Author: naturehydro

It is interesting to note that the region is actually quite weak in environmental and conservation employment, despite the presence of strong water policy nonprofits. This is largely due to the majority of the State’s environmental organizations being headquartered in the Bay area, or in the state capital, Sacramento. These organizations, such as the aforementioned influential Pacific Institute, are very active in Southern California water policy. The Pacific Institute is a global water policy research center producing reports an all matters of water and issuing a yearly report, The World’s Water, which comprehensively documents the state of the world’s water. Dr. Peter Glieck is an international water policy celebrity who lectures throughout the world. Despite having less environmental organizations then the San Francisco bay, Southern California does have some very important environmental organizations. For example, Heal the Bay and Surfrider have been extremely successful in educating the public and government agencies about storm water pollution into the Ocean. Southern California does not receive very much rain, a little over 17 inches a year, but thanks to the extensive flood control infrastructure—most obviously the concrete drainage tunnel that was formally the Los Angeles River—the majority of rain it does receive is quickly flushed into the ocean—usually within 24 hours . In addition to education and pushing the policy debate, organizations like Heal the Bay, Tree people, Surfrider, The Council for Watershed Health are helping to bring about so-called soft path approaches to integrated water management. Surfrider for example, runs a very successful ocean friendly garden landscaping program which promotes on-site water storage, . Tree people has been at the forefront in developing studies—including a pioneering study on the value of urban trees —and promoting low impact development in the Los Angeles region. The Council for Watershed Health not only conducts research but also builds model low impact development urban designs that promote water conservation.

These organizations are working to build support for local integrated water management techniques but they have a long way to go. These organizations also work actively with firms and industrial networks in order to serve to further promote knowledge diffusion. All nonprofit organizations by definition do not generate profits,flood table or if they do the must reinvest their profits into operations. These entities are organized under the IRS 501 tax code and pay less, or in the case of 5013 organizations, pay zero Federal taxes However, 5013 organizations are extremely limited in the activities that they undergo. First, they can not make a profit that benefits share holders or a private entity. Second, they are limited in their ability to influence legislation may not participate in any campaign activity for or against political candidates . However, in practice, the ban against political campaigning is very narrowly defined, and nonprofits are free to dedicate themselves to working on behalf of a cause which may have political ramifications. It is very notable that in contrast to these case studies, there are very few organizations dedicated to building the water industry for economic reasons. There is no “Californian water sector” like there is a “Dutch water sector” nor is there a true equivalent to the Singapore Water Association or a Milwaukee Water Council. In fact, on the Sectary of State’s web page listing California’s trade associations, not one mentions water . The important networking and knowledge diffusion functions are often filled by existing industry trade associations, various water agencies—most importantly MWD, LADWP, and OCWD—or universities such as the ones highlighted in Chapter 4.5, Knowledge Creation. Despite numerous entities marketing the region’s water expertise, there are not very many independent organizations focused on trumpeting the regional water industry as a jobs creation tool. Although to be fair, California is one of most well known brands in the world.

Thanks to Hollywood’s movies, much of the world might have an inkling that we have water problems—anybody who has watched one of the numerous car chases filmed in the Los Angeles River could probably venture a good guess. Still, the lack of a regional economic water industry message is somewhat surprising, and even the general regional trade associations like Foreign Trade Association of Southern California or the Los Angeles County Economic Development Corporation —who operates the Los Angeles Long Beach World Trade Center—do not loudly advertise the region’s water industry expertise. In fact, on the LACEDC website, one can’t even search for “water” . A notable exception to this general observation is the partnership of California water companies and public agencies who formed the non-profit, CalDesal, to lobby for the passing of the Carlsbad Desalination Plant which would become the nation’s largest desalination plant. The project was stalled in planning for over a decade before being finally approved in 2009 . Construction industry unions, particularly the plumbing union are also very active in promoting local water construction projects. For example, the plumbers union recently helped sponsor the Los Angeles County Economic Round Table’s water industry job analysis and participates in public campaigns on behalf of the water industry . They, along with other construction unions, have been very active in promoting the State’s various water bond measures. Table 9 highlights some of these groups. These organizations produce reports, pass industry information among members and likely hold thousands of events per year. In short, they are at the forefront of knowledge diffusion. These groups do participate in the promotion of the local industry. It is only that the region appears to lack a coordinated regional economic strategy oriented around its water expertise. This differs from the examples profiled in the case studies. These organizations are invaluable in diffusing knowledge. Additionally, California also has a large number of specialized journalist organizations such as the San Diego based, Environmental Business Journal, which is focused on environmental industries. The state is also home to numerous clean technology organizations such as the aforementioned Bay Area Next Ten or the Clean Tech Group, although these organizations tend to be clustered in Norther California. There are also many other active business development nonprofits in California like the aforementioned Imagine H20.

Imagine H20 holds annual technology competitions and teaches inventors how to commercialize. Although, it should be noted that there are relatively few of these organizations, and they are not specifically regional in focus. For example, Imagine H20‘s competitions are open to national and even international entrants .Although this would agree with our observations,rolling benches it would be surprising if it were true, given that others have not noted this function before. Alternatively, glycosylation has been proposed to function in flagellar stabilization and lubrication in P. syringae pv. tabaci, where non-glycosylated flagella formed stiff flagellar bundles. If lack of glycosylation makes the flagella more sticky and prone to breakage, then non-glycosylated mutants might still have functional flagella, but these flagella might break more easily, requiring an enhanced supply of fresh flagellin and/or a lubricating surfactant. Given the co-regulation of BRF with class IV flagellar genes, it was tempting to speculate that FliA, the sigma factor that activates transcription of Class IV genes, might also be directly responsible for regulating brfA expression. However, a disruption of fliA did not abolish surfactant production, and all Class III mutants still produce at least small quantities of the surfactant, indicating that flagellar regulation of BRF production occurs at multiple levels. It remains to be determined exactly how flagella are acting to affect surfactant production. It is also curious that flagella have less of a role in regulating surfactant production in broth conditions, where the surfactant is relatively highly produced. If the function of this surfactant is to lubricate the flagella at surfaces, then why would P. syringae produce such high quantities in broth culture? Although we do not have any evidence of a role for this surfactant in broth cultures, some clues about the surfactant’s properties can lead us to hypothesize possible functions. When large quantities of this surfactant are produced in broth culture by constitutively expressing BrfA we see that this surfactant imparts a milky appearance to the culture supernatants. This is indicative of a surfactant with low water solubility, which most likely associates with surfaces such as the bacterial cell surface, instead of the bulk medium. Therefore, when this surfactant is produced, it likely coats the cells and changes their surface properties. The role of this surfactant in aqueous environments and its effects on cell surfaces and the adhesiveness of cells need to be addressed. As a counter example, syringafactin, a water-soluble surfactant which readily diffuses away from P. syringae, is down-regulated in broth cultures. Thus BRF might best be considered a surface-associating surfactant that modulates the surface properties of either the producing bacterium or the surfaces over which the bacterium must move. Another important clue for the function of BRF was the finding that multiple stress pathways apparently strongly impact its production.

