Tag: vertical agriculture

The community’s rebirth began with the 1990 revival of the Sociedad de Beneficencia Israelita de Iquitos by community member Víctor Edery in response to his own personal desire to rekindle Jewish community life in Iquitos among the remaining practicing families. Without a Torah or other sacred texts, a solvent community fund, much collective knowledge of Jewish ritual outside the home, or a synagogue, the new community officers and the thirty-three members they represented felt outside help was needed to make a group of individual families of varying levels of independent practice into a cohesive community. They wrote letters to the Lima community asking for such help, and reached two important figures: the Argentinian-born Conservative/Masorti rabbi Guillermo Bronstein and Federación Sionista del Perú member Debora Frank. While current members of the community remember little of Debora Frank, Guillermo Bronstein continues to be active in the community, despite recent illness. It is already notable that Edery and the community sent these letters to a non-Peruvian rabbi and an Israel-focused organization as well as to Lima. The Iquitos community, which, despite its isolation,hydroponic net pots has always been diasporic by virtue of being Jewish, realized that a transnational action was needed to get the support they desired, and did so through intra diasporic networks.

Although Lima’s community of 1,900-2,000 Jews is by far the largest and closest Spanish-speaking Jewish community in Peru , internal dynamics, such as colorism and citified prejudice against far-flung Loretanos, made Lima’s Jewish community a less viable option for aid, which explains why the letter moved from the Limiñosynagogue to Bronstein and the Federación Sionista. Meanwhile, transnational forces in the Jewish diaspora ensured that international institutions like the Conservative/Masorti movement and pro-Israel organizations like the Federación were most likely to respond. A jump from Lima to Argentina is also unsurprising; Argentina boasts South America’s largest Jewish community, at 180,300 individuals , with attendant resources. Rabbi Bronstein’s connection to the Conservative/Masorti movement is significant, as it is linked to member synagogues throughout the world, including Israel, and has strong relations with the Jewish Agency for Israel , an organization relevant to the post-2011 Iquiteño experience. When the Iquitos community broke its long isolation, it reconnected itself to the webs of organizations that crisscross the Jewish world. The organizations that were most eager and financially ready to engage with the Iquiteño community, therefore, were institutions that centered Ashkenazi practices and the modern state of Israel as foundational to Jewish identity writ large, which thought of diaspora as requiring transnational integration. At this point, it was practically certain that the influence of Israel-focused groups, who make up a significant percentage of Jewish philanthropic organizations working internationally, and Ashkenazi groups, where there is the most money and influence, would have an out sized impact on the future of Iquitos. Bronstein especially responded with alacrity, encouraging the Iquitos community to draw up congregational statutes and begin to observe various major holidays.

He also reached out to his contacts in Buenos Aires, Miami, and New York. Among them was Ariel Segal, then a PhD student at the University of Miami, who visited Iquitos in 1995 and involved himself intimately in projects to convert Iquiteños and help them make aliyah. Meanwhile, Debora Frank made the Iquitos community familiar to the Federación Sionista, which boasted many connections to Israeli officials and other Zionist groups across South America. The stage was set. The impact of this is immediately visible in the circumstances of the two first mass conversions in August 2002 and December 2004. Rabbi Bronstein, in a 1993 letter reproduced in full in the fifth appendix of Ariel Segal’s book, argues that conversion should be granted only to “those who pledge to make aliyah” and who will undergo a second, Orthodox, conversion once in Israel.10 From the very beginning, then, not only was emigration to Israel dependent on conversion, but conversion was dependent on emigration to Israel. To be as clear as possible: in order to make a spiritual commitment, a practical action was seen as necessary. In order to make a practical move, a spiritual commitment was necessary. Regardless of whether these migrants continued to practice as they were taught in Israel, a question that is beyond the scope of this thesis but which deserves attention, it is clear that mixing together transitional actions with religious and diasporic identity was useful to actors invested in a particular transnational outcome. The conflation of Jewish identity and Israeli citizenship in Iquitos begins to show itself most clearly here, with no thought to standards of living, improved education, or other practical concerns. The practical and the religious converge here, and they do so because transnational activity was used as proof of diasporic conviction.

Segal speculates that the emigration stipulation was meant to weed out those who were not sufficiently devoted to Judaism, a concern that appeared again and again in my interviews, ironically twisted to a fear that some individuals would “fake” a conversion in order to emigrate. The state/institutional level, therefore, allows us to understand that,blueberry grow pot for the first several years of the Iquitos community’s revitalized existence, Jewish education, community support, and conversion depended on external institutions which happened to strongly support connections between Jewishness and Israel. Before examining the individual/community-level through data from the interviews Iquiteño Jews and I conducted in 2016 and 2019, I wish to remind readers of the timeline of this community. While states and major international organizations were instrumental in setting up the initial conditions for conversion and migration, they no longer play as important a role in influencing the individual decisions Iquiteños make in response to global or personal factors. Between the second conversion, in December 2004, and the third, in August 2011, there was a great shift. The presidency transferred to the Abramowitzes and the synagogue moved into its current location in 2009, and leadership attitudes towards conversion and migration have practically reversed — a notable change and one that proves, in case there was any doubt, that despite strong interference from non-Iquiteños, a great deal of the changes happening there come from within the community. “I do not want people to see us as a travel agency,” Señora Francisca Abramovitz told me sharply over lunch one afternoon. The de facto female leader for the community, Sra. Abramovitz is also the primary bookkeeper, synagogue caretaker, programming coordinator, outreach specialist, Internet publicist, and general macher of the Iquitos community. She makes the arrangements with the rabbis, solicits donations for, selects, and distributes prayer books and Hebrew primers, sets up the synagogue for services and cleans it up afterwards, conducts the entrance interviews for people interested in joining the synagogue, organizes outings and children’s classes at the synagogue in everything from liturgy to karate, collects and records dues and donations received, and reaches out to Jewish youth groups in Argentina to come visit. Although she and Sr. Abramovitz are technically two of the five-member Directiva , they do almost all the work of maintaining the community’s organized behaviors themselves. Congregants often refer to them as a unit simply as “la Directiva” or “los presidentes.” Sra. and Sr. Abramovitz are also the intermediaries between individual congregants and the rabbis of the Batei Din and the Jewish Agency for Israel, which currently handles the emigration paperwork for Iquiteño olim.

The process is not easy, and Sra. Abramovitz’s travel agency remark came at the end of a long demonstration of the mountains of complex paperwork involved in journeying from non-affiliated Iquiteño to new Israeli citizen. If one does not know the community’s history with Israeli immigration, the option seems no better than emigrating to, say, the United States or Argentina if gain is the primary motivation. Nonetheless, the fear that people might be taking advantage of Sra. Abramovitz’s hospitality —the synagogue, after all, is literally inside her home— and fierce Jewish faith to simply pursue a selfish financial end permeated many of our conversations.They must find it, first of all, and then make it past Sra. Abramovitz’s entrance interview. Then, there is an application to become a “Miembro Activo de la Asociación Judía de Beneficencia y Culto de Iquitos”. The new members must then take classes in Hebrew, liturgy, and “Jewish life,” and regularly attend Friday night and holiday services, as well as other community events, for at least two years. This educational programming is done in conjunction with Rabbi Bronstein and Rabbi Rubén Saferstein of Buenos Aires, who began his involvement with the Iquitos community in 2004. These two rabbis, and a rotating cast of others, eventually conduct a formal conversion in the Masorti tradition, complete with a brit for the men, and present the new converts with a stamped and signed certificate of conversion, a shtargeirut. Those who wish to make aliyah must provide three copies of that document, their birth certificates, passports, and National Identity Cards and those of their spouses and children, certificates of completion for all the classes they have passed complete with syllabi, their marriage records, proof of circumcision for the men, and a letter from the presidents affirming that they have been “good and active participants” in the community. That enormous packet of documentation passes from the Abramovitzes to the Jewish Agency for Israel, and then, through some alchemy that the Abramovitzes themselves do not fully understand, to the Israeli Bureau of Immigration, where applications are tentatively approved or denied. Potential olim must then travel to Lima, a matter of either an expensive plane flight or a days-long journey by boat and bus, to be followed by an also-expensive stay of multiple days in the capital, for an entrance interview. Only then may they officially be granted Israeli citizenship and have their travel and resettlement expenses taken on by the state. Those with family already in Israel may choose to live near them; those without are settled according to the dictates of the state. Most in the latter category are sent to the mixed Jewish-Arab city of Ramla. All this, and even then, theolim and their children will need to undergo a further, Orthodox conversion to access the full rights of an Israeli citizen11 and may also be required to serve in the Israel Defense Forces. At the time of writing, this process is for people who very likely have practiced a form of Judaism for much or all their lives, identify as Jews, have Jewish parents , and frequently have relatives already living in Israel. If this is a travel agency, it seems like one of the most redundant, possibly insulting, and certainly difficult tours I have ever heard of. Why do this? In 2002 and 2004, aliyah was a prerequisite to conversion. That is no longer the case; converts in 2011 and 2018 made no such commitment. Sra. Abramowitz ensured that this would no longer be the case, hoping that it would prevent more Iquiteño Jews from leaving Perú. On the one hand, if migration is undertaken for practical personal gain, why undergo a long, difficult, expensive, and, frankly, annoying process to go to a country where one does not speak the language and may not be able to control even where one lives? On the other, if aliyah is undertaken out of a deep spiritual desire to connect with a particular facet of perceived Jewish identity, why submit to the indignity of being told your Jewish practice is insufficient, inauthentic, and invalid? Although not in as many words, these were the questions I posed to my interviewees in 2016 and 2019. It is in the content of Iquiteños’ responses and the eagerness with which they gifted them to me that I have found something approaching an answer. I argue that, despite changes in leadership conversion method over a long period of time, Iquitos’ Jewish education has always conflated Jewish authenticity with Israeli citizenship because of its early influence by the Federación Sionista, Rabbi Bronstein, and Ariel Segal, creating an environment that suggests the endpoint of a successful conversion is aliyah. Furthermore, there is now a self-sustaining transnational dynamic between immigrants from the 2002 and 2004 conversions now living in Israel and friends and relatives in Iquitos. Lastly, when deciding whether to migrate or not, Iquiteños do not draw clear distinctions between practical and spiritual reasons for migration, because those pro-Israel individuals and institutions framed Israel as central to a valid diasporic Jewish identity.

Yellow leaves resemble green ones in that, in contrast with brown leaves, they have an intact cuticle, and their cells are essentially still alive. California bay leaves infected by P. ramorum have been shown to senesce and abscise from trees more frequently than uninfected leaves. In infested forests, a great portion of senescent leaves probably enters the stream already colonized by P. ramorum. As leaves that fall into the water do not dry out, their cells likely remain alive for an extended period, allowing further colonization by P. ramorum. However, stream resident clade 6 Phytophthora species also compete for this substrate and may limit the extent to which P. ramorum can grow on, persist, and reproduce from them. As dry, brown, senesced California bay leaves begin to make up a greater proportion of leaf litter in late summer and fall, the ability of clade 6 Phytophthora species to exploit these, while P. ramorum cannot, may be one explanation for why the latter is recovered less regularly and with lower frequency from these and other California streams in the fall and early winter. Moreover, as the summer progresses, green and yellow leaves will be more decomposed and less suitable for P. ramorum. The warming of streams late in the summer may additionally favor clade 6 Phytophthora species that are known to have generally higher optimal growth temperatures than most other species. We maintained temperatures constant for experimental purposes, but the persistence and sporulation of these Phytophthora species, and P. ramorum in particular,dutch buckets may be significantly affected by temperature fluctuations and extremes.

We have isolated P. ramorum and clade 6 Phytophthora from leaf litter of other tree species in naturally infested streams, including leaves of coast redwood, madrone, white alder, big leaf maple, and coast live oak. Occasionally, we have found portions of other submerged riparian plants, such as chain fern or elk clover , to be colonized. It is well-established that California bay leaves are an optimal substrate for P. ramorum, and though clade 6 Phytophthora species are known from a great variety of vegetative litter, it is uncertain how conducive other vegetative litter would be to survival and sporulation of either species. Stamler et al. recovered primarily clade 6 and 9 Phytophthora species from rivers in the southwestern USA using leaves of Salix and Populus species, common asriparian trees, as bait. It would be expected that natural leaf litter in such ecosystems would also harbor these organisms. Themann et al. recovered primarily P. gonapodyides but also P. cinnamomi from vegetative litter in sediments in an irrigation reservoir. Therefore, leaf and other vegetative litter should be considered as potential sources of Phytophthora, including pathogenic species, whether they are found in natural streams or other surface waters. Alternatively, the suitability of local vegetation may be a determinant of what Phytophthora species become established or prominent in streams.Current farming and food systems confront and are implicated in multiple challenges and unsustainable changes, including biophysical dimensions such as climate change , environmental pollution, escalating losses of biodiversity, and deteriorating ecosystem services. 

