough mixing and incubation for 5 min, the absorbance at 412 nm was measured . Membrane lipid peroxidation, a typical indicator of stress phytotoxicity , was estimated by measuring the concentration of malondialdehyde, according to the reaction with thiobarbituric acid as described in Sun et al. .All treatments were performed in triplicates. A procedural blank and a sample duplicate were included in every batch of 10 samples to monitor background contamination and reproducibility, and the calculated relative standard deviations were b10%. The d4-Acetaminophen was used to account for any loss during sample preparation, matrix induced ionization effects, and variations in instrumental response. Authentic standards were used to confirm the target analytes. A six-point calibration line was used for quantification with the r 2 values of at least 0.99 for all analytes . No acetaminophen or any of the conjugates was detected in the solvent or treatment blanks. Limits of detection and quantification for individual acetaminophen and the conjugates were calculated as 3 and 10 times the signal-tonoise level from the low-level spiked samples . The recoveries in all samples were within acceptable limits ranging from 75 to 110%, demonstrating good method accuracy and precision.

Y79 cells derived from a retinoblastoma tumor have been shown to have 50 – 100 copies of the N-myc gene transcript per cell. We first tested whether N-myc is preferentially expressed during specific parts of the Y79 cell cycle. Y79 cells were synchronized with double thymidine blocks at the beginning of S, then released from the block and collected for isolation of total cytoplasmic RNA and for analysis by flow cytometry. N-myc DNA probes hybridized with dot blotted RNAs and RNAs from Northern blots ,of agarose-formaldehyde gels showed no differences in the numbers of transcripts as the cells traversed 0 1 and entered the S phase over a period of 12 hrs. Protein synthesis inhibitors added to randomly growing Y79 cells did, however, show a dramatic increase in N-myc transcript number over a period of 4 – 6 hrs; control genes showed no such increase. Inhibition of RNA synthesis by actinomycin D treatment led to a rapid decrease in N-myc transcript number which was partially prevented by protein synthesis inhibitors. Thus, N-myc transcripts have a short half life whose stability may be increased by inhibition of protein synthesis. These results confirm that similarities between c-myc and N-myc go beyond their partial sequence homology and suggest that N-myc may serve the same function in neuronal cells that c-myc plays in non-neuronal cells. The gene copy number is normally a highly conserved factor during DNA replication. It is clear that on an evolutionary scale genes have increased in number sometimes forming functional gene copies; however, often the amplification results in the formation of pseudogenes.

Recent studies have indicated that the control of gene copy number may break down on a shorter time scale. Numerous example have been found in which the number of gene copies for a particular selectible marker has increased when cells are grown in the presence of a selecting agent. Generally,hydroponic gutter after applying a selecting agent these amplified genes appear within a few cell generations in the form of extrachromosomal gene copies and may progress after long exposure to form reintegrated tandemly arranged multiple gene copies. Whether the selecting agent is inducing the amplification of genes or selecting for pre-existing cells with amplified genes is not known. It has been shown, however, that agent which damage DNA can induce either the stabilization of amplifi¢ genes, or the amplification process itself. As a model system for the study of gene amplification, we have been studying the regulation of copy number of SV 40 viral sequences present in human cells after transfonnation. Our hypothesis is that there are cellular factors that regulate the copy number of these sequences and that we can identify these factors by developing an in vitro DNA replication system. We have begun by characterizing the number of copies of SV40 DNA present in human cells transfonned ‘ by this virus. In our initial studies of two human cell lines we obtained from the American Type Tissue Culture collection that were transformed with wild type SV 40 virus we discovered that the cells contained many copies of SV 40 that were extrachromosomal elements. The cell lines we are studying are G,M637, a human fibroblast cell line from an apparently normal individu31, and XP12RO, a line from a Xeroderma pigmentosum patient containing a mutation in a DNA repair gene. Both of these cell lines were transformed with wild type SV 40 by other investigators.

The aim of these’ studies has been to determine the involvement of DNA repair processes in gene amplification. The presence of the extrachromosomal copies of SV 40 in human cells has been observed by other investigators. The origin of this extrachromosomal DNA and its relationship to the imput transforming DNA i~ not known. To understand the system better we began to characterize the DNA. We found that in an actively growing population of GM637 cells the copy number, of SV40 DNA is approximately 400 per cell. The extrachromosomal DNA is also present in XP12RO cells. Other human cells we are carrying in the lab that are not transformed with SV40 virus do not have the extrachromosomal SV40 DNA. On agarose gel electrophoresis, the extrachromosomal DNA from both cell lines has a lower molecular weight than wild type DNA. Restriction enzyme digestion indicates that the lower molecular weight is the result of a deletion in the A-gene coding for the T-antigen. In both cell lines the deletion maps near the boundary of the intervening sequence and the small exon of the T -antigen. The difference between the two DNA’s seen so far is that the size of the deletion for the DNA from GM637 is about 300 base pairs and for the DNA from the XP12RO cells is about 250 base pairs. We have cloned the extrachromosomal DNA from GM637 into a pUC vector and then subcloned a Hind III fragment containing the deletion into M13 for sequencing.

The sequence indicates that the deletion has eliminated one of the splice sites for the intervening sequence and has stopped just short of the translation termination site for the small t-antigen. The deletion stops out-of-frame so that presumably the translation machinery reads through the normal termination signal, but encounters another termination signal in the new frame just upstream from the deletion. The prediction from this data is that the mutant DNA encodes a mRNA that is similar in size to the message for wild type T-antigen, but that the premature translation termination signal would result in a protein being made that is just slightly smaller than small t-antigen. We are presently isolating the mRNA and the proteins from the cells to determine what is made from the mutant DNA. At this point we know nothing about the function of this extrachromosomal DNA,u planting gutter or about its origin from the wild type virus used to transform the cells. Its curious that two cell types that originated from separate transformation events give rise to very similar extrachromosomal deleted DNA. We have been developing a system for the study of the replication of cardnogen modified DNA in mammalian cells. For this assay we have contructed a shuttle vector that can replicate both in bacteria and mammalian cells. This vector was contructed by cloning of the SV 40 origin for DNA replication into an M13 vector. M13 virus contain a single stranded DNA which replicates efficiently in E. coli, and is ideal for sequencing DNA placed in the Messing cloning site. The construct we have made contains the SV 40 origin in the I region adjacent to the cloning site and the ,B-galactosidasegene. The assay for replication in mammalian cells has been to transfect the double stranded replication form into mammalian cells at approximately 50 ng per 106 cells. The DNA is harvested at different times after transfection, digested with Dpn I to eliminate the DNA that has not replicated, and then used to transform E. coli. The surviving and replicated DNA will form plaques on a lawn of E. coli. M13 DNA which has been mutated to block the active production of ,B-galactosidase can be distinguished from input DNA by a color assay. Our studies so far have indicated that the DNA we have constructed can indeed replicate in Cos 7 cells which provide a functional T-antigen from an integrated copy of SV 40.

So far we have not been able to demonstrate replication of our vector in human cells; however, we have only tested the GM637 cells which already contain many copies of their own extrachromosomal DNA. We plan to construct a transfection recipient of human cells by utilizing an approach similar to that used to construct the Cos 7 cell line. Wild type SV 40 will be inactivated for replication by cutting at the origin with Bgi I followed by. treatment with Bal I to digest away the ends. The deletions generated will than be transfected directly into untransformed human fibroblast cells and transformed variants will be isolated. The transformed cells will be tested for extrachromosomal DNA, and only those clones with integrated SV40 will be used as transfection recipients. We intend to carry out this technique with different human cells so that we can have recipients to test the effect of such things as differentiation state of the cells and DNA repair capacity on the apility to replicate transfected DNA both with and without carcinogen modification. UsiI1g the M13 vector containing the SV40 origin we contructed we are testing the effect of carcinogen adducts on the replication of DNA. This project has come out of our extensive work characterizing the effect of benzo[a]pyrene on the replication of SV40 viral DNA. The conclusions of these studies has been that the carcinogen causes a build up of replicated intermediates which were unable to complete the final steps in separating the daughter molecules into mature completely replicated forms. One difficulty with these studies has been the random location of the adduct and the inability therefore to locate the replication block relative to the adduct. By site specifically placing the adduct we hope to get around this difficulty and to also provide a vector for studying the kind of DNA sequence changes that occur when adducted DNA is replicated. The vector does replicate in ~onkey cells containing an active gene for production of Tantigen with a low mutation frequency as judged by the plaque assay on E. coli described above. We have treated the DNA with benzo[a]pyrene at a level which reduces the yield of plaques by 50%, and have obtained vector lacking the ability to induce ,a-galactosidase from DNA replicated in the monkey cells. We are presently characterizing these vectors to determine precisely what mutations have occurred.This work is being done in collaboration with the Prof. John Hearst’s laboratory. In our approach, we have made two M13 vectors containing the SV40 origin. One vector contains a synthetic oligomer in the cloning region, and the second lacks this region. Hybridization of these to single strands creates a double strand DNA except in the region of the cloned synthetic oligomer. The approach is then to place a psoralen adduct on another synthetic oligomer that hybridizes to the oligomer cloned into the vector and then t,o seal the fragment in place with ligase. We have completed the work to the formation of the gapped intermediate and presently are determining if the synthetic oligomer will hybridize into the gap and be ligated. Our, future work will then be to transfect this DNA into cells with different capacity to replicate and/or express the DNA and detemine how the adduct affects these processes. Our experience working with DNA transfected back into mammalian cells has shown us that the DNA is greatly modified during the transfection procedure. In order to overcome these difficulties we have begun to develope a system for conducting the DNA synthesis experiments in vitro. Until recently it was not possible to get any mammalian cell DNA replication to occur in vitro., Recent work in other laboratories have shown that it is’ possible to replicate SV 40 based DNA in complex mixtures of factors from human cell lines. Not only does elongation of preinitiated replication occur, but multiple rounds of initiation are possible. We are setting up this _ assay in our lab to look at the replication of the psorlen modified vectors described above. This system will also be used to investigate the cellular factors required for DNA synthesis and how carcinogen pretreatment of the cells affects the levels of DNA replication. Chemicals and radiation in the environment often interact adversely with DNA of living cells resulting in abnormal behavior and or death of the cells if the damage is not repaired. To understand how the damaged DNA is recognized and repaired one needs to know, among other things, the structures of the damaged molecules and their intermediates at the molecular level. Two of the most studied damaged DNAs ‘are those containing UV induced thymine dimer and psoralencross-link. Combining the structural information from the crystal structures of a double stranded DNA fragment and the psoralen-thymine monadduct we have recently constructed a molecular model for the psoralen cross-linked DNA.

Whether in the US or India, improvements to energy efficiency can often be attained through no-cost or low cost ECMs that lower the first costs of construction and equipment. Optimizing building loads can lead to lower first costs and operating costs. By targeting low-hanging fruit through early-stage ECMs, the first costs saved through these can be applied toward more expensive technology solutions like high-quality glazing or sensors that can further the energy and cost benefits later in the building life cycle. Hence, it is important to decide which measures to prioritize initially, and then what to cross-subsidize eventually. For example, if one is able to save costs by reducing the number of lighting fixtures and taking advantage of high daylight levels in a space, then those savings can be used to install daylight sensors. The latter can provide a large cost benefit with a relatively short payback time by driving down the operational hours for artificial lighting. The ECMs at the whole building level using systems integration can greatly benefit the EPI of a building. Table 1 shows whole building energy use metrics, using Standard , Better , and Best Practices at the whole building level.Displacement ventilation delivers the air at low speeds using the principle of air stratification. Here, air is delivered at close to floor level for primarily conditioning the occupied volume and extracted at the ceiling height rather than conditioning the unoccupied higher volume first.