However, where it has been most studied, high osmolarity environments repress motility, and an OmpR knockout is associated with increased flagellar synthesis, increased motility, and also increased production of virulence factors. Such findings are opposite to our observed loss of BRF production in an OmpR mutant of P. syringae. Alternatively, the OmpR homolog in P. aeruginosa, termed AmgR, has been described to function more like the protein conferring membrane stress response, CpxR in E. coli. An examination of the AmgR regulon in P. aeruginosa revealed that it had much less in common with that of E. coli OmpR regulon than that mediated by CpxR, which has been coined a surface sensor. Anecdotally, we have observed that a mutant in the ompR homolog in P. syringae grows well on fresh agar media but exhibits impaired growth on relatively old plates with dried surfaces. In contrast, our mutant screen also revealed the role of two members of the AlgT extracellular stress pathway, both of which when knocked out resulted in an up-regulation of BRF production. The AlgT stress pathway controls the production of alginate in response to membrane stress , and was recently found to similarly influence syringafactin production, with loss of the pathway resulting in up-regulated syringafactin synthesis. It remains unclear why these potentially overlapping stress responses have apparently opposite effects on production of BRF. Further examination of their roles in surfactant production should help elucidate the complex interaction between these two pathways. It might turn out that the combination of these two pathways allows the cell to determine the difference between subtly different stressful situations, only some of which would benefit from surfactant production. It is significant that BRF is produced by an RhlA homolog, which is responsible for the biosynthesis of the rhamnolipid precursor HAA in P. aeruginosa. In P. aeruginosa, HAAs serve to repel neighboring tendrils and maintain an outward motility during swarming. Such a behavior would tend to maximize the ability of a bacterial colony to explore a given habitat by suppressing inward movement, and thus enhancing only outward movement away from colonized areas, and surfactin in B. subtilis has been similarly indicated to have this role. It appears that BRF shares this ability, but it remains to be determined if the swarm repulsion observation has a true physiological function, or is just a laboratory phenomenon that is merely a result of a fundamental physical property of the surfactant. While a syringafactin mutant of P. syringae DC3000 did not apparently make any surfactant and was incapable of swarming motility , we find that such a mutant in strain B728a produces BRF. An examination of the DC3000 genome reveals a close homolog to brfA, but one having a stop codon at the 13th amino acid, apparently accounting for the lack of its production in strain DC3000. P. syringae DC3000 is a poor epiphyte, with low rates of survival on the leaf surface ; it is intriguing to speculate that BRF is not made in DC3000 because it is primarily useful for epiphytic colonization of plants, or alternatively it might be detrimental and/or induce a host response in the apoplast. Restoration of BRF production in P. syringae strain DC3000 should reveal if it can change its virulence or epiphytic fitness. In this study we have utilized an atomized oil assay to identify the biosynthetic and regulatory pathways leading to production of a biosurfactant expressed in a strongly context-dependent way in P. syringae B728a.

The engineers of the Orange County Water District clearly demonstrate this with their willingness to investigate water reuse technologies as a solution to groundwater problems. Hence, the presence of a conservative technocratic civil service of water engineers is potentially a plus for the future adoption of a water reuse infrastructural projects.Institutions are the sets of common habits, routines, practices, rules, or laws that regulate the relations between individuals, networks, and organizations . It is difficult to write comprehensively of institutions as so many of the rules that underlie our conduct are often unwritten or simply established practices or cultural norms that generally go without saying. But the most important institutions are the legal institutions, the laws or ordinances that define the agents of our society and determine the rules of our markets. These laws define the actors—who is a firm, a non-profit organization, a special government agency, an inventor—and identify what rights actors have and how they can legally interact.The Federal and State governments use legal ordinances and tax codes to classify organizations according to legal status. A for-profit corporation has different rights and responsibilities than firms that are organized as partnerships, S-Corporations, sole proprietor ships, or nonprofits. Corporations have limited liability, so employees may lose their jobs and shareholders may lose their investment,nft channel but neither will be liable for the corporation’s debts. Such an organization has long been thought to encourage entrepreneurship and risk-taking . Nonprofits can file under the Federal tax code’s 501 status, granting them tax benefits which can allow these organizations to pursue appropriate goals without the additional burden of potentially onerous taxes. However, these rules also restrict some freedom of action and limit political activity .

Federal and State governments also arbitrate important legal instruments such as patents or financial tools like stocks and bonds. Our system of patent protection grants inventors a right to exclude others from making, using, selling, offering for sale, or a whole manner of other activities of their patented product or process for twenty years . This gives the patent holder a monopoly ensuring they have ample opportunity to profit from their invention. Financial instruments, such as the tax exempt municipal bonds discussed above, are another legal instrument that have a profound impact on the working of our economy. Generations of legal philosophers have composed libraries debating the theories of our various legal tools, and I have no interest in attempting to wade into such ground here. Here I will turn briefly to water rights—and particularly ground water rights. These legal instruments have a direct bearing on our study of Southern California’s burgeoning water reuse industry. In fact, they were central to Orange County’s groundwater reuse project, and will be critical for the potential growth of additional projects. Since the adoption of the 1928 reasonable use use doctrine, California water is protected for the use and benefit of all Californians . Water cannot be outright owned by individuals, groups, businesses, or governmental agencies. The state issues permits, licenses, and registrations to give individuals or organizations the right to use “reasonable amounts of water” . Like our collective legal system, water rights in the United States largely evolved from the English Common Law system. However, California has a long tradition of water litigation which has led to a “California Doctrine” that differs significantly from prevailing US norms . The California doctrine recognizes rights as valid if they are based on one of three factors: riparian rights—ownership of land near watercourse; appropriative rights—prior use of water, and groundwater rights—title of water rights underneath land. English common law traditionally valued riparian rights and granted land holders near absolute groundwater rights.

Appropriative rights privilege early water users over later users and became a common practice as settlers moved into arid American west to maintain peace. They are without basis in traditional English Common Law . California further broke with the English Common Law tradition by finding that groundwater was not solely the property of a land owner. Instead, California has a complex system based on both the rights of the landowner and a correlative rights tradition. Correlative rights are similar to riparian rights in that they are based on proximity to water, or in the case of groundwater, physical contiguity.Although Statewide there are only 22 adjudicated water basins, meaning that the rights have been settled and every individual or organization with a claim can use water but only in a sustainable manner, most of these are in urban areas . Adjudication can limit the freedom that a managing entity has over groundwater basins, often requiring additional negotiations. Sometimes these negotiations turn into difficult court cases . This is even further complicated if the groundwater basin is polluted, such as Los Angeles’s heavily industrialized San Fernando Valley where legal mandates have been handed down requiring that the various water rights holders must clean up the basin . Adjudicated basin agreements were primarily designed to control the allocation of water supply, not replenishment or the removal of contaminants, so this is still relatively unchartered legal territory . Overall, Southern California is rich in groundwater potential, with a total estimated available capacity in MWD’s service area of 3.2 million acre-feet. , however these reserves have been regularly over drafted . California law allows the creation of special districts to manage or replenish water sources. Today several districts, most notably the Water Replenishment District of Southern California , have plans to purchase excess MWD water and inject it into groundwater basins . However, there are complex legal issues involved in groundwater replenishment, particularly in adjudicated basins. WRD’s plan to inject 450,000 AF over two wet years when MWD water was plentiful was blocked in 2009 by a lawsuit from Central Basin Municipal Water District and the cities of Downey, Signal Hill, Cerritos, and the Tesoro Oil Company who protested the plan . In May 2012, the State Supreme court voted in favor of WRD. In 2012 water prices have risen, reflecting the drier weather of 2011,hydroponic nft which will make the groundwater injection plan much more costly. Additionally, untold millions in legal fees have been squandered on such inter agency lawsuits over groundwater.

Furthermore, attempts by the State to adopt new groundwater regulations have been strongly opposed by land owners, particularly agricultural interests and local water districts who are fearful that new rules will limit freedom of water use . In short, regional groundwater management is at best, inefficient, and at worst dysfunctional. Efforts to introduce truly comprehensive water management solutions, such as low impact development ordinances that mandate onsite storm water management that promote groundwater replenishment through natural processes of soil percolation are further hindered by additional institutional complexity. Cities control planning and land use, yet the state transportation department oversees the roads, while various public and private entities control land. It is very unlikely that California will ever see anything as far reaching as Netherland’s or Singapore’s recent reforms that created single ministries responsible for comprehensive water management. The complex legal structure that underlies groundwater rights in California has already had a major impact on the water reuse industry, and continues to in three primary ways. First, the Orange County Water District would not exist without being set up to protect the groundwater basin, and therefore never would have built Factory 21. Water districts with groundwater basins that are over-drafted, under threat of seawater, or are contaminated have an incentive to use the cheapest water that they can to replenish their supplies, therefore they may turn to water reuse as a source. Secondly water rights are one of the largest determinants of water price, which is the largest determent of private investment in innovative technologies . How an individual or a utility obtains its water supply is a huge factor in the price it pays for that water. For example, if a utility has ample high-quality groundwater sources available, its rates will be lower than if the utility needed to purchase or import its water supply . Conversely, if a utility does not have groundwater and needs to import its water supply, the cost of that water will almost always be higher. In Southern California, a water district or company with easily accessible groundwater has the freedom to choose its sources; it can purchase imported water from MWD when prices are low and pump when prices are high; these districts are unlikely to give up this low cost advantage. The legal complexity over adjudicated ground basin might persuade some districts to turn to water reuse. It is fairly clear that OCWD’s groundbreaking GWRS project would not have been undertaken without the alignment of groundwater water rights, storage capabilities, and clear compelling need. In my investigation I found that GRWS managers were certain that their complete control of the aquifer was critical to moving forward with the project .