Social forces and structures as well as unsustainable socioeconomic processes also strain present capacities to manage growing population pres sure, unplanned urbanization, food and nutrition insecurity, dietary shifts, and health disparities associated with poverty, and growing inequality among multiple stakeholders, including women, youth, migratory workers, and indigenous peoples. Both urban and rural actors are impacted in relation to land ownership and land use change issues and drivers underpinning global industrial agriculture and connected food systems. Human activity has approached critical limits over an increasing number of the so-called Planetary Boundaries , beyond which the functioning of ecosystem ser vices may be substantially altered, increasing the risk of destabilizing life on our planet. Agriculture and food systems are both a villain and a victim in approaching or breaching PBs, and this is already impacting the ability to farm and produce food. How can humanity sustainably grow nutritious food and return to a safe operating space within the PBs? As an alternative to this scenario, a growing number of studies and reports indicate significant potential gains from transitioning toward agroecological agriculture as a way of nourishing current populations sustainably while allowing for future generations to support their livelihoods. One core quality of transitioning to agroecological farming systems is the regenerative trend of increased “outputs” per unit “input” for a more efficient agriculture for using and conserving diversity on a long-term basis, through the use and combination of different agricultural techniques in ways which restore and nourish the soil and enhance the local environment, instead of continuously degrading it.

In addition, the diversification strategy makes food producing systems resilient to external shocks and influences, such as floods or droughts, using, for example, approaches built on the principles and science of agroecology. There is growing evidence that such production systems allow for lower cost and more diverse fruit and vegetable supply. Furthermore, conventional thinking about food is increasingly being challenged, shifting from being regarded only as a commodity toward becoming acknowledged for its nourishment, social and cultural values, the links it creates between people, and its deep connectedness with ecosystems, ecosystem services, and natural resources. The current globalized industrial food system exhibits the same drivers which impact and shape farming industries and food production, and underscores the importance of focusing on how food flows into food systems, and which structures and related policies are shaped to support and reinforce current farming as well as food systems. It is not only conventional and industrial production of animal feed, genetic material, or major commodities such as wheat, rice, coffee, sugar, maize, and chicken which are controlled and shipped across continents by large trans-national corporations. Our globalized industrial food systems sometimes also include food which originates from farming systems based on organic farming regulations and principles like the IFOAM principles, calling for more coherent, equitable and holistic food systems,grow bucket and applying agroecological farming methods. In other words, the intentions behind such farming systems and their contributions to agricultural and environmental sustainability are not always extended to food systems, which generally contribute to out-competing local produce, distorting prices and producing huge amounts of food waste and other waste. This can be seen as a contradiction and emphasizes the importance of thinking of not only organic and agroecological production, but also has consequences for thinking the principles into the entire food systems. At the same time, there are many examples of organic farming and food as well as agroecology presenting alternatives to the industrial farming and food systems , and by increasing and emphasizing this, we can move toward a food system that falls within the PBs. This calls for profound analyses of how agroeco logical food systems function, and how they can contribute to coherent, resilient and equitable production and exchange of food, while human and social capitals are built up throughout the food systems, and resources are cycled rather than transported through, from or to disconnected parts of the systems. How can such food systems meet challenges such as losses of complex and system-oriented, context-relevant knowledge about farming and food, and how can they contribute to re-connect consumers and the food that they eat across urban-rural settings in city-region food systems? An increasing number of papers and reports link agroecology and food systems , referring to the fact that agriculture and food systems are intricately linked, and to a large extent driven by the same global structures.

Given the intricate and mutually-reinforcing relations between agriculture, food, and socioeconomic systems, the present article aims to characterize and explore how the concept of agroecology stimulates the conceptualization of agroeco logical food systems, or perhaps even a more inclusive term like “socio-agroecological food systems.” Food systems following the principles of agroe cology calling for resilience, multi-functionality , equity, and recycling of resources face particular challenges and have significant options for impacting sustainable development in city regions. This needs to be seen in a light where an increasing amount of the global population lives in urban areas, from smaller towns with a few thousand inhabitants, to mega-cities of millions of people. Urbanization has changed diets and nutrition, while food consumption has become detached from food production worldwide. Taking a systems approach to reconnecting these gaps requires major changes in consumption patterns, resource management and social responsibility, if everybody is to be nourished in agroecological food systems. We aim to explore the connections and linkages between the concepts of agroecology and food systems, and focus particularly on how the food system framework can locate and ground the concept of agroecology within a rural– urban landscape setting. This exercise requires us to critically examine the reciprocal flows and the multiple environmental, social, and governance related connections needed for an agroecological food system transformation.A food system is a system that involves activities, social and institutional structures, and processes related to the production, distribution, exchange, and consumption of food. Agricultural systems are part of food systems, integrated in ecosystems, and constituted socioecological systems. Over the past few decades, the understanding of food systems has clearly developed as result of the development of a more and more globalized food system. Ericksen compared some features of “traditional” versus “modern” food systems, and addressed the governance of different food systems, with or without support for local production, and Foran and co-authors point to the existence of different concepts of how food systems are constructed, with examples from so-called developing countries. The structure and governance of the food system clearly influences consumption patterns by providing both producers and non-food-producing consumers with options of availability. The range of social and environmental welfare outcomes stemming from food system activities were also discussed and visualized in Ericksen , and Jennings and co-authors analyzed how planned and well gov erned city-region food systems could contribute to different aspects of food security for different groups of citizens, stable incomes, circular economies, and resilience at various levels. Characterizing a food system can follow through its different social aspects and arrangements, like the type and degree of contact between those who grow and produce food and those who receive and eat the food without participating in the production of it, or who and how many people are involved in the cycle between the soil and the plate. Where local food systems with short supply chains have potential for involving resource feedback loops, raising collective awareness among different actors within the food system, and give possibilities for mutual learning , a larger and decoupled food system lacks the direct interaction and feedback, reduces exchange of experiences and knowledge, or the embedded ness inherent in a localized food system. A decade of research on New York’s Chinatown produce economy gives an example of the importance of this con nectedness: the studies revealed that 80-plus produce markets offered an incredibly diverse assortment of lower-cost produce because they are connected to a web of nearby, independently-run small farms and wholesalers.In a food chain , a product flows through different steps, where various forms of transformation may occur, and connection and feedback loops between these different steps may not necessarily exist. In such systems, farmers or industrial food producers can risk becoming producers of “food from nowhere,” as expressed by Bové and Dufour , and later unfolded by Campbell , and “consumers” can become reduced to a non-informed and non-responsible person, only “consuming food no matter of origin,” as a contrast to so-called “food citizens” defined as a consumer who makes decisions that support a democratic, economically just and environmentally sustainable food system, with a possibility of being actively involved in the food system at different levels. The call and practice of re-localizing of food systems is similarly seen as a harbinger of rural– urban reciprocity as consumers and producers are re-embedded physically and socially in the food system while raising awareness of their respective impacts on one another.Agroecology is widely acknowledged equally as a science, a practice and a movement. Its academic roots go back nearly 100 years, drawing on the fields of agronomy, horticulture, and ecology.

Sediment and plant samples were also collected at the PCW inlet, midpoint, and outlet. Sediment samples were collected as described above.All samples were transported on ice prior to laboratory storage at 4 °C. Sediment samples were dried and ground with a mortar and pestle before extraction. Plant samples were thoroughly washed with DI water to remove any attached sediment particles and biofilms. The cleaned plant samples were dried before pulverizing the tissue in the presence of liquid nitrogen using a mortar and pestle. The TSS, sediment, and plant samples were extracted by pressurized fluid extraction on a Dionex ASE 350 using a method similar to Brennan et al. and Maul et al.. Briefly, sample cells were filled with 1:1 dichloromethane:acetone , heated to 100 °C, and extracted at 1500 psi for two 5 min cycles before being flushed with 60% solvent for 1 min. For the TSS samples, the entirety of the solids from each water sample was extracted. Aliquots of the prepared sediment and whole plant samples were extracted and subjected to in-cell cleanup with Cu powder packed between cellulose filters. All extracts were evaporated to approximately 2 mL under a gentle nitrogen stream. Each sample then underwent clean-up on a Florisil cartridge preconditioned with hexane and was eluted with 9:1 hexane:acetone. Cleaned extracts were again evaporated under a gentle stream of nitrogen to approximately 0.5 mL and reconstituted in 1.0 mL hexane for final analysis. Samples were analyzed using an Agilent 6890N/5973N GC/MSD equipped with a 30 m x 0.25 mm x 0.25 µm DB-5MS column.

Additional analytical information is provided in the SI. Following sample quantification,ebb flow tray statistical analyses were performed using SAS® 9.4. Numerous steps were taken to ensure the accuracy and quality of analysis. Instrumental controls during analysis included running a calibration standard every 10 samples, adding 13C4- 15N2-fipronil and d5-bifenthrin as internal standards to each extract, and determining method detection limits using EPA Method 40 CFR Part 136, Appendix B. Calculated MDLs were 2 μg L-1 for all analytes except for fipronil desulfinyl, which had an MDL of 1 μg L-1. In addition, several procedural controls were utilized. Reagent blanks were analyzed with every set of 7 liquid-liquid extraction samples and 10 pressurized fluid extraction samples. Reagent blanks revealed no presence of the target analytes. Matrix spike samples were analyzed to measure extraction efficiencies, which are listed in the SI. In addition, ethiprole and decachlorobiphenyl were added to all samples prior to extraction to assess surrogate recoveries, which were 105 ± 23% and 121 ± 18% for liquid-liquid extraction, respectively, and 108 ± 21% and 116 ± 18% for pressurized fluid extraction, respectively. Additional quality control measures are described in the SI. The concentrations of fiproles and pyrethroids in water samples collected from the PCW are depicted in Figure 3.1. In general, mean whole water concentrations progressively decreased on a spatial level when moving from the inlet to midpoint to outlet for all compounds of interest. In addition, mean outlet concentrations were always lower than mean inlet concentrations. Furthermore, statistically significant differences existed for the majority of inlet and outlet concentration comparisons. When the differences between inlet and outlet concentrations were not statistically significant, inlet concentrations were found at low levels and with relatively high variability.

Since water and the associated TSS is all that enters and leaves the PCW, these findings provide evidence indicating that statistically significant removal of fiproles and pyrethroids occurred as a result of treatment in the PCW. Previous research by agricultural and WWTP effluent-polishing CWs has similarly shown removal of fiproles and pyrethroids. The mean whole water concentrations of fiproles and pyrethroids also exhibited distinct temporal trends during the sampling period. Mean fipronil concentrations peaked in June 2018, gradually decreased until October 2018, and then increased until January 2019. Mean whole water concentrations of fipronil desulfinyl, fipronil sulfide, and fipronil sulfone incrementally increased from June-August 2018, decreased, and then increased until January 2019. The peak mean whole water concentrations for fipronil desulfinyl and fipronil sulfone occurred in January 2019 while the peak for fipronil sulfide occurred in August 2018. Taken together, these trends correspond to high fipronil application rates in the summer months, gradual decrease via degradation of the parent compound and delayed formation of degradates, and increased transport of applied and degraded residues due to rainfall events in the winter months of 2018-2019. The mean whole water concentrations of bifenthrin and cyfluthrin, on the other hand, steadily increased from June-August 2018, gradually decreased until November 2018, and then increased in December 2018 and January 2019. The mean bifenthrin concentrations peaked in August 2018 while mean cyfluthrin concentrations peaked in January 2019. These trends indicate high use of pyrethroids in the summer months, followed by a period of reduced use and transport, and finally an increase due to runoff from the rain events in the winter that could have transported additional residues through the CW and/or caused resuspension of residues from the sediment bed. Fipronil and cyfluthrin were detected at the highest levels in whole water PCW samples, with mean concentrations ranging from 13.5-369 and 13.8-455 ng L-1 , respectively. 

Fipronil sulfone and bifenthrin were detected at moderate mean concentrations of ND-32.5 and ND-63.9 ng L-1, respectively. The compounds detected at the lowest levels, fipronil desulfinyl and fipronil sulfide, had mean whole water concentrations of ND-2.48 and ND-4.09 ng L-1, respectively. The water concentrations of fiproles and pyrethroids measured in this study were similar to previous measurements in California in urban runoff. Figure 3.2 shows the sediment concentrations of fiproles and pyrethroids in the PCW. As was the case with mean levels in whole water samples,flood and drain tray mean sediment concentrations for all compounds followed a decreasing spatial trend from the inlet to midpoint to outlet. Since the same spatial pattern was observed for mean water concentrations, this suggests that sediment binding was partially responsible for the dissipation of fiproles and pyrethroids from the water. This finding was to be expected due to the hydrophobicity and strong affinity of these two insecticide classes, particularly for pyrethroids. This was in agreement with previous studies where sediment binding was shown to be an important removal mechanism for pyrethroids in flow-through wetlands receiving agricultural drainage. However, based on this spatial trend alone, it was unclear whether the contaminants were temporarily retained by the sediment and available for partition back into the passing water or if they were subsequently degraded in the sediment. The mean sediment concentrations of fipronil desulfinyl, fipronil sulfide, and bifenthrin followed a similar trend over time. These contaminants generally increased in concentration from June-August 2018, when they reached a peak for the entire study, followed by a decreasing trend until November 2018 before gradually increasing until January 2019. Fipronil in the sediment increased from June-July 2018 when it reached its peak level, decreased until November 2018, and then increased until January 2019. Fipronil sulfone and cyfluthrin peaked in January 2019 and June 2018, respectively, but they followed the same trend as the other compounds. An inspection of Figure 3.1 and Figure 3.2 reveals similar seasonal temporal trends for fiproles and pyrethroids in both whole water and sediment samples. In other words, when analytes were present at high levels in whole water samples, they tended to also be present at high levels in the sediment. This finding, combined with the fact that sediment concentrations did not continuously increase over the duration of the study, indicates that fiproles and pyrethroids were likely actively degraded once partitioned into the sediment phase. Another possibility is that the contaminated sediment particles underwent resuspension and were carried out of the PCW; however, the generally lower whole water concentrations at the outlet relative to the inlet suggested that the contribution of this process was likely negligible. Among the six compounds, fipronil and cyfluthrin were detected at the highest mean sediment concentrations during the study period. Fipronil sulfone and bifenthrin were present at moderate levels ranging from 0.166-4.42 and ND-5.40 ng g-1, respectively.