Well designed DV systems provide better indoor air quality since the air in the occupied zone is generally fresher than that for mixing ventilation. There are no perceived air drafts. Any released pollutants rise rapidly to above the occupied zone. Large cooling energy savings are possible, as it uses a higher supply air temperature at 18°C,rolling bench which also increases the efficiency of mechanical cooling equipment and lowers equipment requirements. Underfloor Air Distribution technology uses the underfloor plenum beneath a raised floor to provide conditioned air through floor diffusers directly to the occupied zone. A thoughtful design can overcome the usually cited challenges of uneven floor surfaces, difficulty in providing added airflow to the perimeter of the building, and perceived control difficulty. The advantages of a well-designed UFAD system are: improved thermal comfort, occupant satisfaction, ventilation efficiency and indoor air quality, reduced energy use and the potential for reduced floor-to-floor height in new construction. Radiant Cooling works on the principle that water can store 3,400 times more thermal energy per unit volume than air. It offers the potential to reduce cooling energy consumption and peak cooling loads when coupled with building thermal mass. Some radiant systems circulate cool water in dedicated panels; others cool the building structure . Because radiant surfaces are often cooled only a few degrees below the desired indoor air temperature, there are many opportunities for innovative cooling energy sources, such as night cooling and ground-coupled hydronic loops. The heating and cooling supply water temperatures for radiant systems operate at higher set points compared to traditional systems.

The radiant cooling system supply water temperature would typically operate at 15°C–18°C for cooling, whereas typical supply water temperatures for a traditional forced air system are around 5.5°C–7.5°C. The central cooling equipment can operate more efficiently at these temperature set points.In a typical office space, the airflow required to cool and ventilate the space can be three to four times greater than that required to just ventilate the space. If the space cooling is decoupled from the ventilation, especially through a hydronic system, the central air handling system and associated distribution system can be downsized accordingly. A system called a Dedicated Outdoor Air System is typically used to serve the ventilation needs. A DOAS also allows for the effective use of energy recovery on the incoming outside air to further reduce the associated heating and cooling ventilation loads. Localized demand control ventilation can also be implemented to turn off the ventilation air when the space is not occupied, which further reduces the total system energy. The efficiency gain of this demand-control ventilation strategy needs to be weighed against the additional system complication, cost, and the additional fan energy necessary for the required air terminals. Also, the traditional air distribution system has air terminal devices to modulate the cooling capacity to each individual space. These air terminals add additional pressure drop and increase the associated fan energy. With a DOAS, the air terminals are not required for proper system operation. The space saved by using a DOAS can be used to install a low-static air-side distribution system to further reduce the associated fan energy. Therefore, consider decoupling the cooling and ventilation. Separate the process load and the regulated sensible load from the latent load . Serve different types of loads by various levels of cooling relevant to the specific need. Soil salinity adversely affects crop productivity and agricultural sustainability in many areas of the world, especially in arid and semi-arid regions .

Plant growth can be inhibited by high salt concentrations through osmotic stress, nutritional imbalance, and specific ion toxicity . It is known that the growth inhibition and the adverse effects induced by salinity can be alleviated by proper use of fertilizer and water management, depending on plant species, salinity level, and environmental conditions . Nonetheless, over fertilization with N may contribute to soil salinization and increase the negative effects of soil salinity on plant performance . In addition, the potential for NO3 leaching may increase where moderate to high amounts of salts are present in the soils because plants under salt stress can not absorb and or utilize the applied N as efficiently as the plants not subjected to salt stress . Further, as the salinity of irrigation water rises, the leaching fraction must increase to control root zone salinity. Higher leaching fractions combined with lower N use efficiencies represent a worst case with regard to groundwater pollution . Therefore, judicious fertilizer and water management is essential in salt-affected soils to sustain yields and to minimize the degradation of soil and groundwater. In the arid Xinjiang province of China,grow table hydroponic cotton is being grown widely because of its high salinity tolerance. In these regions, water sources are frequently brackish and high-quality water for agricultural purposes is increasingly scarce due to rising demands from urban areas . As a result, the proportion of crop production under deficit irrigation with poorer quality water is increasing. Drip irrigation is thought to be the most efficient irrigation method . With fertigation , the application of fertilizer can be controlled to match the plant needs at each physiological growth stage, which can enhance plant growth and increase fertilizer and water use efficiency while minimizing environmental pollution. Plant responses to salinity change with plant age, plant development, and growth stages . It is important to study plant growth response to N and soil salinity during the whole plant life cycle to reveal whether the amount of N applied alleviates or aggravates the detrimental effects of salinity during a specific growth stages . In addition, examining plant growth during the whole growing season provides information about crop salt tolerance over time. The objective of this work was to determine the influence of different soil salinity levels and N fertilization rates on the cotton growth, including the root development, plant height, and above-ground mass. The uptake of N, K, Ca, Na and Cl were measured to understand the combined effects of N and salinity on cotton growth.The experiments were conducted in a greenhouse from May to November at an agricultural experimental station at Shihezi University, Xinjiang, China . Cotton was grown in plastic pots with a volume of 84 L.

The minimum and maximum air temperature was 17°C and 32°C, respectively. The relative humidity ranged from 40– 62%. A clay loam soil taken from the station field was passed through a 2-mm sieve and packed in the plastic pots with 0.1 m increments to 0.5 m. Each pot was filled with 96 kg air dry soil. The bulk density of the packed soil was 1.2 g cm−3 and the gravimetric water content of the saturated paste of the soil was 45.6%. Selected physical and chemical properties of the soil are presented in Table 1. The experimental design was a 4×4 factorial with four salinity treatments and four levels of nitrogen. Soil salinity was created with applying NaCl and CaCl2 to the soil before the experiment. The resulting ion compositions in the treated soil were similar to those observed in the local saline soils. The soil salinity levels were 2.4, 7.7, 12.5, and 17.1 dS m−1 , referred to as non-saline , low , medium , and high saline, respectively. The low and high salt treatments were set based on the 100% and 50% yield threshold values for cotton, respectively . The amount of N was estimated with a population density of 221,000 plants ha−1 according to common field practices used by local farmers. The N application rates were 0, 135, 270, and 405 kg ha−1 , which correspond to 0, 2.65, 5.30, and 7.95 g N per pot in terms of plant population density, referred to as no , low , medium , and high fertilization. The medium fertilization level was set based on the common field practices used by local farmers. The experiments followed a completely randomized block design with four replications for each treatment. Each block included 16 treatments. Water was applied through drip irrigation units with a discharge rate of 1.1 L h−1 . Drip laterals were installed on the top of the pots, and the emitter was fixed in the centre. Each pot was irrigated by one emitter. Cotton seeds were planted at 5 cm distance from the emitter in each pot on 28 May, and then the top of the pot was covered with a polyethylene film to reduce evaporation. Each pot received 9 L of water to help germination and seedling establishment. At the three-true-leaves stage , the crop was thinned to four plants per pot, yielding a population of 221,000 plants ha−1.Fresh water was used for all treatments. Two pots for each treatment were weighed every 2 days to keep the soil water content between 50% and 80% of the field capacity during the growing season. For each pot, the amount of water to be applied was determined by gathering water discharged from the emitter close to the pot. There was no drainage or leaching through the pots. Triple super phosphate and potassium sulfate were applied as base fertilizers when filling the pots, while nitrogen fertilizer was applied through the drip irrigation system during the cotton growth period. Urea was used as the N source and applied in five equal splits at 50, 65, 78, 90 and 108 DAP, according to common field practices used by local farmers. The fertilizer solution was stored in a plastic container of 100 L volume and pumped into the irrigation system. The experiment was terminated at the boll-opening stage .Plant height was measured weekly during the growth season. The shed were collected daily for each pot. At the end of the experiment, plants were cut at the soil surface and separated into leaf, stem, bur, and seed. Roots were collected after the soil was passed through a 0.5-mm sieve with the aid of a water jet. Debris, weeds and dead roots were sorted by hand from the ‘live’ roots during washing, based on visual observation that the ‘live’ roots appeared light in colors . Each plant component was washed with distilled water, dried in an oven at 70°C for 72 h, and weighed. Dry masses of each component were measured and samples were grounded to pass through a 1-mm sieve. Soil samples were taken with a 0.03 m diameter tube sampler from three randomly chosen replicates of each treatment, at distances of 0−0.05, 0.05−0.1, 0.1−0.15 and more than 0.15 m from the emitter. The soil cores were divided into 0.1 m increments to a depth of 0.5 m, soil cores from the same treatment and depth were pooled, air dried, and a sub-sample was fine-ground with a mortar and pestle. The gravimetric water content of the soil was measured via drying in an oven at 105°C for 48 h.

Plant seedlings when inoculated with endophytic diazotrophs showed enhanced growth. Both above ground shoot and below ground roots increased almost 50% relative to uninoculated controls. We were able to locate fluorescence signal from the strain R1Gly cells bearing the reporter gene gfp localized in the certain plant tissue. This indicated that strains were active inside seedlings, and fixing Nitrogen in-planta. In order to identify carbon substrates that potentially attract N2-fixing bacteria to plants, diazotrophic isolates were grown in root exudates collected from hydroponically grown plants, and analyzed for substrate uptake/release. Carbon compounds that are consumed by endophytic bacterial strain and carbon compounds that are excreted by this strain have been identified. The goal of this project is to establish an x-ray footprinting program at the Advanced Light Source. XF is a technique that allows determination of protein structure and dynamics in the solution state at the resolution of a single amino acid. With this technique, the probing reagent – hydroxyl radicals – are produced by energetic photons alone, and no extraneous reagents produce additional unwanted protein modifications. After exposure, samples are digested and analyzed with mass spectrometry to determine which amino acids were modified by the hydroxyl radicals; this results in a map of the solvent-accessible areas of a protein, indicating sites of protein-protein or protein-substrate interactions. Since the necessary exposures are on the order of milliseconds,led grow lights mixing reactions can be performed to give snapshots of protein dynamics or protein-protein interactions as a function of time.

The methodology of data collection, buffer calibration, and mass spectrometry analysis for x-ray foot printing has been previously developed and used very successfully at the NSLS over the last decade. Establishment of an XF program at the Advanced Light Source serves two purposes: 1) it allows continued support of projects currently underway at the NSLS beamline X28C and 2) it further develops and extends the technique of XF into the sub-millisecond time regime.With the previous two years of LDRD funding, an XF program was established at the ALS; experimental feasibility was established using protein and fluorophore standards, and resulted in a publication . Further, results from ALS beamline 5.3.1 demonstrated that foot printing can be conducted in the microsecond domain when using a white-light focusing optic from an ALS bend magnet source, representing a significant extension of the technique. These results were published . An MOU with the ALS Experimental Systems Group was established, allowing access to beamlines 5.3.1 and 3.2.1 on a regular basis. In addition, permission has been obtained from ALS Management to commission beamline 3.3.1 as a dedicated foot printing beamline at the ALS, and initial top-off calculations of the 3.3.1 aperture show that the beamline can be brought online without further shielding work. In addition, we have established an MOU with the NSLS to support NSLS XF users starting in Oct 2014 using beamlines 5.3.1 and 3.2.1 at the ALS. Several important systems were investigated following establishment of the technique at the ALS, and one example is the orange carotenoid protein . In cyanobacteria, this 35 kDa protein is involved in a photoprotective mechanism, undergoing a conformational change under high light conditions that results in a cascade of protein–protein interactions to control the blue green algae photo system. XF experiments on OCP at ALS beamline 5.3.1 showed a significant rearrangement of residues on one side of the carotenoid under blue light illumination.