Additional lawyers of complexity would have added costs and risks which might have imperiled the project . Market demand is the primary motivation behind new products or technologies, and the water industry is no different. If sufficient demand exists to justify investment, investment will often be made. In a market economy, if the cost is high then firms will seek out innovations—and it is hoped—be rewarded with market share. Specialized demand conditions often distinguish a regional industry and meeting that demand will often impart an advantage to the region. Water is not a product that is manufactured, nor is it strictly speaking, a commodity that is dug from the ground, but rather it is more a hybrid product that is both a renewable resource in the manner of agriculture that needs to be collected and transported, but also a product that can be derived from a recycling process. Furthermore, the water economy is not truly a market economy, for water is a public good that is critical to the functioning of our region. Water will often be delivered regardless of costs, and benefits greatly from implicit public subsidies. Water delivery is also a natural monopoly as the majority of its costs are infrastructural, the cost of physically placing pipe to carry water is very high and thought to account for up to 80% of the price of water , which is a significant barrier for potential competition. But like other markets, the primary driver behind innovation in the water technology industry is the cost and/or availability of water. If supply is limited and price is high, then investments will be made in technologies and/or process innovations. The four principle factors in the Southern California region are the availability of groundwater, weather , infrastructural capabilities, and legal constraints . The first three are fairly straightforward; rain needs to fall, the water needs to be accessible and the infrastructure must exist to collect it, clean, and deliver it. Legal constraints include considerations of legal water rights—who has claim to the water—as well as government mandates such as the Owen’s Valley court decision that limited water withdrawals for environmental causes, or the conservation mandates imposed during droughts. In practice, the diverse circumstances faced by the myriad of Southern Californian water agencies—regional wholesalers, distributors, local retailers, reclamation districts, replenishment districts, and private water companies—results in equally diverse pricing schemes making it difficult to identify the true cost of water in Southern California, and hence potential savings from investment in efficiency or technology improvements. Industry analysts have repeatedly cited the lack of transparency in water pricing, and particularly, the hidden subsidies, as impairing potential investment in water technologies . In my case studies, both Singapore and Australia made identifying the true cost of water a central platform in their respective water reforms to both encourage conservation and efficient allocation . Few Californians know how much it really costs to deliver water. All Interviewees working in the water industry agreed that California pricing schemes were confusing, and nearly all expressed a desire for prices to rise in order to increase investment . One notable exception worked for a public agency that delivered water. They agreed that truer pricing regimes would increase investment but also pointed out that cheap water was in keeping with their agencies public mission .

Japanese technology conglomerates such as Mitsubushi, Toray, Kutuba, and Asahsi dominate many of the most advanced water treatment processes and therefore would merit further study. Among wastewater treatment suppliers, Madrid, Spain, also has a striking concentration of firms , and like Israel, Singapore, and Australia, Spain also recently embarked on major water market reforms . French and British Multinationals such as Veolia and United Water dominate the global privatized municipal urban water delivery services market , a curiosity that would also invite further inquiry.Two locations, Israel and Singapore, consistently top the water industry literature as having the most technologically developed industries, a fact which was further verified through several of my industry interviews. The Netherlands is home to the world’s largest and most important water services engineering firms, as well as hundreds of technology companies and several globally prominent research centers . In comparison, Australia has a much smaller impact on the water technology industry. It is also more than a region being a large country, the only one to encompass an entire continent, although 85% of the population lives in the temperate south eastern coastal region. I chose to include it for three principle reasons. First, Australia shares many similarities with Southern California. Both are largely arid climates. Both share Anglo Saxon legal and business traditions, as well as strong industrial histories based on natural resource extraction, and both are now heavily urbanized. Like southern California, Australia has recently witnessed major growth in water technological innovation—through industrial investment in water reuse technologies ,ebb flow table infrastructural investment in desalination and reuse plants and huge growth in domestic water technology patent production . In 1994, Australia began a series of major reforms to its water industry, removing water subsidies and separating regulating utilities from service delivery firms.

Since that time, the country has witnessed an increase in water productivity of more than 50 percent. In contrast to southern California, however, Australia has also embraced an economic growth strategy centered on the exportation of goods and services. Finally, I also felt it important to explore domestic centers of water technology. Milwaukee, Wisconsin has embarked on a strategy to become a global center of water technologies led primarily by the private sector. In 2008, the US Federal government targeted the Tri-State region around Cincinnati for the development of a water innovation system focused on water cleanliness. During the course of my research, I discovered that California’s own San Joaquin Valley has actively pursued a water technology investment cluster strategy targeting water flow technologies and I would be remiss if I did not include a discussion of the efforts to build this water technology innovation system into an economic growth engine. I found that many of the irrigation firms active in San Joaquin Valley are also active in Southern California. It is important to point out that the following summaries are only a cursory exploration of the water technology innovation systems active in each of the case studies. They are not meant to be comprehensive, but simple summaries of the key components and economic strategies of the respective water innovation systems. renowned in irrigation, water treatment, and flow control technologies. The country currently boasts over 70 emerging water technology start up firms . The water sector generates over $1.1B in xports, but is targeted to generate more growth in the future . The Israeli government also has a strong history of strategic economic development, targeting research and development and setting out business incubation. Since 1994, Israel also has had an active economic clustering program known as MAGNET—the Hebrew acronym for Generic Pre-Competitive Technologies and R&D . Today the country has 41 active economic clusters, including a water technology cluster centered in Ashekelon . The occasion of Minister Ben Eliezer’s speech was the unveiling of a new national government initiative, Novel Efficient Water Technologies program, or simply “NEWTech” . NEWtech is designed to increase coordination between government agencies, academic institutions and private firms in order to increase water technology innovation and exportation.

The program calls for the water technology industry to become a main growth engine of the Israeli economy . This is laid out explicitly in the government mandate “New Governmental Decision Number 157: Development of Water Technology in Israel” which calls on the country to recognize “the latent potential that water technology could have on the Israeli economy; and on the importance and centrality that government and private companies have in this market, in Israel and in the exporting industry.” The declaration continues by calling for the government to “examine the methods and the means that different governmental offices can use to support the development of water technology, from the R&D stage to the manufacturing and exporting stages impacting irrigation practices around the world” . Israeli water economy boosters like to point to the biblical story of Moses drawing water from the rock to show the historical importance of water to Israel , and it certainly has been important to the modern state. The early Zionists that left central Europe for the arid British protectorate came to build a community of Jewish agriculture communes, Kibbutzim . In Palestine, traditionally water was considered a right for all residents ; however, in contrast to central Europe, the region did not have abundant water supplies. Furthermore, native residents were often fearful of the new Zionists and hostilities occasionally broke out. During the post World War II British decolonization period, this enmity escalated into a full-scale regional war when the State of Israel was declared in 1948. The young state prevailed and then expanded again after another regional war in 1967. Today, the ongoing Israeli/Palestinian dispute continues to define the Middle East, leaving Jewish Israel largely isolated among its Muslim neighbors. Unable to cooperate with neighbors to secure additional water supplies, Israel has had to rely entirely on its own territory for sufficient water for agriculture, a fact that likely contributes to Israel’s ongoing military occupation of the internationally recognized Syrian territory of the Golan Heights—the source of the country’s largest river, the Jordan—and continuing occupation of the Palestinian territories,hydroponic grow table where large groundwater aquifers exist . Additionally, Israel is blockaded from importing oil from Muslim producers, which has put a significant damper on the country’s energy capabilities and likely unleashed a frantic search for alternatives.