The lowest mean levels were found for fipronil desulfinyl and fipronil sulfide, at ND-0.740 and ND-0.718 ng g-1, respectively. This pattern was also reflected in the whole water concentrations. These results again suggest that fiprole and pyrethroid residues in PCW water partitioned into the sediment and underwent degradation on site, rather than accumulated over time. Fiprole and pyrethroid concentrations in whole plant samples are shown in Figure 3.3. Mean plant concentrations follow the same spatial trend as whole water or sediment concentrations, with levels generally decreasing from inlet to midpoint to outlet for all compounds. Since the spatial trends of fiproles and pyrethroids are the same in whole water and plant samples, it appears that plant uptake played a role in the removal of these insecticides in the PCW. Fipronil is a systemic insecticide, so some degree of plant uptake of fiproles was to be expected. Detection of pyrethroid residues in whole plant samples was an unexpected finding due to the hydrophobicity of these insecticides and their consequent affinity for sediment. However, there are studies that have documented detection of pyrethroids in plant samples, either by uptake or by apparent irreversible sorption to plant tissues. Different temporal trends were observed for fipronil, fipronil degradation products, and the pyrethroids in plant tissues. Fipronil mean plant concentrations initially decreased from June-July 2018, increased to peak levels in August 2018, gradually decreased until November 2018, and progressively increased until January 2019. This trend of fipronil concentrations over time coincided with the temporal trends of fipronil in whole water and sediment samples, providing more evidence that plant uptake contributed to the removal of fipronil in the PCW. Moreover, it is likely that fipronil initially adsorbed to wetland sediment and was then absorbed into macrophyte roots. The mean plant concentrations of fipronil desulfinyl, fipronil sulfide, and fipronil sulfone all gradually increased from June 2018-January 2019, indicating some degree of accumulation in plant tissues over time. However, since fipronil did not follow this temporal trend of accumulation in wetland macrophytes, it is likely that some of the parent compound was metabolized into these derivatives upon uptake. Fipronil sulfone was present at higher concentrations in plant samples than the other degradation products, which was in agreement with previous studies showing that in vivo plant oxidation is a major metabolic pathway for absorbed fipronil. Bifenthrin and cyfluthrin displayed no temporal trend in plant tissues since they were both only detected in inlet samples at one time point. This suggests that plant adsorption or absorption did not play a major role in the removal of pyrethroids by the PCW. Fipronil was detected at the highest levels in PCW plants, with mean concentrations of 4.70-194 ng g-1. Moderate mean concentrations of ND- 17.7 ng g-1 were observed for fipronil sulfone. The lowest mean plant concentrations were measured for fipronil desulfinyl , fipronil sulfide , bifenthrin , and cyfluthrin. The results of plant tissue analysis reveal that plant uptake played an important role for the removal, degradation, and storage of fipronil, but did not contribute substantially to the removal of fipronil degradation products or pyrethroids. However, it must be noted that the dense vegetation was essentially slowing down the flow and filtering off suspended solids, contributing greatly to the removal through sedimentation. In addition, microbial activity in the rhizosphere of plant roots likely facilitated the degradation of these chemicals in the sediment, further contributing to the overall pesticide removal. The concentration-based removal values of fiproles and pyrethroids from water flowing through the PCW are given in Table 3.1. Over the entire course of the study, removal values for fipronil desulfinyl, fipronil sulfide, fipronil, fipronil sulfone, bifenthrin, and cyfluthrin were 100%, 99.7-100%, 57.8-88.1%, 75.6-100%, 74.7-100%, and 36.6-82.2%, respectively. The compounds with the highest removal values were fipronil desulfinyl, fipronil sulfide, fipronil sulfone, and bifenthrin, while fipronil and cyfluthrin showed the lowest removal. It is important to note that only fipronil, bifenthrin, and cyfluthrin were detected every month, and fipronil and cyfluthrin were detected at higher levels than all the other compounds. Previous studies have similarly shown that the average CW removal rates of fipronil and pyrethroids were 44% and 52-94%, respectively, for other CW systems. 

Observations of the regulation of production of this surfactant in various culture conditions suggest a role for this surfactant primarily in fully hydrated environments. Its coordinated expression with flagella suggests an intimate role between surfactant production and flagellar motility, but the identification of many other regulatory elements reveals a complicated mechanism of regulation. Examinations of the interaction of this surfactant with the bacterial cell, its flagella, and with the surfaces that this bacterium colonizes should illuminate its role in the epiphytic lifestyle of P. syringae. Biosurfactants, or biologically-produced surface active agents, are a broad group of natural amphiphilic compounds that are capable of lowering the interfacial tension between two phases. Although many different types of natural products have been identified to function as biosurfactants, one of the most intriguing classes of these compounds is the lipopeptides. Lipopeptides have a peptide head group attached to a lipid tail, and the peptide moiety is unique in that it is synthesized non-ribosomally; ie, it is not translated from an mRNA. Rather, lipopeptides are generally synthesized by non-ribosomal peptide synthetases , large enzyme complexes that catalyze the sequential assembly of a small peptide, as well as direct the addition of a carbon tail. Additionally, these assembly units can specify the incorporation of unusual amino acids,ebb and flow bench modifications, and/or cyclization. Because of this flexibility and ability to create unique peptides, NRPSs have found widespread use in bacteria and fungi for the synthesis of a variety of compounds including antibiotics, siderophores, pigments and many others. 

NRPSs specific for lipopeptide synthesis contain an initial condensation domain that catalyzes tail addition to the first amino acid of the peptide, and thus can be readily identified bioinformatically in genomes. The most familiar example of a lipopeptide is that of surfactin produced by Bacillus subtilis. This cyclic lipopeptide is composed of seven amino acids linked to a 12- to 16-carbon tail; the seven amino acids being somewhat variable in composition due to the low fidelity of many NRPSs. Although generally linked to biofilm formation and motility, surfactin has recently been revealed to act as an autoinducer signal, wherein surfactin production is sensed by non-surfactin producing cells, inducing them to produce an extracellular matrix. Other Bacillus lipopeptide families include the iturins and fengycins, while Pseudomonads have been found to produce an even broader range of lipopeptides. Two of the most extensively studied Pseudomonas lipopeptides are syringomycin and syringopeptin produced by Pseudomonas syringae, which have been noted for their membrane-disrupting and resultant phytotoxic properties. These cyclic lipopeptides contain 9 and 22 to 25 amino acids, respectively, and contribute to the virulence of this microorganism. Recently, production of syringafactin, an 8-amino acid linear lipopeptide, was also described in P. syringae DC3000 and B728a. With the continued identification of new lipopeptides and the sequencing of their genetic loci, an interesting pattern has emerged; many of the NRPSs for lipopeptide production in Pseudomonads possess divergently transcribed LuxR-type regulators both upstream and often also immediately downstream, of the biosynthetic cluster. When they have been characterized, disruptions in the upstream and sometimes downstream regulator results in blockage of lipopeptide production. These LuxR-type regulators have a characteristic C-terminal helix-turn-helix DNA-binding region, but form a distinct family separate from other characterized LuxR-type regulators. 

Classic LuxR homologs have an autoinducer-binding domain, while other LuxR-type response regulators have receiver domains typical of two-component systems.However, the NRPS-associated LuxR-type regulators have neither domain, and thus form their own group of regulators. SalA and SyrF are the best characterized of these LuxR-type NRPS regulators; they control and are located in close proximity to the NRPS loci for syringomycin and syringopeptin in P. syringae B301D. They have been shown to dimerize, and that a dimerized SalA binds the promoter region upstream of syrF, while dimerized SyrF binds to the promoter region of syringomycin. Thus, similar to V. fischeri LuxR, they become active after forming a multimeric complex. However, while LuxR must first bind an autoinducer to dimerize and become an activate transcription factor , it is unclear what if any factors contribute to the activation of the NRPS associated LuxR-type regulators. In addition to SalA and SyrF, a third LuxR-type regulator, SyrG, also exhibits partial control over syringomycin synthesis in P. syringae, although it operates independent of SalA and SyrF. Furthermore, P. syringae B728a possesses two additional regulators of this type , which flank the syringafactin biosynthetic cluster on both sides. SyfR, the regulator physically upstream of the cluster, was previously demonstrated to be required for syringafactin production in P. syringae DC3000. However, the LuxR homolog downstream of the syringafactin biosynthetis cluster had no effect on syringafactin production when deleted, and remains unnamed. No further characterization of SyfR has appeared. Although it is clear that these LuxR-type regulators often control lipopeptide synthesis in Pseudomonads, there has been little investigation of how environmental signals feed into this regulation. Some plant signals have been shown to induce lipopeptide production in plant associated Pseudomonads, supporting their proposed roles in virulence. Additionally, lipopeptides are regulated in a manner dependent on quorum sensing and cell density in a few Bacillus and Pseudomonas species. 

We recently found that expression of syringafactin in P. syringae is dependent on contact of cells with surfaces. The current study was undertaken to investigate the role of SyfR in such contact-dependent syringafactin production in strain B728a. We will show that SyfR controls more than syringafactin production,4x8ft rolling benches and is involved in a complex web of cross regulation between other LuxR-type regulators and other lipopeptides in P. syringae. Neither of the truncated SyfR constructs restored syringafactin production in a syfR– mutant. Either SyfR164-257 apparently did not include the correct regions of the DNA-binding domain, or SyfR has a different structural organization than LuxR. We also introduced these truncated SyfR variants into the wild-type strain to test for dominant negative interference which would indicate that SyfR forms multimers similar to SalA and SyrF. While the wild type strain expressing SyfR164-257 retained full syringfactin production, the wild-type strain constitutively expressing SyfR1-198 produced only the same size small surfactant halo as a syfR– mutant strain. Additionally, when we introduced the pPsyfA-gfp reporter fusion into these strains, we observed a similar pattern of GFP fluorescence as production of syringafactin in these strains; overexpression of the SyfR binding domain has a repressive effect on syfA transcription. This supports the hypothesis that this regulator forms a multimeric complex in order to induce syringafactin transcription. We tested the hypothesis that SyfR might be involved in conveying the preferential production of the surfactant syringafactin when cells were cultured on agar plates compared to broth cultures. Initially, we determined if constitutive expression of SyfR is sufficient to induce high levels of syringafactin production in broth culture. We grew the wild-type strain, a syfR– mutant, and a wild-type strain that over-produced SyfR by expressing syfR constitutively on the plasmid p519n–syfR, in both plate and broth conditions, and tested for surfactant production by the drop collapse method. Similar to the oil spray assay depicted in Figure 1, the water drop collapse assay indicated that relatively large quantities of syringafactin were produced in both the wild type and wild-type harboring plasmid p519n-syfR strains on agar plates, while a syfR– mutant was deficient in surfactant production. In contrast, while syringafactin production was low in a wild-type strain when these strains were grown in shaken broth cultures, constitutive expression of SyfR induced sufficient syringafactin production to enable drop collapse under these culture conditions. This suggested that low levels of SyfR might be responsible for the low levels of syringafactin production seen in broth cultures. We thus hypothesized that the surface regulation of syringafactin is at least in part mediated by SyfR.We determined if the apparently low levels of SyfR in broth culture stemmed from low levels of syfR transcription. To test this model we constructed a bioreporter in which a gfp reporter gene was expressed under the control of the promoter of syfR in plasmid pPsyfR-gfp. When a wild type strain carrying pPsyfR-gfp was grown in broth media, apparent syfR transcription was about 3-fold lower than when grown on agar plates. 