The results point to an opening of one domain of the protein during light exposure, allowing access to the carotenoid for interaction with other proteins in the complex. This is a significant contribution to understanding the mechanism of activation for this protein, especially given that the “red” state of OCP has been intractable to crystallization. These results have written up and submitted to Nature. The purpose of this project is to understand light-induced water splitting in green plants, algae and cyanobacteria; organisms that are responsible for producing most of the oxygen in the atmosphere. An important application of this knowledge will be in the design of future fuel production schemes based on artificial photosynthesis. The splitting of water, creating oxygen and hydrogen, is accomplished by the protein complex photo system II, which contains a catalytic center containing four manganese atoms. To drive the reaction to completion, four sunlight photons sequentially oxidize the Mn atoms after which the catalyst returns to the reduced state. Traditional methods for studying structure and function, such as X-ray crystallography, have been hampered by the high sensitivity of the Mn center to probing X-rays, which reduce the metal atoms to the Mn valence state. We have an unprecedented opportunity to map out the detailed reaction mechanism using X-ray free-electron laser experiments at the Linac Coherent Light Source .X-ray probe pulses at LCLS are short enough that all observations can be made before reduction and other damage processes occur.

As the probe pulses are extremely intense, the sample becomes fully ionized after the observations are recorded; therefore, a continuous stream of new crystals is required for a full data set. Atomic structure of the protein will be probed by X-ray diffraction, while simultaneously the electronic state of the Mn atoms will be measured by X-ray emission and spectroscopy. The experimental team is a collaboration between many groups at different institutions. This LDRD is specifically targeted at developing the requisite computational methods to handle the large data sizes, with the involvement of NERSC resources. In it’s third and final year , the project published XFEL-derived crystal structures of photosystem II in the dark-equilibrated state, as well as in three illuminated states along the reaction coordinate. While our published structural data extends to 4.5 Å resolution only, our latest experiments with optimized crystal growth conditions indicate that 2.5 Å data are achievable. We published extensive computational methods for XFEL diffraction experiments applied to structural biology. The unique data collection conditions required us to develop analysis methods to optimally model the crystal orientation and internal physical properties. Our software is general and widely applicable to XFEL-based protein crystallography. In collaboration with Axel Brunger we created procedures for organizing large datasets where the diffraction quality is heterogeneous, explored systematic correction methods for converting the Bragg spots to reduced Fourier coefficients for calculating the electron density map, and contributed to the development of a new microfluidic trap device that will hopefully reduce the amount of sample required for a full dataset. With David Eisenberg we developed new algorithms to process very sparse crystal diffraction patterns from small molecules. We’ve used XFEL diffraction to investigate small peptide fragments that determine the β–sheet protein structure in numerous amyloid diseases,vertical grow system and in particular the mechanism of a single-amino acid mutation causing Parkinson’s disease. As data sets grow larger, there is an increasing need across multiple LBNL divisions for tools to simplify “High Throughput Computing” — the efficient processing of many thousands or even millions of small independent tasks. For example, users need to apply the same analysis program to many thousands of images or spectra or gene sequences, or run the same simulation with thousands of different input parameters. The traditional batch model of 1 task = 1 script = 1 job becomes impractical for both the batch queue system and the human management of such scripts. At the same time, pre-bundling multiple tasks into a single batch script lacks flexibility, e.g. for processing new data as it arrives. We are developing an alternate model to simplify HTC workflows at NERSC and elsewhere, based upon separating the queue of tasks to perform from the batch jobs that process those tasks.

This work will simplify HTC workflows at NERSC, attract new users, and reduce the software development costs for future projects with big data HTC processing requirements. In the first year of the project we submitted a wafer design to Teledyne DALSA, and the CCD wafer fabrication was successful. The second year of the project described here utilized these wafers in the MSL for QE improvement development and for testing the low noise CCD designs. The figure below shows the improvements in QE at both blue and red wavelengths that have been demonstrated. The measured QE on recently-fabricated CCDs is shown by the red symbols, and that is compared to the standard QE shown in the blue symbols. The improvements are due to the use of a thinner backside contact layer and the use of improved antireflection coating materials, specifically the use of ZrO2 in the AR coating. We also demonstrated improved noise performance. In addition to the use of a direct connection between the CCD sense node and output transistor polysilicon gate electrode that we had described previously, we also explored the use of a thinner gate-insulator layer in the output transistor. This allowed for the use of a smaller gate length in the output transistor, and resulted in a reduction of the noise floor from about 1 e- rms to about 0.8 e- rms. We also explored the use of mixand-match lithography to reduce the size of the sense node to 1 × 1 µm2 from 2 × 2 µm2 . Although this did not result in a noticeable noise improvement, the ability to utilize the finer-line lithography could be useful for future noise improvements. These enhancements were included on both small format CCDs, and on 16- channel CCDs to allow for improved statistics. See the listed publication for more details. We have also made progress on single-photon detection via impact ionization in a charge multiplying CCD, with the first demonstration for us of the multiplication of sub-electron light levels with realistic gain values in our p-channel technology. A first prototype chip was designed in FY12 and fabricated in FY13. This chip contains an active pixel matrix. Working devices were received in February 2013 and then tested to validate the design methods used and simulation results. These tests were successful, leading to submission and presentation of results at the International Image Sensor Workshop in June 2013. Following this, devices were irradiated in September wiht protons to a maximum dose of 30Mrad. Testing of the irradiated devices took place in FY14. In FY14 devices were also characterized for minimum ionizing particle detection efficiency using the SLAC test beam facility. The conclusion from these studies is that the concept works, but cannot achieve the desired 99% detection efficiency for MIP detection. Instead, the MIP efficiency of the tested devices is between 50% and 60%. Irradiaiton did not degrade the performance. To increase efficiency one must reduce capacitance and/or increase the collected charge. A test structure chip was fabricated in FY14 to explore the process parameter space for the charge collection implant to minimize capacitance and maximize breakdown voltage. This characterization was successful, but the results disappointing, showing very little room for improvement. Finally, in FY14, a redesign of the pixel geometry was carried out to reduce the size and therefore the capacitance of the pixel. However, this design was not fabricated. Even with these improvements, simulations show that the efficiency would reach just 80%. A higher substrate resistivity is needed to boost the signal and reach the desired efficiency . At the conclusion of the LDRD no higher resitivity process with the needed deep implant feature was available. The purpose of this project is to develop detector technology for next-generation Cosmic Microwave Background polarimetry experiments. Primordial gravitational waves produced during inflation and gravitational lensing by large-scale structure produce “B-mode” polarization patterns on the sky which have a handedness. A detection of primordial B-modes would be a “smoking gun” for inflation as the origin of the universe and would determine inflation’s energy scale. A detailed characterization of the lensed B-modes would also allow us to constrain the sum of the neutrino masses and the evolution of dark energy by cross-correlating with BAO experiments such as DESI. Just last year, experiments such as POLARBEAR detected B-modes from gravitational lensing directly for the first time. Current CMB experiments are deploying of order 1,000 detectors and experiments with order 10,000 detectors will deploy within few years. The community is discussing a possibility of stage-four CMB experiment to definitively characterize B-mode polarization with a order 500,000 detector count.

Our goal is to examine the complex coupling of oxidative ageing chemistry with environment- and history dependent viscosity, and generate a comprehensive model that can be applied to a range of aerosol transformation chemistries. Our model design strategy is to develop key components of the model separately, and merge them after validation. This approach is uniquely enabled using stochastic simulations. The main components required are a description of uptake of gas reactants, a description of the free radical chain over many product generations, and a description of dynamic perturbations by environmental factors such as temperature, pollutant plumes and humidity and internal factors such as immiscibility of products.In the first year of this LDRD we have made significant progress on the first two components: modeling uptake and the free radical chain. We have focused on a system for which there are extensive experimental data in the literature, the reaction of squalane aerosols with OH over a broad range of pressures. Using only kinetics from the literature we have shown that the current understanding of uptake is at best incomplete – it is not simply the sticking probability of reactants to particle surfaces followed by reactions.

Rather,hydroponic gutter the uptake coefficient is inseparable from the intrinsic internal transport rate of the aerosol itself relative to the gaseous reactant-aerosol collision rate, and is therefore an emergent property of the system. This represents a fundamentally new insight that promises to impact thinking on transformations of aerosols and liquid films. The free radical chain reaction that ensues after the initial reaction between OH and squalane aerosol is complex, involving many generations of addition of oxygen-containing functional groups forming hundreds of distinct products as well as fragmentation that transform the composition of the aerosol and cause it to shrink. In work led by the postdoctoral researcher in this project, Aaron Wiegel, we have developed a compact description of this chemistry that fully reproduces experimental observations. The free radical reaction model, which uses only literature kinetics, describes molecules as collections of functionalities and is therefore inherently general. It is the core of a universal free radical oxidation scheme that can be used to apply to a broad range of molecular systems. We aim to massively accelerate multi-modal data analysis to enable real time data explorations in order to speed the discovery and hypothesis generation process in neurosciences. We plan to precisely quantify changes in network structure, especially those that lead to neurodegenerative diseases. Our collaborative research project promises to significantly improve the fidelity and scope of neuroimaging analysis using high-performance computing methodologies via state-of-the-art graph analysis, image processing, and visualization techniques.

The research developed for these techniques will be applicable to a variety of evolving big data domains of interest to the DOE. Overall, our work will address the 3V components for big data neuroscience problems. Performance improvements in the segmentation of structural MRI and high speed computation of adjacency matrices will allow the end user to process more data in a shorter time, with expectations to view and analyze data in real time to speed the discovery and hypothesis generation process . By enabling researchers to integrate and interrogate data from multiple data modalities at the same time, we address the issue of variety. Additionally the volume problem is also addressed via the significant acceleration of comparative data analysis from multiple measurements for the same or multiple patients. We developed a data driven method for functional parcellations of brain regions as well as an adaptive hierarchical community detection method to fine tune parcels. This is a very challenging problem with more than 100K dimensions and relatively smaller sample size. The data is also very noisy due to fMRI measuring indirect blood oxygen levels as opposed to direct neural activity. Consequently, using global correlations is known to give unsatisfactory results because many nearby “voxels” have very similar time series data. CONCORD is the first method with provable global convergence properties that performs sparse inverse covariance estimation. The accelerated version of this algorithm, CONCORD-ISTA, uses block sparse linear algebra instead of coordinate-wise updates. What is needed is the partial correlation between voxels, i.e. the correlation of pairs of voxels after removing for the effects of all other voxels. We apply CONCORD-ISTA to our problem as it provides the best theoretical guarantees for estimating partial correlations between voxels.