In 1937, over a decade before the country itself was formed, the settlers formed the predecessor to today’s state water company, Mekorot Water Company, in order to manage water supplies. Today Mekorot provides Israel with 90% of its drinking water and 80% of total water . In the 1950’s the country passed a series of water management laws requiring: metering , drainage and flood control , permits for it’s use —all of which consolidated the public’s ownership of water and the government’s role in management . In the 1970’s, Israel embraced water demand management as a technique to further promote efficient use of water, and to promote the use of water and energy saving technologies. Appropriate pricing regimes were implemented to discourage waste. Having central government control over water and water infrastructure made this an easy policy to implement. At the same time, the country’s water research institutions continued technological invention, improving drip irrigation, advancing techniques such as reverse osmosis, and improving filtration methods critical to desalination and water reuse . In 1997 Israel built the world’s first Reverse Osmosis plant that was designed to treat both seawater and discharged brine, which it received from an adjacent desalination facility at Elilat . In 2001 Israel sought international help in order to scale up and build the world’s largest RO desalination plant. The country contracted with VID, a special purpose joint-venture company made up of two Israeli firms, IDE Technologies and DanknerEllern Infrastructure, and French water services multinational Veolia , previously Vivendi, to design, build, and operate the Ashkelon desalination plant . IDE Technologies is itself a partnership of Israel Chemicals and the Delek Group. After 25 years the plant will be transferred to Israel. In addition to the required facilities for seawater pumping, brine removal, water treatment, and membrane desalination units, the complex project required laboratory buildings for ongoing testing purposes. Today, Ashkelon generates nearly 6% of Israel’s water supply, most importantly its utilization of advanced RO technologies and economies of scale have given it the cheapest water in the world. It is one of several plants that have been planned for the coast . The international partnership with Veolia further embeds Israeli firms into the advanced water technology scientific and business networks, enhancing knowledge creation and diffusion and increasing market opportunities. In addition to Veolia, Israel has business relationships with global water technology firms Siemens, GE, ABB, Dow Chemicals, and Jain Irrigation Systems. Israel also has formed strategic partnerships with public water entities throughout the world with governments and municipalities in China, Singapore, Beijing, Madrid, Melbourne, Sao Paulo, Buenos Aires, and several others . They are also in close contact with California entities such as the LADWP and the OCWD . Outside of exceptional infrastructural projects like the Ashkelon plant, the state water firm Mekorot owns the majority of the water infrastructure, over 3,000 water works that encompass all phases of the water process, most notably wastewater management . Throughout much of its history, Mekorot partnered with The Tahal Group, an engineering services firm, to build the water infrastructure. Today The Tahal Group is an international firm headquartered in the Netherlands. Prior to the mid 1990‘s when it was privatized, Tahal was an Israeli military contracting and engineering firm specializing in infrastructure . This type of partnership between the state, military, and private firms is common in Israel . It continues today with Mekorot often partnering with local firms to build infrastructure and test technologies. Many of these firms have either been spun off from the military or are closely involved with military projects. The close collaboration between the state, the military, and technology firms, has long been a central feature of the Israeli technology economy. Dan Senor and Saul Singer recently wrote the best seller book Start-up Nation that attempts to explain how Israel has thrived in the high technology economy . They argue that the entrepreneurial culture of market savvy independent thinkers bound by close connections wrought through shared military service, and honed by the military’s appetite for advanced research technology, has created the conditions for the explosion in high technology companies. Israel has over 70 companies listed on the US NASDEQ market—the most of any non US country . Additionally Israeli scientists have been at the forefront of a long list of technological innovations, such as CT scanners, advanced Intel microprocessors, AOL’s instant messenger, zip compression technology, the aforementioned drip irrigation, and many more . Israel also greatly benefits from the vast knowledge and international connections of the global Jewish network, most notably in the United States where Jews are well represented in science, business, academics, law, media, technology, and professional fields. Israel is central to Jewish identity and many American Jews make great efforts to ensure its safety and economic vitality through tying the country tightly into our global economic system. The close connection between the military and entrepreneurs has also been deliberately fostered through an economic development strategy of national technical business incubators as well as the creation of a venture capital industry. The government effectively jump started the venture capital industry in the early 1990’s with the creation of Yozma program to invest in high tech ventures . The country also targeted “clean technologies”, such as water technologies and the renewable energy industry. This is partially due to its limited resources, but also due to the oil embargo implemented by oil rich Muslim nations.For example, Kinrot Ventures was founded in 1993 as part of the Israeli Incubator Program as the world’s first clean tech water technology incubator . Today Kinrot Ventures is one of the most important water venture capital firms in the world. Kinrot targets emerging companies with potentially disruptive water technologies, with a specific focus on advances in purifying, filtering, desalination, transporting, and storing water.

A common pathway in most organisms for detoxifying xenobiotics is through glutathione conjugation catalyzed by glutathione Stransferases . It has long been recognized that GSTs play an important role in normal cellular metabolism in plants . In addition, GST mRNA responds quickly to exposure of xenobiotics , protecting the cell against oxidative stress or chemical toxicity . Previous research on xenobiotics such as herbicides shows that differences in GSH availability and in the portfolio of GST isoenzymes are associated with xenobiotic resistance . Conjugation with various biomolecules was observed in plant metabolism of different PPCPs, including diclofenac , ibuprofen , triclosan , benzotriazole and acetaminophen . However, GST-mediated conjugation may play a predominant role, but has been relatively understudied so far . In particular, the entire process of GST mediated detoxification, from GSH conjugation in the cytoplasm to vacuolar accumulation and processing of conjugates has yet to be elucidated in vivo. Acetaminophen and structural analogs are phenolic compounds that are among ubiquitous environmental contaminants . Acetaminophen is one of the most used pain and fever relief medicines, and many billions of doses of acetaminophen are consumed each year . Acetaminophen was detected in the aquatic environment at 0.01–0.3 mg L−1 in the South Wales region of the UK .Cucumber is a crop plant, widely consumed, with high economic and ecological relevance,hydroponic grow system and recommended for use in phytotoxicity studies by U.S. Environmental Protection Agency . In this study, we used acetaminophen to elucidate the mechanisms of GST-mediated detoxification in cucumber plants.

Findings of this study may provide evidence for PPCP detoxification via GSH conjugation, and will likely prompt further exploratory investigation on the role and value of this pathway for the numerous emerging contaminants.Cucumber seeds were obtained from Fisher and transferred to a 72-hole plate filled with a mixture of vermiculite and perlite . Plants were cultivated under controlled conditions for 1 week and irrigated with half strength modified Hoagland nutrient solution, which is adequate for the seedlings’ energy demands for cucumber growth and the low ionic strength nutrient solution permits a more precise computation of acetaminophen speciation than do more complete nutrient medium. Details on the solution composition are given in Table S2. Seedlings in the third-leaf stage were removed from the pots, rinsed with tap water, and placed in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution. Each treatment contained three replicates, and each replicate was prepared from a single plant by separating into roots and shoots.After 7 days of adaption, plants were exposed to acetaminophen by spiking 25 μL stock solution to yield an initial concentration of 5.0 mg L−1 .Simultaneously, treatment blanks and plant blanks were included in duplicate to determine abiotic losses of acetaminophen under the experimental conditions. All experimental materials were auto-claved, 121 °C for 30 min, before use. A parallel group trial enrolled with acetaminophen at 60 μg L−1 was included to simulate more realistic levels and to validate the high level treatment. The cultivation lasted for 20 days, with the nutrient solution renewed every other day . Additionally, in order to determine if plants can detoxify acetaminophen via GSH conjugation in realistic field environment, cucumber seedlings were cultivated for 20 days in soil amended with 5% bio-solids containing acetaminophen at an initial concentration of 10 mg kg−1 .

A root exudate control was included to evaluate the effect of plant exudates on microbial degradation and GSH conjugation of acetaminophen in the hydroponic solution. Cucumber seedlings were cultivated in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution, and the plants were removed after 7 days. The solution was brought back to 500 mL with half strength modified Hoagland nutrient solution, and then 25 μL of acetaminophen stock solution was added to yield an initial concentration of 5.0 mg L−1 . The nutrient solution samples were exposed to the same conditions, and were analyzed for acetaminophen and its conjugates. To investigate whether GSH-acetaminophen can be transported from the root to the shoot, two-week-old cucumber plants were exposed to 1 mg L−1 GSH-acetaminophen for 2 days. Leaves were collected and extracts from the aerial parts were analyzed as described below. To test whether cytochrome P450 was involved in GSH-conjugates formation, two P450 inhibitors, 1-aminobenzotriazole and piperonyl butoxide, were used to pretreat the plant on the basis of previous studies demonstrated specific inhibition effects . Cucumber seedlings were pretreated with 1- aminobenzotriazole or piperonyl butoxide for 12 h in 500 mL glass jars with aerated, half strength modified Hoagland nutrient solution, followed by spiking 25 μL of acetaminophen stock solution to yield an initial concentration of 5.0 mg L−1 . After 96 h incubation, tissue samples were collected from both the treated and untreated plants.The specificity of these P450 inhibitors was evaluated by monitoring cytochrome P450 activity, GSH content and GST activity in cucumber tissues after the treatment. For better clarity, all the experiments performed was schematically represented Table S3. Simultaneously, commercially available common bean , tomato , alfalfa and wheat seeds were germinated and transplanted under the same conditions as above. After 7 days of adaption, plants were exposed to acetaminophen at 5.0 mg L−1 . After 96 h, plant roots were collected and analyzed for acetaminophen and its glutathione, cysteine and N-acetylcysteine conjugates.