As a control, similar levels of GFP fluorescence were observed in a strain constitutively expressing the gfp reporter gene in these two culture conditions. It should be noted that rates of syfA transcription itself were more than 10-fold higher in cells cultured on agar plates compared to broth. We attribute the larger effect of broth culture on syfA expression than on expression of its regulator syfR as a consequence of the strong concentration dependence of oligomerization of SyfR that would be expected to contribute to its activation. We investigated the possibility that syfR is subject to autoregulation in P. syringae since LuxR induces its own expression at least 2- to 3-fold compared to that in luxR– mutant strains. Similarly, constitutive SalA expression results in a 2- to 3-fold upregulation of salA. It is noteworthy that this range of autoregulation is of the same magnitude as the differences in syfR transcription observed between broth and plate cultures. Therefore, we investigated the transcription of syfR in the absence of functional SyfR protein. Surprisingly, we observed equally low GFP fluorescence of a syfR– mutant strain harboring pPsyfR-gfp cultured on both agar plates and in broth media. This finding suggested two important points. First, it suggests that SyfR is autoregulated, and is necessary for the induction of its own transcription above a low baseline level. Second, it suggests that the surface regulation of both syringafactin production and SyfR abundance are conferred by a post-transcriptional process that affects SyfR levels or activity. Thus, we hypothesize that broth culture conditions reduce the magnitude of SyfR autoregulation, either through degradation of the syfR transcript or SyfR itself, or by alteration of SyfR. Further biochemical experimentation will be necessary to determine the mode of this control. If broth conditions foster the hypothesized destruction or modification of SyfR, then we might expect that constitutive production of SyfR would nonetheless result in lower promoter induction of syfR and syfA in broth cultures compared to growth on agar plates. We earlier observed that constitutive expression of SyfR enabled syringafactin production even in broth culture, but we did not examine syfA expression per se. Apparent syfA expression in broth culture, as estimated with the plasmid pPsyfA-gfp introduced into a strain constitutively expressing SyfR, was slightly below that observed on agar plates , which might lend support to our hypothesis. However, the promoter activity of syfR in a strain with constitutive expression of SyfR wasslightly higher in broth cultures than in cells recovered from agar plates. We have no explanation for why syfA expression was lower in broth cultures than on agar plates while syfR was higher. Further biochemical work might help elucidate any additional factors that contribute to syfA regulation. Nonetheless, the observation that constitutive expression of SyfR results in a further up-regulation of syfR further supports our claim that SyfR is autoregulated. A test of the self-sufficiency of the autoinduction process of syfR would be to demonstrate that SyfR is sufficient for syfA induction in another bacterial taxa that might lack ancillary components found only in P. syringae. Introduction of luxR from V. fischeri along with its regulated bioluminescence-encoding operon resulted in expression of of bioluminescence in E. coli. We sequentially transformed E. coli strain DH5α with both p519n-syfR and pPsyfA-gfp.This indicates either that additional transcription factors are necessary for syfA transcription, that processing or some unknown activation of SyfR cannot occur in E. coli, that these components were not efficiency transcribed in this E. coli host, or that SyfR does not directly regulate syfA. Additional investigation to distinguish these possibilities is warranted. We investigated the possibility that SyfR functions downstream from other global regulators in P. syringae. There have been multiple reports that the GacA/GacS two-component regulatory system controls lipopeptide production. In P. syringae, it has been further demonstrated that GacA/S controls lipopeptide production through its regulation of SalA. We hypothesized that Gac might also control syringafactin production, and thus tested surfactant production in a ∆gacS deletion mutant using the atomized oil assay as well as determining the expression of various genes involved in syringafactin production using transcription reporters.

Private industry interviewees were the most keen in believing that price increases promote technology adaption. Interviewees working for environmental organizations were most supportive of pricing increases for conservation. One academic interviewee, however, was cautious about promoting water conservation projects purely on a price basis, believing that the financial argument will always be less effective than a moral argument centered on values. All agreed that the recent introduction of tiered pricing regimes, where water is cheap initially but rises rapidly as usage increases, are a positive development that will lead to increased efficiency and conservation. The 1973 initial completion of the State Water Project largely signified the end of the State’s major north-south surface infrastructure projects. The 1982, voter rejection of the peripheral canal served to hammer the point home. The state has been forced to innovate in order to create “new” water sources for the growing southern coast. The most recent California Water Plan projects that new sources will make up over 20% of regional water supply by 2030 . The state has five major “new resource” initiatives: water markets, groundwater banking, urban conservation, desalination, and recycled wastewater. The first two do not actually create a new source, but rather more effectively manage existing sources. Water markets would encourage trade between users, for example cities and farmers might trade water so that each uses only what is necessary. Today California’s water market now accounts for roughly three percent of the state’s water use,hydroponic channel with the majority of trading occurring in the Central Valley, much of it a result of the Central Valley Project Improvement Act that required the restoration of the bay delta and introduced some water market mechanisms .

Trading markets are perhaps the simplest idea to implement technologically, as they often do not require much in the way of new infrastructure or technologies, however markets would likely require major changes to the way our institutions are run. For example, the aforementioned San Diego County Water Authority water purchase from farmers in the Imperial Valley is an example of this already occurring.The region’s institutional framework was designed to deliver water, not to allocate it efficiently . SDCWA has taken to the airwaves in its bitter legal dispute with MWD over both the price it is charged to use MWD’s infrastructure for transferring it’s purchased water and the price increase for MWD in general, which the significant hurdles that water markets face . The fact that MWD and its member agencies have resorted to producing academic studies that show MWD has subsidized San Diego’s consumption for decades further muddies the waters .Economists have advocated numerous institutional reforms to promote water markets . However, the State legislature has shown little enthusiasm for action. To an objective observer, unfamiliar with the various legal complexities, groundwater banking is clearly the most logical idea. In wet years when water is plentiful, it should go back into the ground to save for dry years. The exact opposite of a rainy day fund. Furthermore, aquifer depletion is a major concern for the regions, with estimates as high as 60% of the groundwater already being depleted, this despite being nearly a third of the regions water supply—up to 40% in dry years . But, as evidenced by the numerous conflicts and lawsuits over groundwater rights and appropriation, groundwater replenishment faces significant legal hurdles. Furthermore, it is estimated that potentially up to one third of regional urban ground water may be polluted and cleaning costs are uncertain . Additionally, as San Diego’s lawsuit shows water pricing concerns and rights to use existing infrastructure for transferring water must be sorted out before groundwater banking can become a widespread reality.

Organizational and institutional reform will likely be necessary for widespread adoption of ground banking. Currently California does not monitor groundwater, nor does it have the legal authority to, despite its critical importance to the water supply . Utilizing ground banking most effectively would require changes to our management structures to align incentives with rights. Better functioning water markets with the right to use transfer infrastructure would also help by ensuring that the water will make it to the appropriate regional aquifers and then harvested as needed for appropriate end use markets. Arguably, conservation has been the most effective “new source” as evidenced by the fact that the region uses roughly the same amount of water today as it did in the early 1990’s . However, much of the low hanging fruit in conservation has already occurred. Water wasteful toilets have been replaced. Wasteful water users have been identified and behaviors corrected. Tiered pricing regimes that punish wasteful users have been implemented. Numerous conservation programs have spread the message about good water practices. Furthermore, the state continues to pass progressive legislation. Significantly the 2007 20X2020 reduction has called for utilities to reduce their water consumption by 20% by 2020 . Many utilities have already met the goal partially through implementing additional conservation and efficiency measures, but also due to the prolonged economic downturn . Further reduction, however, will require technological investments and changes to systems. Today, the state’s attention is turning to landscaping, which accounts for over half of residential water use in Southern California. Programs such as “Cash for Grass” a program which pays residents to remove lawns and install water efficient landscaping .

Desalination, which creates freshwater by removing saline from ocean water is the only real “new source” of the five being pursued. There are approximately 20 desalination plants at various stages of planning in the region, with the Carlsbad plant the furthest in development . Desalination, however, is inherently controversial. Desalination is very energy intensive, and therefore an additional strain to an already burdened electricity grid. An estimated 19.2% of all electricity statewide is already used for moving, treating, or heating of water . Desalination also has major environmental impacts on the coastal areas where it is located. Both the drawing of ocean water,hydroponic dutch buckets and the discharging of huge volumes of brackish wastewater that results from the process, dramatically affect the surrounding sea life and can even impact the local currents . California has several nonprofit organizations actively working against desalination plants, most notably the group Desal Response, who is solely dedicated to preventing desalination plants . It is notable that San Diego is currently considering purchasing water supplies from a desalination plant under consideration in neighboring Tijuana, Mexico—which is outside the jurisdiction of U.S. environmental laws . Much of the innovation that has occurred in the last 30 years has been spurred by a combination of natural and legally-generated water shortages: a prolonged drought, from 1988 to 1994, and a series of environmental rulings requiring water use cutbacks . Among the successes have been dramatic improvements in agricultural and urban water conservation. These have been driven by new metering and monitoring requirements, new plumbing codes, pricing incentives, water markets, and regulatory cutbacks—all of which which have spurred technological and managerial innovation . This trend appears set to continue. Recent court decisions protecting the smelt in the Sacramento/San Joaquin Delta mean that if the region suffers another drought, then water for human use will be curtailed to ensure the smelt’s survival . In the near future, there are several additional pressures that will likely spur investment in the creation of “new water” sources. California suffers from a rapidly aging infrastructure, much of which was built 50 to 100 years ago . In a 2005 national study, the EPA estimated that over $39 billion in investment is needed statewide by 2020 simply to maintain current infrastructure . Additionally, state agencies increasingly recognized that an earthquake in the Central Valley would disrupt the State Water Project and devastate Southern California’s water supply. A disruption would effectively remove one quarter of the region’s water supply—not to mention the harm it would do to Central Valley’s powerful agriculture industry . Finally, there is the wildcard promised by climate change. Many current models predict a drying out of the Sierra Nevada snow pack . If this forecast is even remotely true, then an ever increasing share of water will have to come from “new sources.” All of these existing conditions and possible future scenarios point towards increased water innovation. When one further considers that the legal complications that exist in water markets and groundwater management are unlikely to be changed soon, and that the electricity, environmental, and fiscal constraints that exist on desalination, then it seems most likely that innovation will likely come fastest in the development of efficiency, conservation, and water reuse.

Turning to reuse technologies, innovation will likely come in two principle forms. There will be small scale distributed—non centralized—technologies such as residential grey water systems or small localized water cleaning systems like UCLA Water Center’s smart water systems . The second form will be major new infrastructural projects such as Orange County’s GWRS or West Basin’s Edward C. Little complex . Culturally our water agency institutions are predisposed more towards centralized engineering solutions rather than advocating a cultural shift to conservation or multiple distributed reuse solutions. Public health concerns further promote centralized infrastructure solutions over small scale local solutions where there is more risk of potential problems . California’s legislative priorities, institutional incentives, and water agencies are all slowly moving in the direction of additional water reuse. A question that this thesis presents is: Are these developments occurring fast enough for the local industry to maintain its global lead in these fields? Or will Southern California find itself purchasing goods and services from other regions whose water technology innovation systems are functioning more efficiently.“Water reuse,” “water recycling,” and “water reclamation” are terms that often used interchangeably and all refer to water that has been subjected to advanced treatment in order to use again. In 1995, the California legislature chose to embrace the term water recycling and amended regulations and statutes that read ‘reclaimed water’ and ‘water reclamation’ to read ‘recycled water’ and ‘water recycling’ . It was thought that the public would be more receptive to water recycling. Water reuse in California dates back to at least the eighteenth century when it was common for farmers to use wastewater for crop irrigation due to lack of steady water sources . At the turn of the century, San Francisco’s Golden Gate park used city sewage water for irrigation . As early as 1967, the state legislature directed the state to encourage the development of water reclamation plants . In 1974, the legislature passed the Water Reuse Law which stated: “The primary interest of the people of the state in the conservation of all available water resources requires the maximum reuse of reclaimed water in the satisfaction of requirements for beneficial uses of water” . Only one year later, the Orange County Water District’s water recycling plant Factory 21 became operational and water recycling became a truly viable solution to the states drinking water problem. The Fountain Valley facility, the precursor to the GWRS, treated wastewater and injected it into 23 wells along the coast, creating a seawater intrusion barrier. Factory 21 became the world’s preeminent prototype water reuse treatment facility,and arguably ignited the global industry . As the OCWD likes to say, since its founding in 1975, Factory 21 has consistently met all State and Federal drinking water standards . In 1991, the state further committed its dedication to water reuse through the passage of the Water Recycling Act. There, the Legislature set statewide water recycling goals that a total of 700,000 acre-feet would be reused per year by 2000 and 1,000,000 acre-feet per year by 2010 . Today there are nearly 200 water reuse facilities throughout the state . Over 70% of reused water is used outdoors, with nearly half used for agriculture, and another 20% for landscaping. Despite the success of the Orange County GWRS, groundwater replenishment only makes up 14% of water reuse . Industry makes up another 7%, although both of these are poised to expand in coming years as new projects and planned expansions at The Edward C. Little Water Recycling Facility are completed.