We also developed a method for interactive visual exploration of functional magnetic resonance imaging data to analyze the correlation between activities in different human brain regions when resting or when performing mental tasks. Our visualization tool improves visual data exploration by generating multiple coordinated views, supporting the brushing-and-linking concept, and integrating community detection. Our tool provides neuroscientists with a powerful means to comprehend such complex data more effectively and efficiently. Mesoscale is DOE’s next frontier in their effort to develop to control chemical and physical processes that lead to new or more efficient renewable energy resources and approaches to reduce the carbon footprint. Modeling the emergent mesoscale phenomena utilizing computational chemistry methodologies requires the exploration of essential collective variables and order parameters in systems of sufficient size and disorder and with sufficient statistical sampling using accurate and scalable computational chemistry methods. We will improve the performance of key ab initio methodologies utilizing tools developed by the SciDAC Institutes, and by developing new and advanced algorithms for two-electron integrals and planewave FFT for the Intel Xeon Phi. We will develop computational chemistry tools that integrate a Kinetic Monte-Carlo methodology with scalable high accuracy ab initio and Car-Parrinello methodologies available in NWChem. To enable major scientific discoveries of mesoscale phenomena, computational models need to be integrated with a broad range of complex spectroscopic imaging experiments. To enable integration of mesoscale modeling efforts with complementary experimental work, we will develop the semantics and tools for needed to analyze and enable knowledge discovery in scientific data generated from mesoscale experiments and simulations. Our most significant accomplishment is the development of the Global Arrays/GASNet Interface called GAGA. This work was done in collaboration with members of the DEGAS project. The current implementation has been demonstrated to work efficiently on Infiniband clusters and Cray platforms, with significant faster performance and better CPU utilization for the coupled cluster single doubles capability in NWChem.Key two-electron algorithms were analyzed, and in collaboration with the SUPER institute we were able to obtain a 25% increase in performance and we improved the load-balancing algorithm on conventional CPUs. Similar performance improvement was found for Intel Xeon Phi, however the absolute performance is lacking relative to CPUs. We optimized the Fock build capability in NWChem and two-electron integrals to increase their efficiency on Intel’s nextgeneration Intel Xeon Phi processors.

In addition,hydroponic nft channel a distance based screening algorithm to enable O computing was implemented that will reduce redundant computation of expensive multi-center two-electron integrals in large systems. A first version of CML in NWChem has been made available to collaborators in industry . The work was presented at IUPAC in 2014 and we are in the process of writing a proposal to the organization for support to develop a consistent dictionary and ontology for chemical sciences. We are partnering with Kitware, using their MongoChem infrastructure to build a demonstration case for heavy element chemistry, linking computational and experimental data in a semantically rich framework. Our goal is to create a computational framework that will allow the in silico design of metal organic framework materials through a strategy based on structure optimization with respect to property. Efficient global optimum search algorithms will be utilized to efficiently navigate the space of possible structures. By performing this search on a broad space of MOFs, we thereby remove limitations on the search space encountered by currently utilized enumeration-based strategies. We plan to explore two approaches. In the first, we abstract molecular models of MOF building blocks as geometrical – or alchemical – building blocks, defined by a number of continuous parameters, which are optimized using gradient-based techniques. In the second, we perform the search in a discrete space of real molecular building blocks and employ Genetic Algorithm -based search techniques. Although the proposed approaches are general and can be used to design a MOF material with almost any desired property, our work will focus on properties critical for gas separation and storage. We aim to enable design of materials for carbon capture and natural gas storage. Therefore properties of interest include high surface area, large pore diameters and adsorption properties of gas of interest. Our framework for MOF design comprises three components: MOF assembly module; rapid property estimation module; and structure optimization control module. The key aspect of each component is modularity: these components can be substituted or extended to include other building block representation schemes, property estimation modules, scoring functions and optimization/search algorithms.Technological advances in computers and sequencing technology have enabled bio-informatics to develop at an unprecedented rate, especially in terms available data volume that require analysis. However, biologists face significant challenges in effectively studying these data sets due to the complexity of optimizing these classes of computations on modern computational systems. The goal of this project is to deliver unprecedented computational capability to large-scale analytics for key bio-informatics applications, via the development and integration of flexible and high-performance software packages. Recent work is targeting the analyses of high-throughput “next generation” genome sequencing technologies that are producing a flood of inexpensive genetic information, which is invaluable to genomics research. Sequences of millions of genetic markers are being produced, providing genomics researchers with the opportunity to construct high-resolution genetic maps for many complicated genomes. However, the current generation of genetic mapping tools were designed for the small data setting, and are now limited by the prohibitively slow clustering algorithms they employ in the genetic marker-clustering stage.

Our most significant accomplishment focuses on the first step of genetic mapping, which involves clustering markers into linkage groups. This is traditionally performed by various standard clustering algorithms applied to a similarity graph of the markers, which creates a significant bottleneck for large numbers of markers. Our work developed a fast clustering algorithm that circumvents the computation of all similarities by exploiting prior knowledge about the specific structure of the marker data: linkage groups have an intrinsically linear substructure that remains reflected in the similarity measure. After sorting, the algorithm creates a specific sketch that respects both the geometry and quality of the data. Using synthetic and real-world data, including the grand-challenge wheat genome, we demonstrate that our approach can quickly process orders of magnitude more genetic markers than existing tools while retaining — and in some cases even improving — the quality of genetic marker clusters. An important application of our method is in the efficient construction of ultra-dense genetic maps for large and complex genomes that are filled with repetitive sequences that frustrate genome assembly but do not limit the number of genetic markers. The most economically important of these genomes are various grasses, including crops grown for food or as bio-fuel feed stocks . The aim of this project is to develop advanced, high-order accurate, numerical methods for the computational simulation of multi-physics processes involving multiple moving interconnected interfaces. To achieve this, we are developing a new computational framework for complex fluid flow problems based on discontinuous Galerkin embedded boundary methods. The framework capitalises on mathematical and computational advantages provided by implicitly-defined representations of geometry, as used for instance in the level set method for single interfaces, and the recently developed Voronoi Implicit Interface Method for multi-phase physics. In particular, we are developing new projection methods for incompressible fluid flow which are high-order accurate in space and time as well as new techniques for consistent coupling of fluid-solid motion. The goal is to enable high-fidelity simulation of multi-physics interface dynamics, especially when the dynamics of the interface produce small scale features such as boundary layers that would not be captured by existing lower-order methods. The new framework holds promise for enabling high-accuracy simulation in complex geometries and offers advantages over other numerical methods in regards to high performance super computing.

Greater recognition is being given to the need for building sustainable and resilient urban food ecosystems . In Figure 1 we have attempted to illustrate how key concepts of agroecology can stimulate the food systems thinking in a city-region food system context.As highlighted above, the focus on food systems in the discussion about agroecology demands a far more comprehensive and holistic systems approach than, for example, the simple “value chain” or “supply chain,” long prominent in food systems development discourse. Agroecological approaches are based on minimal external inputs and increased recycling of resources. Food in a “chain” traces the steps on the way from production to consumption, with potential for complete detachment of relationships between the steps, and often sees eaters as“end-users” who are called “consumers.” In the current detached system, feed can come from a different continent, and the products can go to a third continent, enabling animal production and consumption literally “without limits,” as is the case for example in current Danish pig production, where the feed comes from South America, pigs are raised in Denmark, and the pork is exported to Asia. The systems approach gets lost in this regime, eliminating the potential for feedback signals to improve resilience and adaptive capacity,ebb and flow bench both regarding resource flows, and consumption patterns.

The question of animal products can reveal the limitations of this chain perspective: if stressing the systems approach, animal feed needs to come from within “the system,”which is also where animal products will circulate. If a systems approach is taken – as is necessary in an agroecological system – production is limited by the need to produce food for people situated within and maintaining landscapes – and closer proximity between animals and crops improves the potential and efficiency for nutrient cycling. A “full agroecological food system” may also have short supply chains, based on recycling and circulation, which will connect “the two ends of the chain” and actors within the food system. Following the emphasis above to constantly align and adjust food production with food consumption, the mere production of food can be seen as a big challenge. Depending on the magnitude of the urban areas, the agroecological food producing systems will have clear challenges in producing enough diverse food. Compared to many current urban food consumption patterns, the consumption patterns of agroecological food systems have to change, toward local food, and animal products of an amount which can actually be supported by each agroecological food system. How can the consumption patterns and the capacity of the food producing rural and urban farms be aligned and adjusted to each other, mutually and iteratively? This will require processes of negotiation, adjustments and development of common understandings, shared knowledge, and collective action to ensure that everybody at all times will have access to healthy nutritious food.One aspect which is rarely explored is how such strongly interwoven food systems can contribute positively and benefit the overall landscape and biodiversity , such as, for example, the findings of Chappell, Moore, and Heckelman , where increased ant biodiversity may have been linked to positive changes in local food security in Belo Horizonte, Brazil.

Another aspect that is rarely explored in detail is how urban-rural food systems will require certain features of the food producing systems, which involve the rural areas. How will it change the consumption patterns? Seasonality can present constraints on the “boundedness” of a food system, as can the desire for convenience in contemporary diets. Depending on growing conditions, rain patterns, and seasons, it can be a huge challenge to produce diverse food all year round for a population in and around urban areas and the rural areas connected to it. These requirements emphasize the qualities which are highlighted in the agroecological food system: resilience and multi-functionality in a well-integrated and complex system. A development toward more diverse, integrated production can lead to a much more diverse all-year round production, as is, for example, seen in agro-forestry and food forest systems. The combination of rural farming and urban farming, where rural farming to a larger extent produces food, roots, animal products and, for example, fruit, and urban farming focuses on fresh vegetables, leafy food, spices, nuts and fruit, can form examples of ways of extending the traditional growing seasons. Innovative processing possibilities, for example, solar-powered freezing facilities, can offer other opportunities for bridging the “production cycle” with the “consumption cycle” in urban-rural areas. Furthermore, the diversity of systems – both within systems and within a web of systems of urban and rural farming – will contribute to resilience and nourishment based on balanced diets all year round.The challenges highlighted above – production at scale, producing diversity, and producing food all year round – will of course vary widely depending on the context. Clearly, tropical areas differ from temperate areas, dry areas differ from very wet areas, and the length of growing seasons varies widely. Vandermeer and Perfecto emphasize the necessity of using traditional and local knowledge in combination with the knowledge and insight of “modern ecological knowledge,” to develop agroecological knowledge which is both deep and broad at the same time, allowing for learning across sites, as well as developing each site. In large parts of Europe and North America, current farming practices have focused on very few types of production with only one yearly harvest of, for example, grain. Many exciting initiatives could serve as examples of urban food strategies involving local food producing systems , and emerging agroecological food systems, viewing rural–urban landscapes as interconnected, and connecting actors through exchange of food and resources .