Sampling, sample preparation and analysis Cucumber seedlings treated with or without 5 mg L−1 acetaminophen were sacrificed at 0, 12, 24, 48, 72, 96, 120 and 144 h. Before sample preparation, roots were rinsed thoroughly with tap water. Harvested tissues were separated into roots and shoots and then frozen in liquid nitrogen. Enzyme activities of GST, glutathione reductase , γ-glutamylcysteine synthetase , and cytochrome P450, as well as contents of reduced glutathione in both roots and shoots, and lipid peroxidation were immediately analyzed after sampling, as described below. The remaining plant samples were freeze-dried and stored at −80 °C until chemical analysis. At each sampling time, nutrient solution samples and the aqueous root exudate samples were collected and mixed with equal volumes of methanol. The mixture was added with d4-acetaminophen as a surrogate, filtered through a PTFE syringe filter and stored at −20 °C before analysis. To quantify acetaminophen and its glutathione,indoor garden cysteine and Nacetylcysteine conjugates, the freeze-dried plant tissue samples were ground to a fine powder with a mortar and pestle, and a 0.5-g aliquot was used for solvent extraction. Each sample was spiked with d4- acetaminophen as the recovery surrogate, and extracted, sequentially, with 20 mL methyl tert-butyl ether , acetonitrile, and 0.5 mM HCl solution in an ultrasonic water bath for 20 min for each extraction. The supernatant from MTBE and acetonitrile extraction was combined after centrifugation and reconstituted in 1.0 mL methanol after drying under nitrogen . The extract was pooled with the supernatant from the HCl extraction. The pooled extract was loaded onto an Oasis™ HLB cartridge that was preconditioned with 6 mL methanol and 12 mL deionized water, and eluted with 15 mL methanol under gravity. The eluate was evaporated to dryness under N2, and the residue was recovered in 1.5 mL methanol:water mixture and filtered before analysis. The instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography coupled to a Waters Micromass Triple Quadrupole mass spectrometer equipped with an electrospray ionization interface . Acetaminophen, and its glutathione, cysteine and N-acetylcysteine conjugates were separated using ACQUITY UPLC BEH C18 column with water and methanol as mobile phases. Electrospray ionization was operated in the positive mode. Detailed information on the instrumental analysis is given in Table S4.Fresh cucumber roots were frozen in liquid nitrogen, and homogenized on ice with 5 mL of 50 mM potassium phosphate buffer containing 1 mM EDTA and 1% polyvinylpyrrolidone. After centrifugation at 15,000 g at 4 °C for 20 min, the supernatant was used for the assay of activities of glutathione S-transferase, glutathione reductase, and cytochrome P450 . Measurement of GST activity was carried out spectrophotometrically after the glutathione and 1-chloro-2,4-dinitrobenzene adduct synthesis reaction . Briefly, GST activity was measured in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM CDNB, 5 mM GSH and 100 μL enzyme extract. The increase in absorbance as a result of GSH-CDNB synthesis was determined at 340 nm for 5 min on a Cary 50 UV– Visible spectrophotometer . The GST activity was calculated using the extinction coefficient of GSH-CDNB .

To determine the activity of GR , a 200 μL aliquot of enzyme extract was added to the reaction mixture of 0.15 mM NADPH and 0.5 mM oxidized glutathione in 50 mM PBS . The absorbance decrease was monitored at 340 nm for 3 min after GSSG-dependent consumption of NADPH . For the assay of γ-ECS , plant tissues were homogenized and suspended in 5 mL of 0.1 M HCl. After centrifugation at 20,000 g for10 min at 4 °C, the supernatant was collected. The γ-ECS activity was measured according to Shan and Liang . An incubation solution was prepared with 800 μL 50 mM TrisHCl containing 0.25 mM glutamate, 10 mM ATP, 1 mM dithioerythritol, 2 mM cysteine, and 400 μL of the γ-ECS sample. The mixed solution was incubated for 1 h at 25 °C, and then 800 μL phosphorus agent was added and mixed. After incubation at 45 °C for 25 min, the absorbance at 660 nm was measured on the UV–Visible spectrophotometer. One unit of γ-ECS activity was defined as 1 mmol cysteine-dependently generated PO4 3− per minute. The levels of proteins in the enzyme extracts were determined using Coomassie Brilliant Blue G-250 . Brieflfly, 100 μL of enzyme extract was added to 5 mL of 0.01% Coomassie Brilliant Blue G- 250. After 5 min, the absorbance at 595 nm was measured on the UV– Visible spectrophotometer. The protein content of enzyme extracts was calculated by comparison with a standard curve using bovine serum albumin as the standard. The protein concentrations in the extracts were estimated using the BSA standard curve. The content of GSH was determined spectrophotometrically according to Jiang et al. . Plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice and centrifuged at 12,000 g at 4 °C for 15 min, and the supernatant was analyzed for GSH. To 0.5 mL of supernatant, 0.5 mL PBS and 0.5 mL of 5′5′-dithiobis-2- nitrobenzonic acid were added. After thorough mixing and incubation for 5 min, the absorbance at 412 nm was measured . Membrane lipid peroxidation, a typical indicator of stress phytotoxicity , was estimated by measuring the concentration of malondialdehyde, according to the reaction with thiobarbituric acid as described in Sun et al. .All treatments were performed in triplicates. A procedural blank and a sample duplicate were included in every batch of 10 samples to monitor background contamination and reproducibility, and the calculated relative standard deviations were b10%. The d4-Acetaminophen was used to account for any loss during sample preparation, matrixinduced ionization effects, and variations in instrumental response. Authentic standards were used to confirm the target analytes. A six-point calibration line was used for quantification with the r 2 values of at least 0.99 for all analytes . No acetaminophen or any of the conjugates was detected in the solvent or treatment blanks. Limits of detection and quantification for individual acetaminophen and the conjugates were calculated as 3 and 10 times the signal-to noise level from the low-level spiked samples . The recoveries in all samples were within acceptable limits ranging from 75 to 110%, demonstrating good method accuracy and precision. Extraction efficiency was N91% for acetaminophen and its conjugates. The plant position was regularly rearranged every 2 days to avoid side-effects.The level of acetaminophen in the nutrient solution without plant, after the removal of plant , or with cucumber plant, all decreased over time . Significant losses occurred in the unplanted control or the root exudate control . Considering that the cultivation system was not a sterile environment, both microbial and abiotic degradation may have contributed to the apparent acetaminophen dissipation in the solutions. This observation was consistent with Bartha et al. , who also reported the occurrence of plant-independent, biotic processes that decreased the available acetaminophen in plant solution.

Plant health and development are influenced by a broad range of microorganisms inhabiting the root rhizosphere, including beneficial associative bacteria. These bacteria are usually minor components of the rhizosphere microbial community but have been shown to significantly enhance plant growth and yield . This ability to enhance plant growth has been attributed to diverse mechanisms, including biological nitrogen fixation, production of phytohormones, enhancement of nutrient uptake , and bio-control of pathogens and pests . Plant growth-promoting bacteria can colonize roots either on the root surface or as endophytes without eliciting a noticeable plant defense response. Unlike some other bacterium-plant interactions that show strict host specificity, many PGPB can colonize a wide variety of plant species, including agriculturally important members of the Poaceae family, such as rice, maize, wheat, and a variety of bio-energy grasses . PGPB strains have been described for several host plants, including Setaria viridis, a model C4 plant, which is a close relative to a variety of bio-energy grasses. In a previous study, we demonstrated that S. viridis under lab conditions can obtain up to 100% of its nitrogen needs through biological nitrogen fixation mediated by diazotrophic PGPB . Among the strains used were the betaproteobacteria Azoarcus olearius and Herbaspirillum seropedicae, which exhibit strong growth-promoting ability . Azoarcus olearius DQS4T was originally isolated from oil-contaminated soil and, based on its genome sequence, this strain shows high similarity with the well-studied A. olearius strain BH72 . Strain DQS4T can colonize the roots of rice and Setaria to high levels and increase below- and above-ground biomass . H. seropedicae SmR1 is a well-studied endophytic bacterium that colonizes several plants, including maize, wheat, and Setaria. Recently,container size for strawberries in situ metabolomic profiling of S. viridis roots colonized by SmR1 demonstrated that inoculation induced a wide variety of plant metabolic changes, including those affecting nitrogen and phytohormone levels .