In 2011, Kinrot Ventures was the most active water venture capitalist purchasing four individual companies Ecochemtech, Hydrosping, Kolmir Water Technologies, TA Count . Kinrot’s advisory board is made of leading academicians and industry leaders. In addition to a close relationship with Mekorot, Kinrot has strategic partnerships with water entities all over the world, including the LADWP . Kinrot claims to be the leading seed investor in water and clean-tech related technologies worldwide. AguAgro Fund LP, another Israel Venture Fund, acquired Kinrot Ventures in 2007.The government also provides grants and scholarships for research in water with potential commercial applicability .Perhaps most critical for future industry growth, Israel has at least 10 venture capital funds that together hold over $250 million targeted for investment in water technology firms . VC funds include Israel Cleantech Ventures , Terra Venture Partners, Tene Capital, and the aforementioned AquAgro Fund which holds over $100 million itself. These firms invest in start ups with technologies from throughout the water process. Examples include Emefcy Limited, with a ‘microbial fuel cell’ industrial wastewater treatment system that generates electricity and hydrogen , Blue Water Technologies which is developing an electro-optic technology for water quality analysis, Aqua Digital, with a remote water metering technology, and SmarTap which develops water efficiency technologies for the home .European Union water and wastewater utilities serve over 390 million people across 17 countries with sales over $65 Billion per year .

European multinationals such as Suez Environment , Gundfros , Veoila are among the most important water technology firms in the worlds Although there are many centers of water technology in Europe,flood tray several countries have targeted the water technology industry for economic growth like Denmark or Germany . I have chosen to focus on the Netherlands as the best example of a thriving European water industry innovation system. Despite its small size, 17 million people occupying a mere 16,600 square miles, the Netherlands has an incredibly strong water technology industry with nearly 1,450 companies with annual sales of $12 billion, or 2% of GDP, in 2007 . The Netherlands has several important water research centers. from world famous agricultural centers like Wageningen University, to institutes like Delft University of Technology. The country has a very healthy heterogeneity of firms working in the water industry from high technology startups such as Berghof Membrane, Dutch Rainmaker, Aqua Explorer, Capilix to major corporations such as global food industry conglomerates Nestle and Unilever, oil giant Shell, and electronics manufacturer Phillips . Additionally, the Netherlands has private foundations such as STOWA and Stichting Well, which are dedicated to fostering innovation in the water industry. Notably, the Netherlands is also home to the world’s two largest water and wastewater engineering service design firms, Arcadis and Grontimji and the global headquarters to the aforementioned Israeli infrastructure contracting firm The Tahal Group . The fact that the Netherlands has become a global water hub is perhaps not surprising considering its position at the delta of four major rivers, most importantly the Rhine, a major drainage and navigation waterway of Western Europe. Nearly one quarter of the country is below sea level requiring a massive series of dikes and waterworks to effectively manage its water resources. Furthermore, the country is a global agricultural powerhouse. In addition to strong production of food commodities such as cereals, the Netherlands is the largest flower exporter on the planet, selling over 52% of all flora products in 2006.

This is somewhat surprising given the relatively poor climate. Porter, in an investigation of the tulip industry, showed that this disadvantage actually served as a spur for the technological innovation . The country’s position on the delta gives it incredibly fertile soils, whose productivity is further amplified by heavy use of fertilizers. Today country is the world leader in many water technologies, such as greenhouses and hydroponic agriculture. The Netherlands’s heavy use of fertilizers and pesticides led to high levels of contamination which further encouraged the development of the water treatment expertise. The country’s unique low-lying geography has fostered a strong history of water management cooperation, and corresponding water engineering prowess. Unlike America, where sewage and sanitation are often administered separately from water delivery or waterway navigation, in the Netherlands all three have been closely linked. Today, local regional water boards work closely with the national and municipal bodies to manage water . Additionally, there is a large and vibrant ecosystem of private contractors and consultants with water management specialties. Nationally, water management regulation is being gradually consolidated under the Ministry of Infrastructure and the Environment, a new ministry that has been formed through a merger of Ministry of Transport, Public Works and Water Management with the Ministry of Housing, Spatial Planning and the Environment in 2010 . The link between agriculture and economic development continues to be very important to the Netherlands. In 2010 when the government revised its national governance structure, it also combined the Ministry of Economic Affairs with the Ministry of Agriculture, Nature and Food Quality to form the new Ministry of Economic Affairs, Agriculture and Innovation, a title which further highlights the importance the country has recently placed on innovation as a key to economic development. Under the act,ebb and flow tray water regulation was also drastically simplified. Today the Dutch government issues a single permit for any water activity that impacts the environment.

Previously, several permits were needed for every individual activity, including clean drainage or groundwater extraction—or infiltration—the drainage of polluting substances, etc. . In 2007 the national government, working in collaboration with the European Union’s Regional Development Fund, designated the City of Leeuwarden in Friesland Province as an official European water technology hub . The national government established Wetsus, Center of Excellence for Sustainable Water Technology. In addition to working with the various scales of government, Westus has partnered with over 80 companies and 25 universities and research institutions throughout the world to pool resources and work on water technology solutions . Additionally, private investment such as Dutch Angel investor, Westt, is involved in building the water cluster. It should be noted that the region is on the northern periphery of the Netherlands and many water related firms are still located in the Randstad, the area between The Hague, Rotterdam and Utrecht . However, the initiative is effectively forming partnerships and spurring new knowledge creation. By 2011 Westus had already generated over 33 new patents in water technology, and brought more than 250 PhDs together to produce over 90 scientific papers . Additionally, multiple new technologies have been licensed and 10 new firms have been spun off including Desah , Redstock , Metal Membranes , and AF&F . For a number of reasons, Europe has traditionally lagged behind the United States in innovation. Scholars have found that institutional and cultural barriers have led to imperfect market integration and hence, lower innovation rates . The United States also has higher rates of venture capital than Europe. The Netherlands has some VC firms, but far less than Israel, where per capita VC spending is more than 10 times the EU average . The Netherlands also has an advantage in that the country has traditionally been very internationally oriented. It its drive to develop its water technology industry, it has continued to build on this tradition. In 2006 the water industry formed the independent organization, Netherlands Water Partnership , to promote the Dutch water expertise worldwide. The organization has more than 160 Dutch members. NWP details numerous innovative Dutch firms and technologies and highlights successful international partnerships with Singapore, Israel, China, India, and Egypt. The NWP sponsors trade delegations, hosts events, and maintains a website and directory of the dutch service firms . Every August, the Singapore Water Association sponsors the Singapore International Water Week—the largest water conference in the world. Last year, over 14,000 people from over 112 countries attended the conference and over $2.8 billion in projects were awarded or announced.

Seemingly out of nowhere—SWA was only formed in 2004—the small tropical island city-state of 4 million people, now sets the gold standard for urban water management and is rapidly becoming the leading international center of urban water research and technology. From 2005 to 2015, the government projects that the industry’s employment will nearly double from 6,100 in 2005 to 11,000 by 2015 . Revenue is expected to nearly triple from SGD 619 million in 2005 to SGD 1.7 billion. The city-state has proudly embraced NEW water and built six new water recycling complexes, catapulting home grown firms like Hyflux into global leaders . Singapore, heavily targeting water research, has built two new research centers, Nanyang Environment & Water Research Institute , with five specialized research sub centers, and NUS Environmental Research Institute. NEWRI aims to be the “most comprehensive and integrated environment and water research institute in the world” . Meanwhile, international water technology global behemoths such as Siemens Water Technologies and GE Water and Processing Technologies, as well as global engineering services firms like Black & Vetch and CDM, have all located their global water research and development centers in Singapore . Singapore actively leverages its gateway position on the Pacific Rim and shared cultural connection with China to become the global water technology center. Singapore has very unique circumstances that precipitated the country’s interest in water technology. Singapore gets plenty of rainfall—over 90 inches annually—but its compact size means that there is a very limited amount of land where rainfall can be stored. In fact, domestic sources only cover half of Singapore’s domestic needs and it makes up the difference through imports from the neighboring Johor state of Malaysia. Singapore is entitled to water under long-term agreements signed in 1961 and 1962 when Singapore was still a self-governing British colony. The country of Singapore was formed in 1965 when the predominately ethnic Chinese city state broke away from neighboring Malaysia. During the country’s early years, water rations were briefly imposed . Relations between the two countries have been friendly but not without the occasional minor tension such as a race riot. The current water contracts run out in 2061, and Malaysia has been negotiating to raise prices for several decades. These unique circumstances have set the stage for Singapore to embrace a comprehensive strategy in order to secure a sustainable water supply, including institutional reform, integrated land use planning, strict legislation, public education, and advanced technology. The country’s official policy is known as the Four National Taps Strategy . The first tap is collecting water from the country’s catchment area, which, in addition to the existing reservoirs, has led to an extensive network of storm water collection infrastructure. The second tap is water purchased from Johor, which is piped in to the country. The third tap is the NEW water program, potable water derived from effluent. The fourth tap is desalination. Singapore has invested billions in increasing the first, third, and fourth taps. Developing clean water from sewage is much cheaper than desalination and can be cleaned to suit individual uses. For example, water used for industrial purposes often does not need to be as clean as drinking water, and therefore can use less costly grey water. Singapore’s two industrial water recycling plants provide discounted reused water directly to neighboring industrial districts. By 2006, the country had achieved 12% of its water supply from reused water . Singapore also uses sophisticated demand management tools in order to promote water conservation. Every end user in the country has a water meter with tiered pricing and water tariffs, notably a conservation tax, are designed to encourage the use of NEW or grey water and to promote a conservation mentality. Legislation mandates that water fees cover the costs of all water delivery and most collection—the exception being some sanitation services and storm water management facilities . NEW water is sold at a lower price than new water. In the early 1990s, Singapore reformed its water governance structure bringing all water related government agencies under a new governmental agency, the Ministry of the Environment and Water Resources . MOEWR oversees the Public Utilities Board and the National Environmental Agency .

Compared to the unplanted controls, however, acetaminophen in the growth media with plant disappeared significantly faster . For example, the average concentration of acetaminophen in the medium with plant decreased from 5.0 mg L−1 at the beginning to 3.3 ± 0.3 mg L−1 at 48 h, and further to 2.1 ± 0.4 mg L−1 at the end of cultivation . The significant differences in acetaminophen concentration between the treatments suggested that acetaminophen was taken up from the nutrient solution into the plant.Time-dependent concentrations of acetaminophen in cucumber roots and leaves are depicted in Fig. 1b. During the short growth period, acetaminophen was detected in both roots and leaves. The accumulation of acetaminophen in leaves suggested its acropetal translocation in the cucumber seedlings. However, acetaminophen concentrations in roots were 1.2–5.0 times higher than those in leaves. For example, after 96 h, 1.7 ± 0.1 μmol g−1 and 0.5 ± 0.1 μmol g−1 acetaminophen were detected in roots and leaves, respectively . The relatively lower levels of acetaminophen in leaves pointed to moderate transport to the above ground and likely different transformation rates in these tissues. The level of acetaminophen reached the maxima at 96 h in roots and 48 h in the leaves, and a significant decrease was observed in both tissues thereafter , indicating active bio-transformations in plant cells or release of parent and conjugated compounds from plant roots to the bathing solution.

In plant tissues without exposure to acetaminophen, neither acetaminophen nor GSH conjugates were detected,hydroponic farming indicating absence of cross contamination.In plants, GSH conjugates have been extensively studied because of their role in detoxification and selectivity of herbicides, such assulfonylureas , triazines and chloroacetanilides . In comparison, only sporadic studies have considered GSH conjugation of other contaminants in plants. Detoxification via GSH conjugation was recently reported for the antibiotic chlortetracycline in plant cells . In the present study, the concentration of GSHacetaminophen conjugate in cucumber roots after 48 h was 1.7 ± 0.8 nmol g−1 , and it remained relatively constant thereafter . In comparison, the level of GSH-acetaminophen in leaves continued to increase during the exposure and reached 1.2 ± 0.1 nmol g−1 at the end of experiment . The relative differences between roots and leaves also suggested that roots were the main location for acetaminophen detoxification. This observation was in agreement with a previous study where extensive transformation of acetaminophen was also found in the root hairy cell culture of Armoracia rusticana .In the follow-up experiments, GSH-acetaminophen conjugate was also found in the roots of common bean, alfalfa, tomato, and wheat plants . The ability to carry out GSH conjugation appeared to vary among plant species, with the overall level of GSH-conjugates following an order of tomato, cucumber, alfalfa N wheat N common bean . The differences in the conjugation of acetaminophen by GSH among plant species may be attributed to differences in GSH availability and the portfolio of GST isoenzymes, differences in Phase I reactions, or the involvement of other Phase II metabolism pathways.

It must be noted that the commercial cultivars used as genotype may make a difference to the present results. Meanwhile, in all acetaminophen treatment crops, elevated lipid peroxidation was observed, with the damage being less pronounced in cucumber, tomato and alfalfa tissues . Combining with the levels of GSH conjugates, this observation implied that the different tolerance to acetaminophen among different plant species may be due to their differences in GSH conjugation ability. When cucumber plants were cultivated in nutrient solution containing acetaminophen at the lower level or in soil amended with acetaminophen-containing bio-solids, GSH-acetaminophen conjugate was also detected, even though the levels were generally lower . This observation suggested that direct conjugation of acetaminophen with GSH in cucumber plants occurred under environmentally relevant conditions. In mammalian systems, acetaminophen is oxidized by cytochrome P450 and peroxidases to a reactive quinone-like compound , which can then undergo GSH conjugation by GSTs. In the current study, it was noted that in cucumber leaves and roots, the total activity of cytochrome P450 , but not GSH content or GST activity , was significantly inhibited by 1-aminobenzotriazole or piperonyl butoxide, two specific cytochrome P450 inhibitors. With the pretreatment of 1-aminobenzotriazole or piperonyl butoxide, levels of GSH-acetaminophen decreased from 0.7 ± 0.1 nmol g−1 to 0.2 ± 0.1 or 0.3 ± 0.1 nmol g−1 , and from 16.5 ± 1.4 nmol g−1 to 1.9 ± 0.9 and 2.4 ± 0.6 nmol g−1 in cucumber leaves and roots, respectively . These results clearly suggested that cytochrome P450 played a role prior to GSH-acetaminophen conjugation. However, there was only a slight difference in P450 activities between cucumber and common bean ; whereas cucumber displayed significantly higher GST activity than common bean .