The visions and practical organization shown in these examples bridge rather than contrast “rural” and “urban,” which opens opportunities for sustainable, agroecological food systems across the rural–urban continuum , which again highlight the importance of contextuality, where smaller towns provide completely different options and challenges than larger cities, seen as contexts for city-region food systems. “Equity” is a cornerstone in relation to systems research and agroecology ,indoor garden and relates to justice in terms of “equitable access to resources” in relation to farming, seed, water, and land, for current and future generations. Many initiatives on justice in the food chain also address equity, for example, “technology justice” building on access, local innovation, and sustainable use of technologies . The term highlights social aspects and includes original populations and peoples’ rights to land, water, and natural resources. It also encompasses the genetic inheritance of humanity, and equal rights to make a living and survive on this planet. It also raises issues of gender equality, acknowledging both women and men’s rights to dignified futures and livelihoods as well as food. It recognizes that women often are responsible for family food, agrobiodiversity, and knowledge transfer between generations regarding many agricultural and food practices. Where agroecological farming systems use methods to nurture the soil and the ecosystems while producing healthy nourishing food, the agroecological food systems takes the very same principles up to the level of the way in which we compose our entire diets and process, sell, buy, and exchange food within the food systems. The concept of nourishment includes nutritional and cultural aspects of food and food consumption, and links to ideas of “sustainable diets,” as defined by FAO: . Furthermore, focusing on nourishment also emphasize the concept of health, which in a more holistic framing can be seen through the lenses of resilience , linking our diets closely to the farming and the food systems. The different understandings of resilience do not only cover social, economic, institutional, and environmental transformation processes of land and food, but also of public health and the health at all levels from soil, plants, animals to humans, and ecosystems.Whether rural areas can benefit from urbanization and can be closely linked to food systems in rural–urban areas depends much on national and international policies on subsidies, land use, trade, and agriculture. Nelson and co-authors emphasized the importance of governments actively promoting and supporting the development of sustainable food systems, although they also notice that in the case of Cuba, this was done primarily for ensuring food for the current generation of humans, rather than for ideological or moral reasons . Schipanski and co-authors outline strategies for realizing resilient food systems in different contexts, and Petersen and co-authors demonstrate a process of increased agroecological governance of the food system in the case of Brazil, strongly influenced by the struggles of rural social movements, helped to gradually form more inclusive and direct rural– urban connections in the food system. Vorley and Lancon call for a shift from “agricultural policies” to more integrated “food policies” involving both agriculture and food in increasingly urbanized areas, and Proctor and Berdegué emphasize the need to deconstruct the rural–urban dichotomy as the first step of creating equitable inclusive rural–urban food systems.

The Kenyan Greenbelt Movement is another example on how land, cities, ecosystems, human livelihoods, and equity issues were combined in efforts for better food security and sovereignty.They are multifaceted and encompass economic, environmental, social, and institutional aspects, requiring deliberation and negotiation within a multi-actor perspective . This is fundamentally different from the current globalized food system that takes little account of the diverse range of perspectives and needs among multiple actors in the production, processing, and exchange of food. Bellamy and Ioris discuss the imbalanced subsidy system, for example, within the EU context from farming to research, where the majority of support goes to industrial farming systems. However, many initiatives are taken on governance levels to stimulate domestic food production and local value chains, for example, Nigeria’s policy to stimulate domestic production of major commodities, and ban of rice imports in 2012 . A considerable effort is required regarding the governance of each agroecological city-region food system to facilitate social interaction and institutional arrangements that can constantly support the processes of recycling and exchange between different levels and elements of the system. Jennings et al. provided a visualization of the concentric city food provenance zones to illustrate how the idea of a “region” might pertain to a political or an ecological region, and to describe how different zones might contribute to a city’s food supply in varying proportions. The importance of planning for change and transition into coherent and efficiently working CRFS is emphasized through innovations in infrastructure and governance, for example, as illustrated in Figure 1 above. Different options for governance of city region food systems are pointed to by Da Silva and Fan , who mention the necessity to coordinate policies for rural and urban areas, promote social protection in rural and urban areas and support inclusive and efficient value chains between rural–urban areas. These highlight the importance of bringing stakeholders, researchers, politicians, and practitioners together, and draw emphasis on the importance of facilitating legal frameworks for these city-region food systems . The city-region food systems need to be organized and supported through governance, among others to allow farmers to plan their strategies and form collaboration efforts , which necessarily must be place-based and complex. Governance is also required in relation to the pricing policy, and external factors surrounding food production are not considered in the current pricing system . Another aspect is the protection of farmers, who are often overlooked or reduced to out-growers or industrial workers on their own land – which is maybe even taken from them – and the governance system around agroecological food systems needs to ensure that the potentials of diverse farms and human as well as social knowledge are fully utilized and valued, and are being described in research efforts taking agroecological principles into account . In current food systems, small-scale producers are particularly often marginalized and have no possibilities to participate to attain a fairer share or distribution of the income, risks, and benefits in these structures of prevailing markets, policies, and related institutions . Agroecological food systems can be essential features contributing to the practical and theoretical realization of initiatives linked to the so-called Milan Urban Food Policy Pact, which was launched in October 2015 and signed by 117 mayors from all over the world .

UA systems may contribute to the reduction of impervious surfaces in urban landscapes, thus increasing the drainage and infiltration potential of precipitation.As mentioned in the Urban agricultural systems and biodiversity provision section, it is important to understand that not all biodiversity is necessarily “good” biodiversity, and there may be a number of disservices that come from UA that can negatively impact the ecosystem functioning and health of cities. In some cases, there is the possibility of negative types of spill over from managed to natural systems or vice versa of weed, pathogen or pest populations, potentially harming native ecosystems and damaging ecosystem service delivery from natural systems . The juxtaposition of natural systems to UA systems also potentially leads to an increased opportunity for biological invasions and detrimental competition to native species . Genetic introgression within natural ecosystems by urban garden plants can negatively alter the genetic composition of native vegetative patches and affect the long term viability of these systems . UA areas may also lead to increased human health issues and disease transmission to urban populations. For example,UA systems provide increased mosquito breeding sites due to the presence of standing water from irrigation, and this may potentially increase the rate of mosquito-borne diseases in certain areas of the city . Additionally, in non-organic UA systems, there is the potential for spillover of chemicals into natural and human habitats,hydroponic grow kit leading to environmental pollution and air- or water-borne health risks .

There may also be potential competition for limited resources between UA and natural systems, such as competition for water in arid environments. All such potential disservices from within and outside of UA systems must be considered in optimizing the overall ecosystem services gained in an urban landscape.There are several research areas that deserve more attention so that urban land use trade offs can be better elucidated and biodiversity and ecosystem services can be promoted through the increased integration of UA in cities. We posit that the three key challenges to improving management of gardens for B&ES delivery are availability of urban space, environmental constraints, and lack of knowledge. Below, we provide three major strategies for addressing the key challenges of enhancing B&ES within UA systems.Increased urbanization will lead to greater competition for space in cities, making it difficult to maintain biodiversity supporting habitats. More research is needed to best take advantage of the limited space available for urban gardens and maximize biodiversity within these areas. Private yards. Private yards make up a significant proportion of green space in a city and do not require the acquisition of new space. Even small-scale, private gardens that present complex vegetation structure can provide appropriate habitats for organisms that have difficulty existing in the urban matrix .In the US, the National Audubon Society’s “Audubon at Home” project offers several management principles to increase bird biodiversity in backyards , and the National Wildlife Federation provides certification for ‘wildlife-friendly’ gardens. More research is needed to understand the effectiveness of these incentives to support native biodiversity and food production as many of the techniques are focused on the augmentation of ornamental or floral plants rather than food crops. Available public spaces.

Because greater housing density has been linked to smaller garden sizes, there is an acute need to better understand how UA can be supported within public green spaces, such as community gardens and easements to enhance ecosystem services . Even within small habitat strips, the conservation of plants known to attract pollinators or pest natural enemies can provide substantial B&ES while utilizing limited space , but more information regarding urban plant–animal interactions needs to be known in order to best augment such spaces effectively. Vacant lots. Vacant lots provide opportunities to create functional green spaces where industrial redevelopment is not likely to happen . UA in these areas can improve B&ES and provide physical and psychological health for people in cities . However, a better understanding of how to successfully rehabilitate vacant lots is needed in order to promote this option. Additionally, creating gardens in abandoned lots has implications on urban land tenure for garden management, and it would be helpful to investigate whether temporary gardens make positive contributions to B&ES and food production in the same ways that more permanent gardens do.A number of environmental changes come with urbanization and affect the agronomic conditions necessary for food production, such as water availability, nutrient supply, soil degradation, and pest pressure . Resilience to climatic change. We need investigations that examine how the choice of garden trees, shrubs, and other plants influence air and surface temperatures in the gardens, and the potential role of garden vegetation to lower energy use and costs in urban environments. Likewise, we need to develop a better understanding of the specific garden plantings that most enhance carbon sequestration in UA. On the flflip side, there is basically nothing known about how different UA respond to climate change or climate extremes, and how the urban environment in which UA is embedded may exacerbate climate effects. Thus, more research is needed to understand how plants in UA will respond to increasing temperatures, drought, changes in rainfall amount, nutrient deposition, and weather extremes. Water use.

Research on environmental constraints related to water use is also needed in UA, as irrigation is often required to provide water necessary for urban farming, especially in arid environments. Rainwater or grey water can be used for garden irrigation, and it is cheaper and at times more available than potable water-based irrigation, but UA gardeners must be aware of the potential pathogens and heavy metal contaminants that can cause human and environmental health problems , especially with water run-off from these sites. Soil ecology. Urban soils are usually compacted, have low levels of organic matter, altered soil moisture characteristics, and sometimes have lead or other heavy metal contamination due to urban environmental processes . A number of methods, such as cover cropping, mulching, producing in raised beds, and changing subsurface drainage through piping, can improve soil conditions to support food production . However, more research must be done to understand how to sustainably rehabilitate urban soils. The use of both organic and inorganic fertilizers in combination with nutrient-rich wastewater can lead to surplus nutrients in these systems . Alternative methods, such as “organoponics”, where organic compost is used as a growing medium instead of existing soils, need to be further explored to develop farming methods that are successful in the urban environment .Two oft-cited barriers to wildlife gardening are the lack of information to alter gardening methodologies for improved biodiversity and the ineffective transfer of knowledge to improve the sustainability of urban gardens . Methodologies for improved B&ES. There is an ever expanding data set on patterns of biodiversity loss in urban areas and the factors that positively correlate with the diversity of plants, arthropods,vertical farming racks and vertebrates in urban landscapes. The time is ripe for a quantitative review or meta-analysis of those specific habitat and landscape features of urban habitats that correlate with increases in species richness and abundance of biodiversity in general, and beneficial organisms in particular. For example, past studies document that urban gardens can increase predator or parasitoid diversity , but we still lack research that documents the garden features that enhance the specific act of predation by ants, spiders, birds, or other predators. Very little research has focused on how management intensity in UA systems will affect biodiversity and ecosystem services provision from these systems. Additionally, landscape level UA connectivity is important for the creation of networked biodiversity refuges and for the improvement of matrix permeability for organisms. There is a lack of knowledge in understanding how the movement of species between landscape elements can allow organisms to carry out functions at different points in space and time and maintain services that would otherwise be isolated and how UA fits into the larger general pattern. More research to understand the effects of garden management on landscape-wide biodiversity and movement will be necessary to determine the most critical management practices for promoting effective landscape connectivity. Knowledge transfer. Increasing our understanding of UA management practices and knowledge spread may be the most important area of research if we are to promote gardens that support B&ES. We need to determine which types of UA contribute disproportionately to food production under different geographic, weather, and socio-economic conditions so that urban gardeners can cultivate specific plant species best suited for their location.