Thus, similar to many other, better-studied plant-microbe associations, PGPB inoculation appears to profoundly affect the metabolism of its host. To identify specific PGPB genes that contribute to plant root colonization, we applied transposon mutagenesis sequencing coupled with random barcoding. This technique assesses gene functionality in mutant strains through the generation of reusable libraries of unique and mapped mutant insertions.TnSeq is a powerful and sensitive approach to identify bacterial gene functions that play roles in bacterial fitness under researcher-defined growth conditions . Starting with information on the location and frequency of each mutation in the population, the change in mutational frequency after passage of the population through an environmental challenge leads to the identification of genes that are either essential, important, or detrimental to growth under that condition. For example, TnSeq of Streptococcus pneumoniae identified genes essential for bacterial basal growth, as well as genes involved in transcriptional regulation and carbohydrate transport . In the PGPB Pseudomonas simiae, TnSeq revealed genes involved in carbon metabolism and motility that could enhance or suppress colonization of Arabidopsis roots . In the present work, we applied standard TnSeq and a variant of TnSeq that uses random DNA barcodes to measure strain abundance to construct mutant libraries of strains DQS4T and SmR1, respectively, and to then evaluate genetic contributions to fitness on the host plant Setaria viridis.Gene essentiality is defined as its importance in maintaining competitive fitness under a condition of interest . For this study, two PGPB bacterial species were used, Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1, both diazotrophic and capable of promoting plant growth . The mutagenized pool of each strain was inoculated onto Setaria viridis roots or a carbon augmented soil control with no plants.

Root-associated bacterial cells were recovered, transposon DNA was sequenced, and resulting reads were mapped to each bacterial genome. The relative incidence of a specific mutation was then determined and used to calculate fitness values. By high throughput sequence analysis of insertion mutants, we covered about 3,692 genes of DQS4T and 3,878 genes of SmR1 distributed throughout either strain’s genome. Thus, regardless of method, very good coverage of each genome was achieved. In the case of DQS4T transposon mutagenesis, 89.2% of 4,135 total genes were mapped to a gene harboring at least 1 insertion event. For the SmR1 library, 81.1% of the 4781 genes had identified mutations. We assume that genes without an insertion in the library likely identify those essential for bacterial growth in culture. Our screening identified 89 and 130 gene mutations that significantly affected the ability of strains DQS4T and SmR1 to colonize S. viridis roots, respectively. Given that our interest was to identify genes that exclusively affected root colonization, we excluded from further consideration any genes that also affected fitness under soil conditions . The genes affecting root colonization were categorized based on the phenotypes as either enhanced fitness value, mutations that increased root association ; or decreased fitness value, mutations that impaired root association . For the complete list of gene mutations affecting fitness under each condition, see Table S2. Unsurprisingly, many of the genes identified lacked a clear functional annotation, especially for A. olearius DQS4T . Even though each strain showed a distinct pattern of mutations that affected fitness, a few common gene functions were identified impairing root colonization in both strains. The most common COG categories important for SmR1 root colonization were those involved in amino acid transport and metabolism, followed by energy production and conversion and coenzyme transport and metabolism. In the case of DQS4Tthe most common COGs were cell wall/membrane/envelope bio-genesis and signal transduction, followed by translation, ribosomal structure and bio-genesis, and inorganic ion transport and metabolism .

Although we cannot rule out that the different mutagenesis methods could contribute to the different fitness profiles, the overall mutational coverage of the genomes of both strains suggest that such effects are likely minor. Hence, the results seem to suggest that any given PGPB strain will have different major requirements for root colonization reflecting the specific metabolic needs of the bacterium.Gene mutations that positively impacted the ability of bacterial strains to colonize roots were defined as those with a fitness score $ 1. We identified 8 and 14 genes in this category in A. olearius and H. seropedicae, respectively . This list included the Azoarcus gene predicted to encode a pyrroloquinoline quinone -dependent alcohol dehydrogenase , which is involved in the oxidoreductase process of ADH. Based on sequence alignment with Azoarcus olearius BH72,container size for blueberries the DQS_RS19730 gene encodes an ExaA5 protein that was previously reported to be induced upon exposure to ethanol as a carbon source . In our study, we found that exaA5 plays an important role in root colonization. Disruption of this gene enhanced root colonization of S. viridis roots . However, the mutant was less competitive in root colonization than the wild type at 1 and 3 days after inoculation but seemed to recover to wild-type levels by 5 days after inoculation . An insertion mutation in the gene encoding a diguanylate cyclase significantly increased bacterial root colonization 3 and 5 days postinoculation and showed the same trend in competition with the wild type . Diguanylate cyclases with a GGDEF activesite motif produce cyclic di-GMP and play a major role in the transition between motile and sessile bacterial lifestyles . We also identified an iron regulator outer membrane protein, TonB , presumably required for Fe31uptake . This protein was reported to interact with ExbA and ExbB, forming a complex that regulates iron acquisition . In SmR1, 6 of the 14 gene mutations that increased root colonization are predicted to play a role in flagellar biosynthesis. FliF, FliG, FlhB, and FliQ are structural components of the flagellar basal body . MotA is complexed with MotB and functions as a proton channel for torque generation . Our testing showed that the absence of flagellar genes in the SmR1 strain conveyed an advantage for root colonization in S. viridis 10 days after inoculation. We also identified a poly-3-hydroxybutyrate -related gene, a poly-3-hydroxyalkanoate depolymerase PhaZ1 , that enhanced colonization ability when mutated . PhaZ1 is a PHB depolymerization enzyme required for the granule mobilization . Furthermore, mutations in the genes encoding transcriptional regulators and ABC transporters also resulted in increased root colonization by strain SmR1 .Mutant strains corresponding to 81 DQS4T and 114 SmR1 genes were identified as significantly reducing root colonization . Therefore, these gene functions are normally required for efficient colonization by wild-type bacteria. Within this group of mutations, we identified two genes present in both DQS4T and SmR1: cheY , which is related to chemotaxis, and ampD , which is involved in peptidogly can degradation. Chemotaxis is directly involved in modulating the movement of the flagellum in response to attractants, helping bacterial orientation and active motion for survival toward favorable conditions .