It is therefore likely that different activities of GST also contributed to the different levels of acetaminophen-GSH conjugates. The results clearly showed that GSH-mediated detoxification of acetaminophen in plants differed from the known detoxification reactions of chlorinated herbicides,such as chlorimuron ethyl , alachlor , and metolachlor . For these chlorinated herbicides, conjugation occurs via a nucleophilic substitution of chlorine by the thiol residue of GSH, forming a polar GSH-conjugate. For acetaminophen, however, the potential site of GSH conjugation may involve alkyl groups and result in the ring opening of acetaminophen. A recent study on ibuprofen metabolism in Phragmites australis also suggested that ibuprofen was not directly amenable to Phase II conjugation; rather, it needed to be first functionalized via Phase I activation . These findings together indicated that conjugation with GSH is a common and important detoxification pathway for xenobiotics of different structures in higher plants. For PPCPs such as acetaminophen, Phase I enzymes may be involved to create favorable reactive sites prior to GSH conjugation.Generally, conjugation to GSH occurs in the cytosol,hydroponic equipment but the accumulation of the conjugates in this compartment may also confer toxicological effects . For example, the conjugate may block GST activity , leading to an accumulation of unconjugated electrophiles in plant cells, or they may encounter cytosolic enzymes , which could result in conversion to toxic metabolites. It is generally assumed that the vacuole serves as a site for the deposition of xenobiotic conjugates in plants and fungi .However, little is known about the biological mechanism underpinning GSH conjugates of organic contaminants across tonoplast. Although vacuole was not collected in this study, GSHacetaminophen conjugate, however, was not detected in the xylem sap of cucumber exposed to acetaminophen . Furthermore, GSH-acetaminophen conjugates were also not detected in the above ground tissues of cucumber exposed to GSH-acetaminophen . These results indicated that the parent compound was not metabolized in the xylem or GSH conjugate produced in the roots was not transported to the above ground. However, all GSH related conjugates were detected in the nutrient solution, ranging from 22.7 ± 4.9 nmol L−1 to 44.6 ± 5.2 nmol L−1 after 5 mg L−1 acetaminophen exposure , suggesting release of GSH-related conjugates from plant roots to the bathing solution. A similar phenomenon where plants released conjugated metabolites was previously observed for triclosan , benzotriazole and 2-mercaptobenzothiazole . Additionally, microorganisms in the growth medium could also convert acetaminophen to GSH conjugates, although this possibility was not explored in this study. Glutathione conjugates produced in plant cells may undergo further transformations to yield cysteine conjugates, as previously shown for animals . Most herbicide-GSH conjugates in plants were rapidly metabolized by dipeptidases to remove the glycine residue of GSH to form cysteine conjugates, which could further yield Nacetylcysteine conjugates .

In this study, cysteine and Nacetylcysteine conjugates of acetaminophen were detected in both roots and leaves of cucumber, and the levels in roots were about 3.3 ± 0.2 and 0.07 ± 0.01 nmol g−1 , respectively, after exposure to 5 mg L−1 acetaminophen for 96 h . Glutathione-acetaminophen derived cysteine and Nacetylcysteine conjugates were also detected in roots of common bean, alfalfa, tomato, and wheat plants , with levels ranging from 0.2 ± 0.1 to 4.5 ± 0.5 nmol g−1 and from 0.02 ± 0.01 to 0.3 ± 0.09 nmol g−1 , respectively. The amount of acetaminophen conjugates tended to decrease after 48 h and 96, in leaves and roots, respectively , indicating that some conjugates were converted further or the conjugated metabolites were released back to the medium. The content of acetaminophen-cysteine conjugate in leaves was consistently much smaller than that in roots throughout the experiment. Previous studies showed that the generated cysteine or Nacetylcysteine metabolites were exported to the apoplast, where they may be bound to lignin and cellulose . Several metabolites of cysteine conjugates have been identified in plant cells , although the precise fate and route of metabolism of cysteine conjugates is still uncertain. Further research is needed to understand the efflux of cysteine conjugates from the vacuole.Conjugation of xenobiotics with GSH is mediated by enzymes that belong to the super family of GSTs, which were previously shown to respond quickly to xenobiotics . An evaluation of changes in GST activity was made to understand the mechanism by which acetaminophen was conjugated by GSH. Dynamic changes in GST activity in cucumber tissues during exposure are shown in Fig. 5a. The GST activity increased in both leaves and roots after exposure to acetaminophen . Compared to the blank control, GST activity was consistently elevated at each sampling point, amounting to 1.07–1.94 and 1.30–1.60 times that in the blank control in leaves and roots, respectively. A similar behavior was reported earlier in tomato leaves exposed to the fungicide chlorothalonil. Although levels of PPCPs in wastewater effluents are usually below the levels used in this study , it may be argued that continuous irrigation, active water evapotranspiration, and adsorption, may render the actual concentrations of PPCPs in the soil pore water considerably higher. Results from the bio-solid amendment experiment further supported the conclusion that GSH detoxification may occur for acetaminophen under environmentally relevant conditions.The GSH availability has been shown to correlate with the adaptation or response to xenobiotics in plants , and is an important factor in determining cell sensitivity. For example, GSH availability has been used in understanding herbicide selectivity . Any perturbation leading to a depletion of GSH can severely impair a plant’s defense against xenobiotics. Here, the concentration of GSH in cucumber roots decreased immediately after exposure to 5 mg L−1 acetaminophen, although it gradually recovered to the normal level at 120 h; whereas GSH content only decreased at 72 h in cucumber leaves. Compared to the control, the level of GSH was reduced by 55.4%in the root after 48 h and by 15.2% in the leaves after 72 h . These results were in agreement with the observation that roots were the tissues where more GSH conjugated metabolites were found. However, levels of GSH gradually recovered to a level similar to the control at the end of experiment , possibly due to rapid recycling via GR or synthesis of GSH by γ-ECS in plant cells . In order to explore the roles of GR and γ-ECS in maintaining GSH homeostasis, their activities during the exposure were further monitored. In leaves, activities of GR and γ-ECS were elevated slightly, at about 1.53- and 1.54-fold after 144 h, respectively . In contrast, treatment of acetaminophen significantly elevated γ-ECS activity in roots and showed a time-dependent response, increasing by 2.32-fold after 24 h . A similar pattern was observed in GR activity in roots after exposure to acetaminophen ; while significant increase in GR activity was only observed at 144 h in cucumber leaves . The finding indicated that activities of GR and γ-ECS were elevated to ensure sufficient GSH turnover in response to acetaminophen induced consumption of GSH through GSH conjugation.Based on the results of this study, we propose a model to illustrate the operation of the GST detoxification pathway, which involves conjugation, accumulation and processing of these metabolites . The present study provided strong evidence that transformation of acetaminophen, one of the most used antipyretic and analgesic drugs worldwide, was likely catalyzed first by cytochrome P450, and followed by GSH conjugation mediated by GSTs.

Interestingly, H. seropedicae SmR1 genes related to PHB metabolism appeared to affect fitness by enhancing or decreasing root colonization as shown in Fig. 4. Among the nine selected genes where mutations impaired colonization by H. seropedicae , six mutants—specifically the opuBC, typA, ampD, purD, exbD, and exbB mutants—showed a significant reduction in root colonization either when inoculated separately or coinoculated with the wild type .Applying the transposon mutagenesis sequencing approach to both A. olearius DQS4T and H. seropedicae SmR1 revealed many genes required for these bacteria to competitively colonize Setaria viridis roots. Our experiments identified 89 and 130 genes where mutations significantly affected the ability of A. olearius or H. seropedicae, respectively, to colonize S. viridis roots, including some genes previously reported to play a role in the plant-microbe associations. This result alone argues that root colonization is not a simple process but one that involves a variety of bacterial functions. General gene classes include those involved in cell wall biosynthesis, motility, chemotaxis and defense, and amino acid metabolism . Colonization assays with the 15 candidate genes selected for further confirmation by insertional mutagenesis showed a strong correlation with the results obtained by TnSeq, giving confidence that most if not all of the genes identified are likely important for root colonization by these PGPB strains. Genes where mutations benefited root colonization can be assumed to normally play a role in suppressing colonization in wild-type cells. Among such genes in A. olearius DQS4T is a homolog of the BH72 exaA5 gene ,large growing pots predicted to encode a pyrroloquinoline quinone-dependent alcohol dehydrogenase involved in methanol oxidation.

Consistent with these findings, previous reports showed that mutation of ADH genes inhibited competitive colonization of rice roots by A. olearius BH72 . Similarly, Methylobacterium spp. mutants defective in methanol oxidation were less competitive for colonization of Medicago truncatula roots when coinoculated with wild type cells . These data suggest that methanol metabolism is important for bacterial growth on the root surface and, perhaps more importantly, colonization is a very dynamic and likely heavily  influenced by the overall microbial community. We observed genes that increased fitness scores clustered within an operon presumably involved in iron uptake in Azoarcus. These genes are predicted to encode an outer membrane, ferric coprogen protein FhuE , and a TonB-dependent siderophore receptor , both of which were previously implicated in the ability of this bacterium to colonize roots . Bacterial iron uptake is complex, perhaps involving multiple bacterial processes, and can also be coopted by plant-encoded mechanisms. Mutation of these genes increased fitness values conveying a phenotypic advantage during root colonization, although less competitive than the wild type. Given the general role that iron availability plays in the ability of microorganisms to thrive and compete in virtually any environment, it is not surprising that iron uptake is also a crucial function for root colonization . Recently, analysis of iron content in maize treated with the PGPB A. brasilense revealed a significant increase in total iron accumulation in seeds and higher yield , suggesting that PGPB can contribute to the iron metabolism of the host plant. Previous studies demonstrated an important role for bacterial genes involved in motility for both endophytic and rhizosphere colonization of host roots.

Many genes involved in cell motility were among the common COG categories that appear to provide a fitness advantage for root colonization by SmR1, specifically mutations in genes related to flagellum assembly. We showed previously that an H. seropedicae mutant in the flagellar regulatory gene fliA was unable to endophytically colonize S. viridis roots, although this mutation did not affect rhizosphere colonization . fliA encodes the sigma factor s28 RNA polymerase that mediates the transcription of genes involved in motility and flagellar synthesis .However, bacterial flagella can be recognized by specific receptors in plant cell membranes and activate a cascade of immune responses controlling bacterial infection . Transcriptome analysis of SmR1 attached to wheat roots showed that the flagellar gene cluster was down regulated, suggesting that the bacteria might switch to a twitching type of motility mediated by type IV pili . Under certain environmental stresses or nutritional conditions, bacteria can use different sources of energy for survival, including mobilization of polymers such as polyhydroxyalkanoates . PHB is the PHA produced by bacteria. The PHB granules act as carbon storage that can be mobilized under different conditions. We found that disruption of PHA depolymerase, encoded by the phaZ1 gene, enhanced bacterial colonization when inoculated individually or in competition with the wild type. According to Silveira Alves et al. , plant biomass was significantly reduced in S. viridis colonized by DphaZ1 mutant despite colonizing roots to the same level as the wild-type strain. In contrast, we identified PhaP1 encoding a phasin involved in the PHB production , where deletion of DphaP1 affected fitness negatively, reducing root colonization. Corroborating our findings, an increase in gene expression of the phasin genes was reported during wheat root colonization . Many genes involved in peptidoglycan and lipopolysaccharide biosynthesis were predicted to impair bacterial colonization of plant roots.