Local ecological knowledge is generally low among urban residents; however, discussion between community members may encourage biodiversity-friendly gardening, either through neighborhood or community exchanges of information . More research is essential to understand how to identify the information most useful to urban gardeners and how to most effectively communicate this knowledge.The ecology of Phytophthora, a genus of fungal-like oomycetes historically erected and known for plant pathogenic species primarily associated with destructive diseases in agriculture, has undergone substantial reconsideration in recent years. The recent emergence of a number of Phytophthora-caused plant epidemics in forests and other non-agricultural ecosystems has clearly shown that many members of the genus have potential as invasive species that can threaten natural ecosystems. As a consequence of research in non-agricultural environments, a surprising diversity and abundance of Phytophthora species have been discovered, many previously undescribed. Incidental to this research has been the discovery that many species of Phytophthora are abundant in natural surface waters, especially in streams. Many such species are so widespread and regularly encountered that they are now considered resident, if not endemic, and characteristic of such environments. Nevertheless, isolates of well-known plant pathogenic species or species complexes are also regularly recovered, often without discernible symptoms or signs of disease on the vegetation. Though the prevalence of Phytophthora in surface waters is now well established, the ecology underpinning this phenomenon is largely speculative. Because these organisms are known primarilyas causes of often devastating plant diseases, the nature of their presence in these environments and its implications for the persistence and spread of pathogenic species are important considerations for disease prevention and management. There is also a growing interest to understand the role of Phytophthora, among other Peronosporales, in decomposition of vegetative matter in aquatic environments. The biology of Phytophthora, a genus of well adapted plant pathogens with a necrotrophic phase, suggests that their ecological role in leaf decomposition should be early colonization and breakdown of relatively fresh, live vegetative tissue. As they colonize leaves newly exposed in streams, they can open the integral tissues for colonization by saprotrophic organisms less able to penetrate the leaf cuticle, in a process analogous to ‘conditioning’ of leaf litter for palatability to shredder organisms. The co-occurrence of both known plant pathogens and primarily stream-associated Phytophthora in aquatic environments also raises the question of whether these taxa have similar or divergent modes of life and whether they compete for resources in these environments. In streams, vegetative litter is the primary source of nutrients for microorganisms, but the quality of vegetative tissues available varies with respect to senescence and degree of decomposition. Coastal forests of northern California largely consist of evergreen trees and shrubs and so green leaves are a regular component of leaf litter introduced into streams, especially in winter and spring when, based on the region’s climate, most rainstorms occur. Nevertheless, much vegetative litter is in the form of senesced leaves. California bay Nutt is a common, broad leaf evergreen component of northern California’s coastal forests and a frequently occurring tree species in riparian zones. It is also a primary source of P. ramorum inoculum in California forests affected by sudden oak death, epidemic mortality of certain species in the beech family resulting from P. ramorum infection of the vascular cambium of the main trunk. California bay leaves are highly conducive to sporulation by P. ramorum which, despite causing localized necrotic lesions and spots on leaves, nevertheless causes little damage to the tree species itself. Additionally, bay leaves are sclerophyllous, as is typical for broad leaf evergreen plants in this Mediterranean climate, and so they decompose slowly. Bay leaves are therefore both very common as leaf litter in northern California forest streams and a highly suitable substrate for P. ramorum.

The activities of several housekeeping genes were also tested. A relatively modest increase in levels of histone H2A and beta tubulin expression in glands compared to leaves was detected. The increase in expression levels of these latter two genes might reflect a combination of the heightened metabolic activity and the unique cellular structure of glandular trichomes. Two different pathways could provide the hexanol required for olivetolic acid synthesis, as shown in Fig. 2. Expression levels provide support for the de novo pathway as a primary source, given that CAN498, CAN82, and CAN915 were much more highly expressed in glands than leaves , whereas the relative expression of genes encoding enzymes in the lipid breakdown pathway were depressed or modestly elevated in glands.The identities of the most abundant ESTs derived from the glandular trichomes of Cannabis sativa are consistent with the protective function of plant glands. For example, the most abundant ESTs encoded a protein closely related to type II metallothioneins. These proteins bind heavy metals such as Cd, Zn, and Cu, and their proposed primary function is the maintenance of Cu tolerance . The second most abundant class of ESTs encoded an RD22-like BURP domain containing protein. This class of proteins contains a hydrophobic Nterminal signal peptide,hydroponic grow systems and an N-terminal conserved region followed by a series of small repeats .

The BURP domain of approximately 230 amino acids is located in the C-terminal region. The function of RD22-like proteins is unknown but some members of this class of genes are induced by dehydration . The third most abundant ESTs encoded a protein containing a hevein domain. Hevein domains contain a conserved 43-amino acid motif that binds chitin and members of this protein class are known for anti-fungal activity . The unique secondary metabolism in Cannabis may also play a role in plant defence. Synthesis of THCA is extracellular and results in hydrogen peroxide production, which has general antimicrobial properties , and a recent report further indicates that THCA may directly inhibit microbial growth . The analysis of gland-derived ESTs has identified nearly all the candidate genes required for THCA synthesis from primary metabolic products. These findings differ from a proteomic study that aimed to identify genes expressed in Cannabis glands but failed to associate any highly expressed proteins with THCA synthesis . This difference reflects the much greater volume of genomic data enabling more robust identification of DNA sequences when compared to proteomics approaches based on the molecular weights of fragmented polypeptides. This is especially true for species such as Cannabis sativa for which there is little amino acid sequence data available to compare with peptide profiles. The present study highlights the utility of using isolated glands as starting material for making EST libraries to study gland metabolism, as was the case in other plant species . In this study more than 50% of the ESTs with NCBI matches were involved in metabolism or cellular activities such as transport and protein translation.

Many other cannabinoids, in addition to THCA, have been identified in Cannabis , and it is likely that many of the genes identified in Supplementary Table 2 at JXB online are involved in the production of these other compounds. In addition to cannabinoids, many other classes of secondary compounds have been found in Cannabis . For example, both monoterpenes and sesquiterpenes have been identified and candidate ESTs encoding activities to produce these compounds have been identified.Synthesis of olivetolic acid from malonyl-CoA and hexanoyl-CoA represents the first committed step toward the synthesis of THCA. Olivetolic acid synthesis is predicted to be mediated by a member of the type III PKS family through a series of three condensation reactions producing a triketide . CAN24, represented by eight ESTs and one of the most highly expressed unigenes in our analysis, encodes a member of the PKS family. This gene was expressed 1600-fold higher in glands than in leaves. CAN1068, another PKS member represented by two ESTs, corresponds to a previously identified Cannabis CHS gene . A third PKS represented by a single EST, CAN383, was also identified. Analyses of PKS crystal structures indicate that the type III PKS enzymes are composed of a dimer with conserved reaction centres and a hollow reaction cavity . All three Cannabis PKS genes encoded polypeptides containing the conserved amino acids, Cys 167, His 307, and Asn 340, that are believed to constitute the reaction centre . In addition, two of the three amino acids that are important for defining the size of the reaction cavity in chalcone synthases are conserved. The third amino acid, Thr at position 300 that is conserved in all chalcone synthases, was missing in CAN24 and CAN383 . Instead CAN24 and CAN383 contained Leu and Iso, respectively, at position 300. Such differences might alter substrate specificity. It has been proposed that either olivetol or olivetolic acid are products of polyketide synthase in the THCA pathway . However assays of plant extracts found that olivetol, the decarboxylation product of olivetolic acid, was not a substrate in the pathway .

Products of the three PKS genes identified in this study were tested for olivetolic acid synthesis in vitro. CAN24 and CAN383 yielded identical products according to HPLC analysis . Because CAN24 was more abundant, this PKS gene was analysed in detail, along with CAN1069, which had CHS activity as shown in Fig. 4. The size of the product produced by the CAN24-encoded enzyme was smaller than olivetolic acid . Further, the absorption spectrum did not match olivetol. A sequence identical to CAN24 has been deposited in the NCBI database and was annotated as having olivetol synthase activity but without supporting data. The product generated in our analyses possibly represents a derailment product in which the enzyme catalyses two decarboxylative condensations instead of three . The failure of in vitro conditions to support the complete synthesis of native PKS products is well documented . The artificial nature of in vitro reaction products is reflected by the failure to find these products in vivo . This may indicate that reaction conditions were insufficient or that accessory proteins may be needed to produce olivetolic acid. Similar results were obtained in a study that characterized a gene identical to CAN1069 where the enzyme could use hexanoyl-CoA as a starter molecule, but only yielded a possible derailment product . That plant extracts used in in vitro assays have also not yielded olivetolic acid suggests that in vivo-like reaction conditions have yet to be imitated . The assay results with CAN1069 also highlight the permissiveness of substrate use by PKSs. CAN1069 clearly had CHS activity in that it could use coumaroyl-CoA as a substrate to produce naringenin. Given that CAN1069 was preferentially expressed in the glands and can act on hexanoyl-CoA, this PKS also may contribute to THCA production. Insights on THCA biosynthesis may be gained by comparison with the secondary metabolism of glandular trichomes in hop,vertical grow table the closest relative to Cannabis. Production of the bitter acid humulone in hop inflorescence glands requires a PKS called VPS. In two independent studies, ESTs representing VPS were among the most abundant in the collections and were at least 5-fold more abundant than other PKS encoding ESTs . Accordingly, the role of CAN24 as olivetolic acid synthase remains tentative pending further biochemical support, however, it is the best candidate based on expression data.A large number of Cannabis gland unigenes encoded proteins similar to transcription factors . Several of these were analysed by qPCR and two were found to be preferentially expressed in glands. This is potentially significant as studies have shown that all but one of the 12 transcription factors required for Arabidopsis trichome formation are preferentially expressed in trichomes compared to whole leaves . In this study it was found that two R2R3-type Cannabis MYBs, encoded by CAN833 and CAN738, were preferentially expressed in isolated glands compared to leaves by 954-fold and 586-fold, respectively. CAN833 and CAN738 are most similar to the Arabidopsis MYBs related to AtMYB112 and AtMYB12, respectively .