In addition, four other genes encoding transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins , were identified in H. seropedicae. The protein CheA undergoes autophosphorylation induced by MCPs, and CheR and CheW acts to modulate the phosphorylation state of CheY . Mutations in these genes impaired the capacity of H. seropedicae to colonize roots, consistent with a previous report .Analysis of the DQS4T TnSeq data identified a gene predicted to encode a two-component sensor histidine kinase containing a HAMP domain involved in signal conversion between the transmembrane-sensing and kinase activity control . One assumes that this histidine kinase is reacting to an unknown factor in the rhizosphere that normally enhances root association in wild-type cells. We also observed genes involved in cell wall formation, such as murI , a glutamate racemase responsible for the conversion of L-glutamate in D-glutamate, and murA , a UDP-N-acetylglucosamine 1-carboxyvinyltransferase responsible for converting UDP-N-acetylglucosamine to UDP-N-acetylglucosamine enolpyruvate. A previous study of H. seropedicae reported the importance of the mur genes for maize root association and also showed that their expression was repressed in the presence of naringenin. In addition, a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD, likely involved in the degradation of peptidoglycan by hydrolysis of muropeptides that can serve as signals for induction of b-lactamase , was identified as important for root colonization in both strains. We also identified a putative transcriptional regulator of catechol 2,3-dioxygenase, a flavonoidrelated compound that might be involved in the degradation of aromatic compounds important for plant-bacterial interactions . Among such compounds are flavonoids that can serve as chemoattractants for rhizobia in legumes . Flavonoids can also mediate PGPB colonization, as reported for H. seropedicae colonization of Arabidopsis and the colonization of rice by Serratia spp. . Interestingly, mutations in genes involved in PHB metabolism—including PhaP1 , a phasin family protein that affects PHB production, and PhbA1 , an acetyl-CoA acetyltransferase— decreased root colonization. The importance of PHB metabolism in bacteria during plant root colonization was reported previously in studies of Setaria-Herbaspirillum and legumerhizobium symbiosis .To more definitively test the importance of individual genes identified in our screen, we selected candidate genes and created insertion mutations in 15 separate genes. These genes were selected to cover a diversity of putative functions representing operons containing multiple genes, as well as single genes, including those associated with both positive and negative fitness scores . Using a gnotobiotic system, a competitive colonization assay was performed in which colonization of S. viridis roots by the individual mutants was measured, as well as their ability to compete for colonization when coinoculated with the corresponding wild-type strain. Since root colonization is a dynamic process and might change over time, we evaluated competition at three different time points. For A. olearius DQS4T , we selected six genes where mutations reduced colonization, specifically a transcriptional regulator, ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU . Mutants defective in genes that resulted in reduced colonization in competition with the wild-type strain showed delayed colonization of Setaria roots. Mutation of the transcriptional regulator gene represents the most extreme case where colonization was completely blocked when inoculated singly onto Setaria roots . This gene encodes an AphS protein known to be involved in the phenol degradation pathway, where phenol is converted into catechol via catechol 2,3-dioxygenase . Analysis of A. olearius DQS4T and BH72 genomes showed two that is produced by the wild type that can compensate for its loss in the mutant. As for the other mutants that impaired A. olearius DQS4T colonization ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU, all showed a significant reduction in colonization that was most pronounced 3 and 5 days after inoculation both when inoculated individually or in competition with the wild-type strain .

Although we have arbitrarily classified surfactants as either causing either a “bright” or “dark” halo in oil drops surrounding a surfactant source, it is most probable that there is a spectrum of contact angles for the oil droplet that is dictated by the expected range of change of the various tensions by various surfactants as discussed above. Similarly, while all of our obtained mutants displayed bright halos, there is a possibility that the contact angles of the oil droplets could be slightly different, especially near mutants unable to produce syringafactin. However, we have not yet found a reliable method for measuring the contact angles of the atomized oil droplets observed with our assay, and no obvious differences in droplet shape were detected during microscopic observation of the droplets. It is not clear if there is an invariant correlation between a surfactant’s hydrophilic-lipophilic balance and the shape it imparts to oil droplets on an agar surface. It is, however, tempting to speculate on the utility of this assay in predicting important characteristics of novel surfactants. HLB values are a scalar factor that reflects the degree to which a surfactant is hydrophilic or lipophilic, with a value of zero reflecting a completely lipophilic molecule, a value of 10 corresponding to a compound with equivalent hydrophobic and hydrophilic groups, and values over 10 for predominantly hydrophilic molecules.For example, common surfactants such as SDS and Tween 20 have high HLB values and are therefore best suited for emulsifying a hydrophobic substance into a water phase. On the other hand, surfactants such as Silwet® L-77 with HLB values near 10 are more suited for wetting, or spreading of a water phase over surfaces such as leaves. These surfactants with balanced water- and oil- loving groups can be very effective as spreading agents, capable of lowering the surface tension of water below 30 mN/m. Rhamnolipid, with a predicted HLB of 9.5,plastic pots for planting which can lower the surface tension of water to 28 mN/m, is a highly effective spreading agent involved in bacterial motility. 

Although there is no consensus on the HLB of surfactin, it is also capable of lowering the surface tension of water to 27 mN/m, suggestive that it may also have an HLB near 10. Surfactants like Silwet® L-77 which had lower HLB values conferred bright halos in our assay. The surfactants with HLB values over 13, which are most ideal for emulsification of oil into water, did not cause the oil droplets to bead, resulting in dark halos when tested by the atomized oil assay. It is interesting that none of the biosurfactants tested conferred dark halos, suggesting that their primary roles are not as emulsifiers.It is noteworthy that the measurements of biosurfactant production using the halo method were strongly correlated with the swarming capability in mutants of P. syringae strain B728a. This suggests that the area covered by surfactants at the air/water interface as measured by our assay reflects a similar distance where swarming movement of bacteria across an aqueous agar surface is facilitated. Moreover, it is significant that drop-collapse activity was not a good indicator of the swarming ability of a strain, which raises the question of what specific properties make a surfactant a good lubricant that facilitates bacterial motility. Because the drop-collapse assay only detects surfactants that are able to greatly lower the surface tension of water, this property appears unnecessary for functions such as swarming. In addition, use of the drop-collapse assay in biological screens may cause a wide array of biologically active surfactants to be overlooked. In view of that, it is interesting that a syringafactin mutant of P. syringae strain B728a appears to produce a second surfactant that can promote swarming but not cause a drop-collapse. This is in contrast to a syringafactin mutant in P. syringae strain DC3000 which does not appear to produce this second surfactant. It is also striking that no mutants were identified in strain B728a that exhibited a total absence of surfactant halo, pointing to differential regulation of syringafactin and the remaining expressed surfactant. 

Furthermore, the disruption of pmpR apparently causes the down-regulation of syringafactin while conferring up-regulation of the other surfactant, suggesting its role in regulating both surfactants. While both P. syringae strains are pathogenic to plants, strain B728a is a much better epiphyte than DC3000. Perhaps this second surfactant is particularly useful for the lifestyle of epiphytes such as strain B728a on waxy leaf surfaces. We are actively pursuing the identity and specific properties of this second surfactant. The phytotoxins syringomycin and syringopeptin have been suggested to possess surfactant activities , although preliminary results have not yet provided support for the identity of either of these surfactants as the second surfactant. It is possible that combining one of the mutations found from this screen with a syfA or syfB mutation could reveal the identity of the second surfactant Some, but not all of the genes found to regulate both biosurfactant production and swarming ability in P. syringae have homologs that influence swarming in Pseudomonas aeruginosa. Disruption of Psyr_3619, encoding an RNA helicase, conferred a similar reduction in swarming as that seen in blockage of its homolog PA2840 in P. aeruginosa. Likewise, disruption of pmpR in P. aeruginosa, a homolog of Psyr_1407, resulted in enhanced swarming in both species. It is significant that P. syringae B728a mutations were not identified in homologs of any of the many other genes found to alter swarming in P. aeruginosa despite the near completeness of the mutant library, emphasizing that the surfactants that contribute to swarming in these strains differ and/or that many factors other than biosurfactant production contribute to swarming ability. It is also noteworthy that relatively few different genes apparently contribute to biosurfactant production in P. syringae B728a. The disruptions of only 12 unique genes, identified from over 7,000 screened mutants,strawberries in a pot were found to alter biosurfactant production. Assuming random transposon insertion, we predict that we have screened a library of approximately 77% of the P. syringae B728a genes. Although we have identified many of the mutations which have an effect on measured surfactant halos, we may have missed a number of mutations which negatively affected syringafactin production but were masked by a compensatory increase in production of the second surfactant.

For life on the leaf surface, Pseudomonads have been shown to employ a variety of traits to grow and survive despite fluctuating water availability. In response to desiccation stress, Pseudomonads produce alginate in order to maintain a hydrated micro-environment. Our finding of multiple components of the AlgT regulatory pathway among mutants of strain B728a with altered biosurfactant production could suggest an intimate relationship between water availability and biosurfactant production. This potential relationship warrants further exploration of either the AlgT pathway or perhaps alginate production itself as a regulator of surfactant production. The role of biosurfactants on the leaf surface is most likely complex, and as such may likely prove to have very complex regulatory networks. The atomized oil assay has revealed a likely diversity of biosurfactants that are produced by strain B728a and their complex patterns of expression, details that would have been difficult to discern using other assays for biosurfactant production. The tools and genetic resources developed here should prove useful in further studies of the roles of surfactants in the interaction of P. syringae with plants. Biosurfactants, or biologically-produced surface active agents, have received wide attention mostly for their potential for hydrocarbon dispersion and remediation.However, a wide variety of roles for biosurfactants have been since described, from biofilm formation to inhibitory activity against pathogenic organisms, sparking a renewed interest in their discovery. Given this interest in biosurfactants, the lack of knowledge of the distribution and frequency of occurrence of surfactant production in the environment is remarkable. Comprehensive examinations of biosurfactant production are lacking, and studies that have addressed this trait in a given environment can seldom be compared with those of other habitats ; both the screening methods used, as well as pre-screening culturing conditions such as medium and incubation conditions usually vary widely between studies. In a recent report we described a high-throughput assay which utilizes the application of atomized oil droplets to rapidly detect biosurfactants produced by bacteria on the surface of agar plates. This method has advantages over other common assays such as droplet collapse assays in that it can be performed for many colonies simultaneously after limited growth, does not require sample preparation of culture supernatants, and thus is suited for high throughput screening for surfactant producing strains. Moreover, this method is capable of detecting much lower concentrations of surfactants than the drop collapse assay, and therefore in principle is capable of identifying biosurfactant producing strains that would escape detection with most other methods. However, since the atomized oil assay has not yet been tested on a broad range of environmental isolates, in this study we address whether the range of strains that it can detect includes all of those detectable by the drop collapse assay.