For instance, mutation of LPS biosynthetic genes or the addition of exogenous N-acetylglucosamine was previously shown to impair H. seropedicae-maize root association . In studies of rice roots colonized by Azoarcus BH72, mutation of an endoglucanase reduced root colonization, suggesting its importance for successful host cell invasion . We demonstrated that a mutation in a transcriptional regulator involved in aromatic compound degradation completely impaired root colonization during single inoculation. AphS was predicted to regulate genes important for phenol degradation in A. olearius BH72 . Only two genes, cheY involved in chemotaxis and ampD involved in peptidoglycan cell wall recycling ,blueberry planter affected colonization of both strains. Our data suggest that bacterial chemotaxis provides a competitive advantage to wild-type cells during colonization of the plant root tissue. This system is well characterized in several motile bacterial species, such as E. coli and beneficial bacteria such as Azospirillum brasilense, S. meliloti, and others . In summary, our data indicate that, rather than a single or small subset of crucial functions, each strain uses differing functions for colonization, reflecting the unique characteristics of each bacterium. Given that our study was focused on bacterial root colonization, mutations related to soil survival were not considered. However, for Azoarcus a deeper investigation of mutations that affected survival in soil could be useful in explaining the different lifestyle and adaptions of each bacterium to their environment, especially considering that Azoarcus sp. BH72, closely related to A. olearius DQS4T , is a strict endophyte and has not been reported to survive without a host . In summary, similar to most plant-microbe interactions, PGPB-plant interactions are complex and reflect the ability of the plant host and bacterial symbiont to profoundly  influence the metabolism of the other. The fact that PGPB have broad host ranges and can enhance crop yield under field conditions has contributed to a continuing interest in using PGPB inoculants in agriculture. This study provides insights to better understand those gene functions involved in PGPB-host interaction and hopefully will contribute to the further development of PGPB inoculants for an efficient, sustainable agriculture.This document provides best practice guidance and energy efficiency recommendations for the design, construction, and operation of high-performance office buildings in India. Through a discussion of learnings from exemplary projects and inputs from experts, it provides recommendations that can potentially help achieve enhanced working environments, economic construction/faster payback, reduced operating costs, and reduced greenhouse gas emissions. It also provides ambitious energy performance benchmarks, both as adopted targets during building modeling and during measurement and verification . These benchmarks have been derived from a set of representative best-in-class office buildings in India.

The best practices strategies presented in this guide would ideally help in delivering high-performance in terms of a triad—of energy efficiency, cost efficiency, and occupant comfort and well-being. These best practices strategies and metrics should be normalized—that is, corrected to account for building characteristics, diversity of operations, weather, and materials and construction methods. Best practices should start by using early design principles at the whole building level. Optimal energy efficiency can be achieved through an integrated design process , with stakeholder buy-in from the beginning at the conceptual design phase. Early in the project, the focus of the stakeholder group should be on maximizing energy efficiency of the building as a whole, and not just on the efficiency of an individual building component or system. Through multi-disciplinary interactions, the design team should explore synergies between systems such as mutually resonating strategies; or sweet spots between inharmonious strategies. Buildings are the most energy efficient when designers and operators ensure that systems throughout the building are both efficient themselves, and work efficiently together. Systems integration and operational monitoring at the whole building level can help push the envelope for building energy efficiency and performance to unprecedented levels. Whole-building systems integration throughout the building’s design, construction, and operation can assure high performance, both in terms of ensures the energy efficiency and comfort/service levels. A Life cycle Performance Assurance Framework emphasizes the critical integration between the buildings’ physical systems and the building information technologies. The building physical systems include envelope, HVAC, plugs, lighting and comfort technology systems. Whereas, building information technologies provide information on the design and functioning of the building physical systems. This can be done- first, by performing building energy simulation and modeling at the design phase one can estimate the building’s energy performance and code compliance; second, by integrating controls and sensors for communications, one can track real-time performance at the building phase, relative to the original design intent; and third, by conducting monitoring-based commissioning and bench marking during operations, one can ascertain building performance compared to peers and provide feedback loops. The next step should be asesssing best practices at the systems and components level along four intersecting building physical systems- Mechanical Systems for Heating, Ventilation and Air Conditioning , Plug Loads, Lighting and Envelope/Passive systems. The qualitative best practices described in this guide offer opportunities for building designers, owners, and operators to improve energy efficiency in commercial office buildings. Although the practices are presented individually, they should not be thought of as an “a la carte” menu of options. Rather, building systems must be integrated to realize the maximum energy and cost benefits. Also, designers and engineers, and developers and tenants need to work together to capitalize on the synergies between systems. Last but not the least, this guide provides tangible quantitative best performance metrics, ready to be adopted by buildings in India. These metrics are concrete targets for stakeholder groups to work together and enable, by providing localized and customized solutions for each building, class, and occupants. Having targets early on in the design process also translates to more-efficient design lead times. The potential benefits of adopting these metrics include efficient operations, first-cost and life cycle cost efficiencies, and occupant comfort and well-being. The best practice strategies, if used thoughtfully provide an approach towards enabling office buildings that would deliver throughout their entire life cycle, a flexible optimization of energy consumption, productivity, safety, comfort and healthfulness. The adoption of the qualitative and quantitative goals, would provide an impetus to scale up and market transformation toward energy-efficient processes, resources, and products- in addition to generating positive outcomes on global warming and societal benefits. This guide’s primary goal is to provide meaningful information on framing building systems performance and guiding important decisions from conceptual design of a building to its operations and maintenance . It focuses on resource and energy-efficient solutions for high performance conditioned offices , with spillover benefits to a diversity of building types, such as retail, hospitality, hospitals, and multi-storied housing. The secondary goal is to initiate a useful, structured repository of design wisdom that can be continually refined and updated over the years in order to time-test the effectiveness of its recommendations and document them. The authors look forward to integrating more ‘Data Points’ and information from buildings in India especially as the bar for best-in-class high-performance buildings is being continually raised. We intend to refine the best practices and metrics, in newer and revised versions of this guide.

The drop collapse assay was performed as according to Bodour and Miller-Maier. 2 µl 10W-40 Pennzoil® was applied to delimited wells on the lid of a 96-well plate and allowed to equilibrate at room temperature. Next, 5 µl of either diluted surfactant samples or supernatant from bacterial cultures or re-suspended bacterial colonies were pipetted onto the oil surface. Drops which retained a spherical shape were scored as negative for surfactant content, while drops which had a visibly-decreased contact angle with the oil and spread were scored as positive for surfactant content. The atomized oil assay was conducted as follows: Bacteria were evenly spotted onto KB agar plates using sterile toothpicks and grown overnight. Alternatively, if visualizing surfactant from broth culture, 1mL of 2-day-old broth culture was centrifuged at 10,000xg for 2 min, and 5 µl of supernatant was pipetted onto the plate and allowed to equilibrate for 30 minutes before assaying.An airbrush was used to apply a fine mist of mineral oil onto the plate with an air pressure between 15 and 20 psi.A collection of 377 bacterial strains isolated from a variety of terrestrial and aquatic sources were grown on agar plates and tested for biosurfactant production using the atomized oil assay in which an airbrushed mist of oil droplets was applied to culture plates.Additionally, cells of each strain suspended from plates into water as well as drops of broth culture supernatants were tested for drop collapse on an oil surface. A total of 41 of these strains exhibited biosurfactant production in at least one assay. The identities of these strains were determined from partial 16S RNA sequences,draining plant pots and all isolates were assigned to described taxa based on 98% BLAST sequence identity. Pseudomonas and Bacillus species were the most common genera identified, in line with previous reports of limited surveys. 

All biosurfactant producers were members of the Gammaproteobacteria or Firmicutes except for a single Rhizobium species. After eliminating duplicate taxa from the same sampling location, a total of 23 unique environmental strains that produced surfactant detectable in at least one assay were identified and further characterized. All 23 isolates produced surfactant detectable by the atomized oil assay, although only 16 isolates conferred drop collapse of either cells suspended from plates or of broth culture supernatants. Furthermore, cells of only 9 of these 16 isolates conferred drop collapse from both culture conditions. Most of the other 7 strains that conferred drop collapse only under one culture condition did so for suspended plate-grown cells. P. syringae strains were typical of this group; cells of four representative isolates conferred drop collapse when suspended in water from plate cultures but not the supernatant of planktonic cultures. While 16 strains of P. syringae, P. fluorescens, or B. subtilis produced biosurfactant that could be detected by both assays, the 7 strains that exhibited biosurfactant activity that was detectable only by the atomized oil assay mostly consisted of a diversity of other taxa. Although not appreciated in most biological studies, surfactants differ greatly in their chemical properties in ways that could influence their ability to be detected by various assays. For instance, a fundamental property of a surfactant is its relative solubility in water and oil, which can be broadly described by its hydrophilic-lipophilic balance value. Some important synthetic surfactants with low hydrophilicity are not readily dispersible in water, and thus have unique functions such as forming inverse emulsions of water into oil. If a bacterial strain produced a biosurfactant with such low water solubility this could account for its inability to reduce the surface tension of water sufficiently to collapse a water drop.

In order for drop collapse to occur on an oil surface, a minimum surface tension reduction at the water/air interface from 72 dyn/cm to around 43 dyn/cm is required. Although a surfactant may be present in a sample of interest, it might not bedetected by the drop collapse assay if it is produced in low quantities or has a property preventing it from lowering the surface tension of water. Because the atomized oil assay can detect 10- to 100-fold lower concentrations of surfactant than that of the drop collapse assay , it is reasonable to hypothesize that the atomized oil assay can detect surfactant production in weakly producing strains. Therefore, it was possible that the 7 strains that did not confer drop collapse may simply produce too little surfactant to be detected with this method. Indeed many of these strains exhibited small halos in the atomized oil assay , suggestive of low surfactant concentrations. However, a few strains such as Bacillus pumilis that did not cause drop collapse produced biosurfactants that conferred halos of de wetted oil droplets around colonies that were at least as large as many strains whose biosurfactants did confer drop collapse. This observation led us to suspect that the surfactant had properties which hindered its ability to be detected by the drop collapse assay. To address the features of biosurfactants that could be detected by the atomized oil assay but not the drop collapse assay, we distinguished the extent to which the hydrophobicity of the surfactants might limit their detection with the later method or whether the higher sensitivity of the atomized oil assay was responsible for their detection. As a test of the relative hydrophobicity of the surfactant produced by B. pumilis we suspended colonies of it as well as P. syringae strain B728a in water to identical concentrations, removed the cells by centrifugation, and then tested the supernatant for surfactant activity using the atomized oil assay. The water soluble material washed from cells of P. syringae B728a, which contains syringafactin and readily causes drop collapse , contained sufficient surfactant to produce a large halo of de-wetted oil droplets when placed on an agar surface. 

However, very little biosurfactant was apparently washed from cells of B. pumilis, since no zone of de-wetted oil droplets was observed. Similarly, the surfactants produced by Pantoea ananatis and Pseudomonas fluorescens strains which were detected only by the atomized oil assay also appeared to have low water solubility when assayed after washing of cells. However, the washings of four other strains that exhibited the ability to de-wet atomized oil droplets but not to collapse water drops,drainage gutter retained the ability to de-wet oil droplets. This suggests that these strains produced only small amounts of a water-soluble surfactant that could be detected by the drop collapse assay if present in higher concentrations. In support of this conjecture was the observation that these later strains exhibited only relatively small halos in the atomized oil assay. The low production of water soluble surfactants in these strains was verified for P. syringae strain PB54 using mass spectroscopy. This strain was observed to produce the same syringafactins as P. syringae B728a, albeit in much lower quantities, confirming that the detection of surfactants in strain PB54 by the drop collapse assay was compromised by its low level of production. In order to confirm our conjecture that the lack of detection of biosurfactant production in our B. pumilis strain in the drop collapse assay was due to its low water solubility, we characterized it using MALDI mass spectroscopy. The mass spectrogram of the material extracted from the cell free region surrounding colonies on the surface of plates revealed a series of prominent peaks in the range of 1050-1130. Several B. pumilis strains have previously been shown to produce a family of pumilacidins in this mass range. The mass spectrogram of our strain shares the same masses of a sample containing a mixture of pumilacidin A, B, C, and D. The masses observed in Fig. 2 are a combination of [M+Na]+ and the [M+K]+ adducts commonly seen in MALDI mass spectroscopy. Therefore, we conclude that our strain is producing a mixture of low water solubility pumilacidins that are capable of readily diffusing away from cells on the surface of an agar plate, but which are not sufficiently water soluble to impart drop collapse. In order to demonstrate pumilacidin’s surfactant capabilities, the surface tension of a broth culture of B. pumilis was measured using a highly sensitive pendant drop analysis. The surface tension of the broth culture supernatant was lowered by production of a surface active compound to 50 dyn/cm; this surface tension is just above the minimum threshold necessary to impart a drop collapse. Since the highly hydrophobic pumilacidins were detectable using the atomized oil assay, we further determined the efficiency with which other characterized synthetic surfactants differing in chemical properties could be detected by this method.