AtMYB112 corresponds to the BOTRYTIS SUSCEPTIBLE1 gene. bos1 mutants are more susceptible to pathogens such as Botrytis cinerea and Alternaria brassicicola, and have impaired tolerance to oxidative stress . A role for CAN883 in tolerance to oxidative stress in Cannabis glandular trichomes is logical, as the last enzymatic reaction in THCA synthesis releases hydrogen peroxide . AtMYB12 controls the synthesis of flavonol secondary metabolites in Arabidopsis and can induce the synthesis of similar compounds in tobacco . Flavonoids have been isolated from Cannabis leaves and flowers, but evidence is lacking for gland flavonoid production . Since flavonols are not predominant in Cannabis glands, it is possible that CAN738 instead plays a role in controlling the expression of genes required for other secondary metabolites in Cannabis such as THCA.The PKSs and many other genes identified in this study are closely related to those from hop.Glandular trichomes located on the inflorescence bracts of both Humulus and Cannabis are the location of unique PKS-derived secondary metabolism . Hop glands produce the bitter acid humulone, which is important for beer flavour, and the prenylated chalcone xanthohumol, which has several potential health beneficial properties . The biochemical pathways leading to THCA, xanthohumol, and humulone have common steps that include polyketide synthases and prenyltransferases. It is probable that these plants share other homologous biochemical pathways given their close ancestry. Information from Cannabis ESTs has the potential to improve the understanding of hop biochemical pathways as well.Urbanization is a major driver of land cover change worldwide and affects the biophysical and socioeconomic landscape. It is estimated that by 2030 >60% of the global population will live in urban areas . Furthermore, in many parts of the world, human development is expanding rapidly at the edge of urban areas and the quality of rural habitat is declining owing to agricultural intensification . Thus, green spaces found within urban landscapes are quickly becoming important refuges for native biodiversity . Urban planners are increasingly interested in maintaining agriculture within and around cities due to food security concerns. Many cities contain ‘food deserts’, where access to fresh produce is limited due to reduced proximity to markets, financial constraints, or inadequate transportation . In response to food insecurity, urban agriculture has expanded rapidly. For example, in the US, UA has expanded by >30% in the past 30 years, especially in under-served communities . This is because urban agriculture can be very productive, providing an estimated 15–20% of the global food supply , and cities can provide good infrastructure, access to labor, and low transport costs for local food distribution . Additionally, interest in UA has escalated recently due to the desire to transform vacant land in post-industrial cities and to address nutrition and childhood obesity issues in disadvantaged urban neighborhoods . Though public and Scientific interest in UA has grown dramatically in the past two decades, there are still significant challenges for integrating UA in an increasingly spatially constrained urban landscape. Much of the debate is centered on land-use trade-offs of UA versus other types of urban development. Although there are a number of socioeconomic considerations that affect the development and proliferation of UA in cities, this review will focus on the ecological aspects of the UA system and how they can be designed to maximize the environmental and health benefits in order to increase acceptance of this particular land use in the urban sphere. One way to encourage the integration of UA is to better understand how planned and associated biodiversity within these systems contribute to urban ecosystem services. However, there are three major gaps in the literature regarding UA status and impacts that limit our ability to increase the range of benefits and ecosystem services that could come from UA systems. First, biodiversity patterns in urban agroecosystems have only recently been documented and require synthesis. Second, ecological communities within UA may translate to the delivery of valuable ecosystem services ; however, the availability of these services within UA has not been well-established. Finally, little is known about the role of UA in mediating resilience to major threats,specifically climate variability .

This type of disjointed management leaves policy makers ill-equipped to provide resilient management strategies. Thus, the success of using the nexus concept to improve food, energy, and water systems will likely depend on how it incorporates issues surrounding resource governance , including how governance and the discourse of securitization become a way to legitimize political agendas. Many have argued that the nexus discourse of security places economic variables over access to resources for the world’s poor, an idea that can be traced back to Foucault’s theory of the linkages between security and the circulation of the global economy . In addition to this discursive ‘securitization’, resource governance outcomes are based on the larger political goals of the government or states involved in policy making . The connections between discourse, policy, and land management, therefore, raise important questions regarding how policy is already impacting nexus outcomes and communities on the ground. Given the recent emergence of the FEW concept, few studies to date have explored the use of the nexus concept simultaneously with an analysis of governance structures . To address this gap, we present a quantitative application of the FEW nexus concept to study resource vulnerabilities and scarcities in the Lower Colorado River Basin in California, Arizona, and Nevada, U.S.A. We analyze the nexus within its sociopolitical, economic,pot with drainage holes and bio-regional context that determine what resources are available, used, produced, and traded .

We take a case study approach, as case studies are best able to translate the on-the-ground nexus realities of a variety of institutions, bureaucracies, and stakeholders across space, time, and scale . Our goal was to understand how the governance structure of the Colorado River constrains the utility of the nexus approach to deal with future stresses. To do this, we first quantified the nexus by identifying the local and global linkages between food, energy, and water as well as the choices confronting water managers, and the Indian Reservations in the study area. We use these findings to look for the emergence of tipping points under two different scenarios: drought and increased demand for alfalfa. We then discuss how the very rigid water laws in the LCRB constrain the ability to improve resource management and respond to these tipping points using nexus thinking. While the main focus of this paper is on how economic and hydraulic factors influence FEW’s nexus governance, in the discussion we also examine the impact of institutional and political factors as well as geopolitics across the transnational boundary between the U.S. and Mexico.The Colorado River Basin has a semiarid to arid climate with an average of 40 cm annual precipitation that originates as snow pack in the Rocky Mountains and contributes to about 70% of the total stream flow in the basin . However, temperatures have been rising for the past century, with winter temperatures increasing more than summer temperatures on average by 2 °C . Temperatures are predicted to rise at least another 1.1 to 2.0 °C by 2050 . These higher temperatures increase evaporation rates and have coincided with a reduction in snow pack and snow melt in the UCRB . In addition, the LCRB has been in a drought since 2002, though at the same time the UCRB has experienced less severe or no drought conditions, outside of extremely dry periods in 2003 to 2004, and 2012. Historically, paleo climatic records indicate pervasive and long-lasting periods of drought occur in the region . However, more recently, the area has received significantly more snowfall. The level of Lake Mead is often used as a proxy for water availability in the basin. Its water elevation has been decreasing since 2000, which can be seen visually in aerial imagery .

The Colorado River is operated under the “Law of the River,” which is comprised of a variety of U.S. federal laws, agreements, court decisions, and regulatory guidelines . These laws apply to seven Western U.S. states: Arizona, California, Colorado, Nevada, New Mexico, Utah, and Wyoming, and Mexico that utilize water from the river. The Law of the River allocates to each basin 7.5 million acre-feet 1 of water per year in perpetuity, with some exceptions that account for water scarcity . High variability in precipitation in the Rocky Mountains results in 5 MAF to 25 MAF of flow in any given year with an average annual flow of less than 16.4 MAF/yr. In terms of usage, on average 91.4% of the allocated water is used by municipalities, for power, or agriculture . The UCRB Indian reservations receive 1 MAF a year, while the LCRB Native American reservations are allotted 0.9 MAF a year. While this water is split amongst the 20 reservations in the LCRB, only six reservations have had their water rights quantified . Five of these six tribes are located in our study area. This has resulted in a disproportional amount of water allotted per person on these reservations compared with the rest of the basin. Specifically, 6.7 AC-FT per person per year is allotted to Lower Basin Indian reservations while 0.9 AC-FT per person per year is allotted to the rest of the LCRB. It should be noted that the water allotments of both basins , Mexico , and the Native American Reservations , adds up to 18.4 MAF, 2 MAF more than the yearly average stream flow of the Colorado River not accounting for climatic fluctuations. The rigid allotments based on the Law of the River have also resulted in most of the agriculture production in the study area taking place on Indian reservations. Prior to the Law of the River, American Indians practiced flood farming practices for thousands of years based on characteristic seasonal rains , as well as dry farming. Today, however, practices are much more water-intensive on Indian Reservations .The Colorado river allows local economies to exist in a semi-arid environment with two-thirds of Arizona’s, California’s, and Nevada’s state gross products dependent on the Lower Colorado River . The river provides 657.5 billion dollars of direct, indirect, and induced GDP to California; 185 billion dollars to Arizona; and 115.4 billion dollars to Nevada . Focusing on food systems, agricultural production in the study area is largely situated on Indian reservations due to their high allotment of river water.

Water is often delivered through an extensive canal system. For alfalfa, one of the most common crops in the region, the water that is used to flood the fields is sent back into the canals. The high-water availability along with the warm climate allows the production of twelve harvests of alfalfa each year . Most of the alfalfa is exported as fodder to China, followed by Japan, Mexico, South Korea, and the EU .A diversity of data sources drawn from the Food and Agriculture Organization of the United Nations , the United States Department of Agriculture ,large pot with drainage and the Energy Information Association were used to quantify the connections between food, energy and water. Water was quantified in terms of consumption types for 2008-2016. Energy was quantified in terms of net generation and cost for hydroelectric, natural gas, solar, and wind generated in the region for 2001 to 2016. Food production was quantified in terms of area of agricultural crops as well as the price that farmers spent on production for 2008 to 2015. Only crops that took up greater than 1% of total area were analyzed. To determine the total impact of one sector on another , we combined the total amount of water used in energy or food, and the total amount of energy used to pump water or produce food .Water-energy linkages were quantified in two different ways. First, the amount of water used in energy production 2001 to 2016 was calculated by multiplying the appropriate water consumption factors of electricity generated from natural gas from Spang et al., 2014 by the amount of energy produced monthly from 2001 to 2016. Water used in natural gas extraction for gas used in the study area was not included since there are no natural gas extraction operations within the basin. Hydroelectric projects have no net water consumption, but reservoirs make evaporation rates higher, especially with higher temperatures. Second, energy used to transport water in the Colorado River Aqueduct was quantified using data obtained from the Metropolitan Water District of Southern California for 2001 to 2016. The aqueduct conveys water from Lake Havasu over the Santa Ana Mountains and to cities in Southern California, including Los Angeles. In addition, energy used to transport water from Lake Havasu to Phoenix and Tucson via the Central Arizona Project was drawn from a previous study by Kleiman .

A final water-energy linkage, the amount of energy need to purify wastewater for domestic consumption was not quantified due to lack of data. Generally, however, wastewater treatment accounts for ∼3% of energy used in the United States.First, information on specific crop water usage was estimated using evapotranspiration rates in ft/growing period and average area cultivated annually for six of the seven most common monocrops in the LCRB. All data came from FAO estimates except for lettuce . FAO evapotranspiration estimates are given in mm/growing period, however, we converted the units to ft/growing period so we could easily translate the measurement to volume of water in AC-FT. Second, data for 2007, 2012, and 2013 from the National Agricultural Statistics Service for the entire Lower Colorado River Basin was used to estimate yearly water used for irrigation . In order to calculate the data at the watershed sub-unit level, we used the United States Agriculture Service 2012 Census and Cropland Data Layer to estimate the percent of irrigated land out of the total acres of cropland . The proportion of cropland that was irrigated varied based on data year, in 2013, 67.74% of cropland was irrigated; in 2012, 75.82% of cropland was irrigated; and in 2007, 71.21% cropland was irrigated. We averaged the percent of cropland irrigated in the watershed sub-unit for 2007, 2012, 2013 to extrapolate to the remaining years from 2008 to 2015. The average irrigation estimate was used in the production scenarios to understand responsiveness of irrigation and crop production to changes in climate.The first scenario depicts how the costs and supply of water, energy, and food production might change in an extreme drought situation. With a decrease in water availability, water and energy prices would increase, but agriculture production would roughly stay the same due to water governance in the region. This would have occurred, for example, if Lake Mead had decreased to 1030.75 ft . At or below an elevation of 1075 ft, Lake Mead is at a critical drought state. At 1050 ft, Lake Mead is below the capacity at which the Hoover Dam can produce hydroelectricity . If it were to stay at this elevation for an entire year, this would amount to a decrease of 36% of the average annual electricity generation from 2001 to 2016. Reduced water availability has previously been shown by Bain and Acker to result in higher operating costs, and higher prices of energy for hydroelectricity in the Colorado River Basin. Additionally, for those that pay for water from a utility, a drought of this magnitude could increase water prices. According to a report from the Public Policy Institute of California, the 2012-16 California drought resulted in an increase in water prices through drought surcharges due to increased supply and treatment costs for suppliers . However, those that rely on water rights for their water, such as on certain Indian Reservations, would continue to receive the same amount of water with no price increase. The Bureau of Reclamation could make a deal with Reservations to hold onto some of their water with some form of compensation. In this case, irrigation will decrease, which was assumed for the drought scenario. However, a decrease in water available for irrigation does not necessarily mean production will decrease as seen in the increase of production of agricultural products during the drought year from 2012 to 2013, with a decrease in the total amount of water used for irrigation.

Several other plant-growth-promoting bacteria, including Bacillus siamensis, Bacillus megaterium, Bacillus subtilis, and Bacillus cereus, had relatively higher IAA yields at temperatures of 2–135 ◦C and pH 7–8. Three different bacteria isolated from therhizosphere of Stevia rebaudiana also exhibited greater production of IAA at a pH range of 6–9 and a temperature of 35 ◦C to 37 ◦C; these bacteria also increased the root and shoot bio-masses of wheat and mung bean. Various carbon sources are used as an energy source for IAA production and could enhance recycling of cofactors in bacterial cells. Our results revealed that IAA yields of B. seminalis strain 869T2 were slightly better when glucose and fructose were used in media. Several previous publications also indicated that the ability of plant-growth-promoting bacteria to produce IAA was different, depending on the carbon source used in the media. Results from these studies and our study demonstrated that IAA production by different plant-growth promoting bacteria can be influenced by various factors, such as temperature, pH, carbon sources, culture conditions, and bacterial species. In this study, we utilized the colorimetric method to estimate the IAA amounts of B. seminalis strain 869T2 when grown in various in vitro conditions and media. Because the available tryptophan in the rhizosphere and root exudates of plants might be relatively lower than the tryptophan used in the media,black plastic plant pots the IAA production of B. seminalis strain 869T2 when grown in inoculated plants shall be determined with more sensitive and accurate methods, such as high-performance liquid chromatography or ultra-performance liquid chromatography systems.

Apart from the IAA production ability of B. seminalis strain 869T2, this bacterium exhibited siderophore production and phosphate solubilization activities. Iron is an important element for many biological processes in plant growth and development. Most iron in soils is present in the highly insoluble ferric form, which is unavailable for plant absorption. Endophytic bacteria can yield iron-chelating agents such as siderophores, which bind ferric iron and help transport it into plant cells via root-mediated degradation of organic chelate, ligand exchange, or other mechanisms. Phosphorus is another essential macro-nutrient for numerous metabolism processes in plants, such as biosynthesis of macromolecules, signal transduction, photosynthesis, and respiration. Most of the phosphorus in soil is insoluble and not available for root uptake to support plant growth. In order to increase the bio-availability of phosphorus for plants, certain endophytic bacteria turn insoluble phosphate into soluble forms via the processes of chelation, ion exchange, acidification, or production of organic acids. Previous studies have also correlated siderophore production and phosphate solubilization abilities with the plant growth promotion traits of other Burkholderia species, such as the Burkholderia sp. SSG isolated from boxwood and the Burkholderia sp. MSSP isolated from root nodules of Mimosa pudica. Burkholderia cenocepacia strain CR318, which was isolated from maize roots, significantly enhanced maize plant growth by solubilizing inorganic tricalcium phosphate. Other studies have revealed that additional Burkholderia species also have the ability to solubilize inorganic phosphate to increase available phosphorous in agricultural soils and improve agricultural production.

In summary, both previous studies and our results suggest that the IAA synthesis, siderophore production, and phosphate solubilization abilities of B. seminalis strain 869T2 may collectively contribute to the growth enhancement observed in the several plant species tested here. We successfully inoculated and reisolated B. seminalis strain 869T2, which was originally isolated from the monocot plant vetiver grass , in several eudicot plant species of the Brassicaceae, Asteraceae, Amaranthaceae, Solanaceae, and Malvaceae families. Strain 869T2 can significantly improve the growth of both the roots and aerial parts of Arabidopsis and several leafy vegetables, including ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. These results suggest that the endophytic bacterium strain 869T2 may have a wide host range. A similar observation was reported for Burkholderia phytofirmans strain PsJN, first isolated from onion roots, which enhanced the growth of Arabidopsis, switch-grass, potato, tomato, maize, wheat, and grapevines. We did not observe significant growth improvement in hot pepper or okra plants after inoculation with strain 869T2; however, we did observe early flowering and better fruit development in these tested plants. These results suggest that the plant growth promotion abilities of strain 869T2might be more apparent in crops with a shorter life cycle or that the latter two tested host plant species might not be fully compatible with this bacterium. The plant colonization process and growth promotion abilities of endophytic bacteria seem to be active processes that are regulated by different characteristics of both the host plants and bacteria. In conclusion, our study revealed the potential of Burkholderia seminalis strain 869T2 for use as a bio-inoculant in agriculture to improve plant growth and production. Broken nutrient and carbon cycles in food systems waste vast quantities of organic resources and contribute to climate change, food insecurity, and soil degradation. Closing these cycles may contribute to global greenhouse gas reduction goals , improve the resiliency of agroecosystems , and advance sustainable development goals . The capture and transformation of human excrement, in particular, represents an enormous and largely untapped resource stream.

An estimated 4.1 billion people do not have access to a sanitation system that includes waste treatment , including 892 million people still practicing open defecation . The release of organic matter and nutrients embedded in untreated feces and urine into the environment impairs water quality and emits GHGs, resulting in public health concerns. Ecological Sanitation is a strategy for improving access to sanitation and full cycle treatment of human waste. EcoSan is implemented throughout the world , and is particularly important in areas where water access, financial resources, and infrastructure are limited . There are several models of EcoSan, with technologies ranging from constructed wetlands to composting toilets and scales ranging from simple household installations to complex decentralized systems . Regardless of implementation, all EcoSan systems share the common objective of the closed-loop management of human waste and a systemic philosophy that connects sanitation and agriculture . Here, we consider EcoSan implementations that combine container-based toilets for capture and transport of waste, with aerobic, thermophilic composting for the treatment process to sanitize human feces and produce an organic soil amendment . EcoSan offers potential solutions to critical sustainability challenges in the nexus of sanitation, water, health, and agriculture . In particular, EcoSan may solve three important sustainability challenges by providing safely managed sanitation for presently underserved communities and reducing the spread of intestinal-born pathogens, returning nutrients and organic matter to degraded agricultural soils,drainage pot and mitigating climate change by reducing GHG emissions compared to alternative waste disposal methods, bio-energy production, and/or promoting soil carbon sequestration in agricultural ecosystems. EcoSan currently makes up a minor fraction of global sanitation services. Current social challenges and technical challenges exist to its widespread implementation. However, as population expands in rapidly urbanizing cities and as water availability declines, cost-effective EcoSan operations, such as container-based implementations we consider here , may grow to serve a larger proportion of the global population . Quantitative data are required to critically evaluate the potential of EcoSan to address these sustainability goals. Here, we focus on the goal of reducing GHG emissions because emissions from EcoSan composting technologies are an important, yet unknown, variable in their overall sustainability. Greenhouse gas emissions vary widely among waste treatment technologies, depending on the bio-geochemical conditions, operating conditions, and associated collection and discharge systems . Wastewater treatment plants are globally significant contributors to GHG emissions . Several sources provide estimates of CH4, N2O, and CO2 emissions from wastewater treatment plants, including national inventories, primary literature, and life cycle assessments . Fluxes of CH4 and N2O are biologically-produced, and therefore fluxes can be reduced by technologies that remove or stabilize carbon and nitrogen during the waste treatment process . Systems that use anaerobic digestion of sludge tend to be a major source of CH4 emissions though some anaerobic digestion systems aim to contain and burn emitted CH4 for energy production . In contrast, GHG emissions data from EcoSan and other non-sewered sanitation systems are sparsely available in primary literature and monitoring inventories. Greenhouse gas emissions from non-sewered sanitation systems, including pit latrines, septic systems, and container-based toilets, are poorly constrained due to their decentralized locations and high level of operational variability.

Direct measurements of CH4 and N2O from septic systems are few and have differed from modeled emissions factors . A recent analysis of pit latrines concluded that globally, pit latrines accounted for 1% of anthropogenic CH4 emissions . The relatively large contribution of pit latrines to global CH4 sources can be attributed to the global extent of pit latrine use e approximately one-quarter of the global population e as well as the wet and unventilated conditions that drive anaerobic CH4 production. EcoSan relies on aerobic conditions to treat waste and has the potential to considerably reduce the GHG footprint of waste management . In aerobic thermophilic composting, CH4 emissions are typically low because of the presence of oxygen. However, anaerobic microsites created by uneven distribution of water in pores and hot spots of labile carbon can create conditions leading to CH4 emissions . The use of bulking agents and pile turning can be used to reduce the occurrence of anaerobic CH4-producing conditions and, when effective, carbon emissions from composting are in the form of CO2, which is considered to be climate-neutral because of its biogenic origin . Composting can, however, produce biogeochemical conditions prime for N2O emissions through nitrification or denitrification, including large sources of reactive nitrogen, dynamic and spatially varying levels of oxygen, and labile carbon sources. Quantifying the magnitude and balance of CH4 and N2O emissions in a given sanitation system is critical as the two gases have 100-year global warming potential values of 34 and 298, respectively. EcoSan systems utilizing aerobic, thermophilic composting are promising because they may mitigate GHG emissions from the waste and agricultural sectors, however these emissions reductions have not yet been quantified. Further, measurements of GHG emissions from management of solid organic wastes are especially limited from tropical climates , where implementation of EcoSan solutions are likely to be greatest. To our knowledge, no direct measurements of GHG emissions exist from EcoSan systems that deploy container-based toilets and thermophilic composting of human excrement. Our primary objective was to characterize and quantify the GHG emissions resulting from the aerobic composting of human waste in EcoSan settings. We considered two operations that employed similar compost practices, but differed in the physical infrastructure that could alter bio-geochemical conditions mediating GHG dynamics. We also compared the GHG footprint of EcoSan with alternative waste management pathways present in the region, including waste stabilization ponds and unmanaged disposal on grass fields. Finally, we undertook an investigation of the effects of compost management options that help reduce EcoSan GHG emissions.Greenhouse gas fluxes were measured from three sanitation pathways in Haiti: two waste stabilization ponds, two EcoSan operations, and a grass field where the illegal disposal of sewage was observed . The waste stabilization ponds were located in Croix ed Bouquets near Port-au-Prince, Haiti and operated by the Haitian government agency, Direction Nationale de l’Eau Potable et de l’Assannissement . Ponds consisted of uncovered concrete basins with effluent pipes connected to secondary overflow ponds. Two ponds were included in the sampling: a pond that received mostly septic tank waste , and another that received mostly pit latrine waste . Solid sludge was scraped out occasionally and stockpiled on-site. Solid and liquid waste from septic tanks and pit latrines were transported to the site and emptied into the waste stabilization ponds. The waste stabilization ponds represent the primary pathway of centralized waste treatment as advanced municipal wastewater treatment technologies are not present in the country.Eighteen and 54 L container-based toilets were collected from households and communities, respectively, in each of the regions.Coarsely ground sugarcane bagasse was added throughout use to prevent flies and reduce odor. Urine was disposed of on-site, and only solid material was transported to the compost facilities. Both facilities used a similar aerobic composting process consisting of a static thermophilic stage, followed by pile turning and maturation in windrows. At the Cap-Haiti€ en operation, hereafter referred to as “Compost CH,” the ground was lined with cement to prevent leaching and an aluminum roof covered the area. Roofs and cement-lined floors were absent at the Port-au-Prince EcoSan operation, hereafter referred to as “Compost PAP”.