The assay was performed on synthetic surfactants that possessed a broad range of hydrophobicities. As seen previously, the atomized oil assay readily detected surfactants having more balanced hydrophilic and lipophilic groups, which were also detected by the drop collapse assay. On the other hand, the hydrophobic surfactants Span® 85 and Span® 80 each yielded large bright halos in the atomized oil assay, but given their low water solubility, could not be detected in the aqueous phase by the drop collapse assay. This is in agreement with our observation that hydrophobic pumilacidins were also only detectable by the atomized oil assay and not by the drop collapse assay. Curiously, the synthetic surfactants not only caused bright halos of de-wetted atomized oil droplets, but those with balanced hydrophilic and lipophilic groups also caused the oil droplets to migrate away from the source of surfactant, traveling at a speed of up to 0.1 mm/minute. Such expanding halos may result from a strong surfactant gradient, such as explored by Angelini et al. , although it is unclear why this should not be also conferred by the hydrophobic surfactants. This property was commonly observed around biosurfactant producing bacterial colonies and might be used to infer the water solubility properties of the biosurfactants. In addition to the surfactants that were only revealed by the atomized oil assay, we also found that many surfactants were detectable in the drop collapse assay only when cells had experienced a particular growth condition. Most prominent among strains exhibiting such growth condition-dependent production of surfactants were strains of P. syringae; cultures of this species never conferred water drop collapse when grown planktonically. The factors determining surfactant production in P. syringae pv. syringae B728a, typical of this species, was thus investigated. While culture supernatants of this strain did not cause water drop collapse on an oil surface, plate-grown cells suspended to the same concentration as the planktonic culture conferred water drop collapse. Suspension of a syfA– mutant blocked in production of syringafactin did not cause water drop collapse, confirming that the drop collapse is due to syringafactin. We thus postulated that enhanced expression of syringafactin production in cells grown on a surface was responsible. In order to link syringafactin production to surface-mediated increases in surfactant production, we examined the transcriptional regulation of syfA using a GFP-based bioreporter. Greater than a 10-fold increased expression of syfA was observed when cells were grown on an agar surface compared to planktonic growth in broth culture. As a control, a strain constitutively expressing GFP exhibited similar levels of fluorescence in both cultures. Since there have been reports that production of some surfactants are influenced by growth stage , we examined syfA expression at a variety of times for up to 3 days during the growth of both liquid and solid cultures of P. syringae. GFP expression was higher in cells recovered from agar plates than broth cultures at all times, indicating that this is not growth-stage dependent phenomenon. Additionally, some reports have documented that surfactant production is activated in more dense cultures by quorum sensing. However, the GFP fluorescence of P. syringae harboring the pPsyfA-gfp fusion in the wild-type and a quorum sensing deficient strain was similar both in liquid and solid cultures, indicating that syringafactin production is not dependent on quorum sensing. Although not previously connected to surfactant production, one of the ways by which bacteria sense surfaces is apparently through monitoring the viscosity of their environment. When PVP-360, a viscosifying agent, was added to broth medium, the expression of syfA was increased to levels similar to that of cells on agar plates. 

The mobile phase consisted of ACN and water containing 0.1% trifluoroacetic acid beginning with 65% and increasing to 100% over 11 min at 1 mL/min. A final wash at 65% ACN for 6 min eluted interfering materials. The tR values for HCHO-DNPH and benzaldehyde-DNPH were verified with analytical standards. Formaldehyde dehydrogenase was used to determine if the HCHO produced was bound or free. Microsomal incubations after 1 h were treated with 0.025 units FDH , 40 mM NAD+ and 80 mM reduced glutathione followed by a 20-min incubation at 37°C then analysis for HCHO as above. Some N-methylol metabolites such as diuron or monuron N-methylol can be detected after methylation to form a N-methoxymethyl derivative. Following the Suzuki procedure, TMX, dm-TMX or diuron-microsomal-NADPH incubations were extracted four times with ethyl ether which was then evaporated under N2 at 25°C to 1 mL and 100 µL of methanol and 20 µL of concentrated sulfuric acid were added. After shaking for 1 min at room temperature, reactions were extracted with ice-cold water and the ether layer recovered. The aqueous fraction was further extracted with ethyl ether. The ether fractions were combined, evaporated to dryness and analyzed as in section 2.2.5 for methylated N-methylol intermediates, but none were identified.TMX is a hepatotoxicant and hepatocarcinogen in mice and its metabolite, dm TMX, is also hepatotoxic in mice with its toxicity exacerbated by dm-CLO as an iNOS inhibitor. Importantly, these unfavorable toxicological features are not evident in rats,macetas 20 litros raising the question of whether mice or rats are the better model for humans. 

Comparative metabolism may be a factor since mouse liver microsomes form dm-TMX and dm-CLO much more efficiently than rat or human liver microsomes. TMX was initially categorized as a “likely human carcinogen” based on the mouse model but this was modified to “not likely to be carcinogenic to humans” based on species differences in metabolism. Initial preliminary studies focused on the in vitro inhibition of two isoforms of NOS by dm-CLO. Reducing the formation of nitric oxide via NOS inhibition is known to enhance hepatotoxicity of other toxicants and may explain the hepatotoxicity observed in TMX-treated mice. However, the tentative conclusion is that dm-CLO does not potently inhibit either iNOS or nNOS in vitro. Therefore the focus of further studies turned to analyzing the formation of other reactive metabolites from TMX and dm TMX. TMX is the only one of the seven commercial neonicotinoids to induce hepatotoxicity or hepatocarcinogenicity in mice or rats. The unique structural feature of TMX and its hepatotoxic metabolite dm-TMX is the oxadiazinane moiety, which is a potential source of HCHO and N-methylol metabolites. In this first study of HCHO as a neonicotinoid CYP metabolite we find that of all commercial neonicotinoids TMX and dm-TMX are the most efficient HCHO generators and much more so with mouse than rat or human CYPs. Our results on species differences in HCHO liberation fully agree with the findings of Green et al.on dm-TMX, CLO and dm-CLO formation from the rest of the molecule. The observed species differences in metabolism of TMX or dm-TMX are likely due to substrate specificity and expression differences of various CYP enzymes, with mice having the highest number of CYP genes compared to rats and humans.

The present study therefore confirms the preference for the rat over the mouse model in TMX human risk assessment. The hepatotoxicant/ hepatocarcinogen candidates from TMX and dm-TMX metabolism are HCHO and N-methylol intermediates. HCHO is a known human carcinogen. To test if free HCHO was formed, FDH was added to TMX- mouse microsomal incubation reactions. Based on FDH-induced HCHO loss, most of the HCHO formed by CYPs from TMX was free, but the remaining HCHO could be protein bound or released from N-methylol intermediates during analysis. A similar result was obtained for HCHO liberated from the NCH2OCH3 moiety of alachlor under comparable conditions. Attempts to detect N-methylol metabolites in the liver of TMX-treated mice were unsuccessful in vivoas well as in vitropossibly due to instability on formation and analysis. The same applies to in vivo detection of HCHO which is very bioreactive. Although not detailed here, white blood cells from the same groups of TMX-treated and control mice were analyzed for DNA-protein crosslinks induced by HCHO production from TMX using the method of Zhitkovich and Costa. These attempts were unsuccessful with the assay method having low sensitivity. Many compounds including pesticides proposed or established to be carcinogens yield N-methylol metabolites some of which may contribute to their toxicity. The inability to detect N-methylol metabolites from alachlor or HMPA in our studies agree with previous literature indicating the difficulty of their analysis due to high reactivity and short half-lives. The candidate N-methylol intermediates from TMX and dm-TMX were therefore synthesized by reaction of CLO and dm-CLO with HCHO and HCO2H. Although individual intermediates were not isolated and characterized, the CLO and dm-CLO reactions with HCHO in HCO2H appear to give two mono-N-methylols and three further addition compounds with m/z equivalent to the addition of two methylols per molecule as the reaction proceeds ultimately ending up as TMX and dm-TMX.

The proposed mono-N methylols from synthesis were found to be stable not only to extraction and LC/MS but also to an additional incubation with mouse liver microsomes. This creates an apparent contradiction wherein the synthetic N-methylols are adequately stable for study but the proposed CYP-formed N-methylols are not observed. This anomaly is rationalized by the different mechanisms and environment of N-methylol formation in the chemical synthesis and enzymatic CYP systems. Perhaps the N-methylols as they are enzymatically formed further react in the CYP active site containing ferrous iron,maceta 40 litros cysteine thiol and imidazole N-H. Cannabis sativa has a long history of cultivation for a variety of uses including food, fibre, medicine, and recreational drugs. Cannabis produces many different secondary compounds such as cannabinoids, flavonoids, stilbenoids, alkaloids, lignanamides, and phenolic amides. D9 -Tetrahydrocan nabinolic acid , a product of the cannabinoid class, is the primary psychoactive agent. This compound is produced as an acid in the glandular trichomes of in- flflorescence bracts and undergoes decarboxylation with age or heating to D9 -tetrahydrocannabinol. Cannabis cultivars differ substantially in economic traits that range from marijuana, arguably the most widespread illicit drug, to hemp fibre derived from the stems of the plant. Marijuana consists of the dried female inflorescences in which the quantity of THC exceeds that of cannabidiol, and potency varies among cultivars by several orders of magnitude. Marijuana cultivars are known to have THC levels exceeding 2–24% of inflorescence dry weight whereas hemp cultivars produce substantially less THC but rather high levels of CBD. THCA and CBDA share the same bio-synthetic pathway except for the last step in which THCA synthase and CBDA synthase produce THCA or CBDA, respectively. 

Recent evidence suggests that the genes encoding the two synthases are allelic. CBD and THC are enatiomers, but only THC elicits psychotropic effects, whereas CBD may mediate anti-psychotropic effects , a difference highlighting the stereo-selectivity of receptors in the human body that bind these compounds. Although classified as a drug without therapeutic value in the United States, ingestion of THC is widely regarded as having effects including pain relief and appetite stimulation, that may, among other things, increase the tolerance of cancer patients to chemotherapy. Dronabinol, a synthetic analogue of THC, is approved for use as an appetite stimulant in the United States as a Schedule III drug. Cesamet , another synthetic analogue, is used as an anti-emetic for patients undergoing cancer therapy. The natural product Sativex is approved for use in the UK and is derived from Cannabis cultivars containing both THC and CBD, and is used to treat pain symptoms associated with multiple sclerosis. Compounds from Cannabis sativa are of undeniable medical interest, and subtle differences in the chemical nature of these compounds can greatly influence their pharmacolog ical properties. For these reasons, a better understanding of the secondary metabolic pathways that lead to the synthesis of bio-active natural products in Cannabis is needed. Knowledge of genetics underlying cannabinoid bio synthesis is also needed to engineer drug-free and distinctive Cannabis varieties capable of supplying hemp fibre and oil seed. In this report, RNA from mature glands isolated from the bracts of female inflorescences was converted into cDNA and cloned to produce a cDNA library. DNA from over 2000 clones has been sequenced and characterized. Candidate genes for almost all of the enzymes required to convert primary metabolites into THCA have been identified. Expression levels of many of the candidate genes for the pathways were compared between isolated glands and intact inflorescence leaves.Seeds from the marijuana cultivar Skunk no. 1 were pro vided by HortaPharm BV and imported under a US Drug Enforcement Administration permit to a registered controlled substance research facility. Plants were grown under hydroponic conditions in a secure growth chamber yielding cannabinoid levels in mature plants as reported in Datwyler and Weiblen. Approximately 5 g of tissue was harvested from mature female inflorescences 8 weeks after the onset of flowering. Tissue was equally distributed into four 50 ml tubes containing 20 ml phosphate buffered saline as de scribed by Sambrook et al. , but made with all potassium salts and mixed at maximum speed with a Vortex 2 Genie for four repetitions of 30 s mixing followed by 30 s rest on ice, for a total of 2 min of mixing. Material was sieved through four layers of 131 mm plastic mesh and the flow-through was split into two 50 ml tubes and spun in a centrifuge for 30 s at 500 rpm. Supernatants were decanted and pellets were resuspended in PBS. The suspensions were combined into one tube and pelleted as before. The resulting pellet was diluted into 100 ll of PBS. Five ll were used for cell counting with a haemocytometer, and the total suspension was estimated to contain 70 000 intact glands. Plant residue was incinerated by a DEA-registered reverse distributor. Quantitative reactions were performed as described previously using primers listed in Supplementary Table 4B at JXB online. Equivalent quantities of RNA isolated from glands and inflorescence associated leaves were used to generate the respective single stranded cDNAs. qPCR reactions containing equal quantities of gland or leaf cDNA were run in duplicate along with reactions containing standards consisting of 100-fold se quential dilutions of isolated target fragments, on a Light cyler qPCR machine. Lightcycler software was used to generate standard curves covering a range of 106 to which gland and leaf data were compared. Two biological replicates were used to generate the means and standard deviations shown in Supplementary Table 4A at JXB online. These values were used to compute the gland over leaf ratios and P-values shown in Supplementary Table 4A at JXB online. Raw relative expression data, means, standard deviations, P-values from gland versus leaf t tests, qPCR primer sequences, and representative real-time qPCR tracings are shown in Supplementary Table 4A at JXB online.Anatomical study revealed that glands located on mature floral bracts of female plants are the site of enhanced secondary metabolism leading to the production of THCA and other compounds in Cannabis sativa. These glands are located on multicellular stalks and typically are composed of eight cells.The capsule contains exudates derived from the gland cells. The weakly attached glands can easily be separated from the bracts and purified as shown in Fig. 1E and F. An EST library was constructed using RNA isolated from purified glands. Over 100 000 ESTs were cloned. Plasmid DNA was isolated and sequenced from over 2000 clones. Because of the directed orientation of cDNA insertion, sequences are expected to represent the coding strand. After the removal of vector only, poor quality sequences, and sequences obviously originating from organelles or ribosomal RNA, the remain ing sequences were clustered into 1075 unigenes. Overall, 111 of the unigenes were contigs containing two or more closely related ESTs. Only 14 contigs lacked a similar sequence in the NCBI database. Nine hundred and sixty four of the ESTs were only found once and of these 710 were similar to sequences in the NCBI database.