Furthermore, although the atomized oil assay has proven effective at detecting surfactants on agar plates, traditionally broth culture supernatants are screened for biosurfactant activity using the drop collapse assay. Depending on the properties of the surface-active compound and its biological role for the producing strain, its production may depend strongly on whether the producing cells are situated at a surface or not. Since a large difference in the transcriptomes of bacteria grown planktonically versus on surfaces have been described, with about one-third of genes differentially regulated , it seems likely that biosurfactant production itself may be strongly influenced by cell culture conditions. Surface sensing is an important cue for many species to transition to surface-associated behavior such as swarming, whereby cells move across a moist surface utilizing flagella and surfactant. Although the surface regulation of flagella has been well documented , the regulation of surfactant production by surfaces has not yet been explored and will be addressed in this report. Insight into the role of biosurfactants would benefit from a better understanding of the numerical distribution of surfactant producers in different environments. A variety of isolated reports have described collections of biosurfactant producers from aqueous environments, polluted/unpolluted soils, and even clouds, with estimates of their frequency in culturable bacterial communities ranging from less than 3 to as much as 50%, but typically around 10%. However, no encompassing model that describes the selection for such a trait has emerged from these studies, perhaps because few comparative analyses of habitats have been performed. We hypothesize that hydrophobic surfaces are habitats that would be particularly selective for bacteria that produce surface active compounds. The surface of leaves that are usually covered with wax would constitute such a habitat, although surfactant production in this habitat has seldom been investigated. In order to survive on leaf surfaces, epiphytes must be able to access limited and spatially heterogeneous nutrient supplies and endure daily fluctuations in moisture availability on a water-repellent surface. Epiphytic bacteria could potentially use biosurfactants to increase the wetability of the leaf, to enhance diffusion of nutrients across the waxy cuticle, and/or aid in motility to favorable growth sites. Despite the substantial potential role of biosurfactants on leaves, only a few studies have examined their production in the phyllosphere, all of which have focused on their possible ecological role in only specific strains and have not addressed the frequency of surfactant producers on leaf surfaces. A comprehensive examination of the phyllosphere inhabitants might reveal strains and biosurfactants not normally encountered in other habitats, and would address the hypothesis of surface enrichment of producing strains. In this study we compare the frequency of surfactant producers in the phyllosphere to those in soil and water environments. We compare the atomized oil assay with the drop collapse assay to characterize surfactants made by a collection of environmental strains, further demonstrating the usefulness of this assay in high-throughput screening and its much higher sensitivity for all types of biosurfactants encountered, many of which are hydrophobic and poorly detectable by the droplet collapse assay. We also investigate the influence of planktonic versus surface-associated culture conditions on the production of biosurfactants from our environmental isolates, and find evidence for frequent contact-dependent production of surface active compounds.

Worldwide use of neonicotinoids continues to expand as pest populations develop resistance to the once-widely-used pesticide classes including the organophosphates and methylcarbamates. The most commonly used neonicotinoids are IMI and TMX, the primary focus of these studies. The overall goal is to further understand the metabolism of neonicotinoids relative to: in vivo importance of AOX, metabolic pathways of the novel neonicotinoid, cycloxaprid and mechanisms of TMX hepatotoxicity and hepatocarcinogenicity. CYPs have been shown to be involved in in vitro and in vivo neonicotinoid metabolism, but the relative in vivo importance of AOX is unknown. AOX is implicated to play a role in the nitroreduction of N-nitroguanidine neonicotinoids, the most prominent subclass. There is considerable variability in the activity of AOX between species and individuals which may be reflected in differences in neonicotinoid metabolism and detoxification. Secondly, CYC is a new neonicotinoid that is under development to control IMI-resistant pests. However, its metabolic pathway has yet to be determined, particularly in reverting to its potent nAChR agonist precursor, nitromethylene-imidazole. Finally, TMX is the only neonicotinoid to produce liver toxicity and tumors in chronically-treated mice, but not rats. Earlier studies concluded that formation of dm TMX and iNOS inhibition by dm-CLO is likely the mechanism of TMX toxicity. Furthermore, differences in metabolic rates between species may explain the mouse specific toxicity. However, the molecular mechanism of TMX or dm-TMX hepatotoxicity/ hepatocarcinogenicity remains unclear. It is critical to fully understand the metabolic/ enzymatic pathways of neonicotinoids and mechanisms of toxicity as their use continues to increase and for future pesticide design.The nitro substituent on neonicotinoids is important relative to their potency and selectivity for the insect nAChR. From the seven commercial neonicotinoids, approximately 100 metabolites have been identified in plants and mammals, some of which are bio-activated and can interact with the mammalian nAChR. 

A number of studies have demonstrated the importance of CYPs in neonicotinoid metabolism in vitro and in vivo. However,macetas plastico cuadradas the role of AOX in neonicotinoid metabolism has yet to be established in vivo, especially in the oxidative- and CYP-rich environment of the liver. AOX is important in xenobiotic metabolism. This enzyme is expressed mainly in liver but is also present in many other tissues with variations in activity depending on species, gender, age, drug usage and disease states. Tungsten or hydralazine in the diet or drinking water results in reduced AOX activity in guinea pigs, rabbits and mice. There are even notable differences in AOX activity between strains of mice , e.g. compared to CD-1 mice, the DBA/2 strain is deficient in the expression of AOX homologue 1 and homologue 2 and has reduced expression of AOX1. Since AOH1 and AOX1 are the primary AOX genes expressed in mouse liver , DBA/2 mice are an appropriate AOX-deficient model for studies on in vivo mammalian xenobiotic metabolism. The wide range of inter- and intra-species AOX activity may result in different rates of neonicotinoid metabolism and detoxification in mammals and insects. Despite the increasing significance of AOX, there have been very few studies examining the in vivo contribution of this enzyme to xenobiotic metabolism. Mice can serve as a surrogate for humans since AOX activity in IMI nitroreduction in vitro is comparable between these two species. This study uses chemical inhibitors and genetic deficiency for mice and Drosophila melanogaster to evaluate the relevance of AOX in neonicotinoid metabolism in vivo. Mouse liver cytosol and microsomes were prepared by homogenizing liver in ice-cold PBS using a Sonic Dismembrator followed by centrifugation of the homogenate at 1,000g for 10 min and then the supernatant at 10,000g for 30 min. An aliquot of the 10,000g supernatant was recovered for AOX activity analysis and the remainder was centrifuged at 100,000g for 1 h to collect the CYP-containing microsomal pellet fraction which was resuspended in PBS for protein measurement and the CYP activity assay.

Mouse liver cytosol was added to 50 µM DMAC solution and the reaction monitored by an absorbance decrease using a VersaMax microplate reader at 398 nm for 5 min with an average control value of -18.4 mOD/min. 7-Ethoxycoumarin is a broad-specificity substrate used to measure the activity of many CYP enzymes by monitoring the oxidation to 7-hydroxycoumarin. Microsomes were mixed with 50 mM 7- ethoxycoumarin in assay buffer glycerol and 0.1 mM EDTA and prewarmed at 37°C for 5 min. After addition of 10 mM NADPH , reactions were incubated at 37°C for 30 min in a shaking water bath.Samples were extracted with chloroform , briefly vortexed, then centrifuged at 3,000g for 5 min. The organic phase was removed and added to 30 mM sodium borate and vortexed. Following centrifugation at 3,000g for 5 min, the upper layer was recovered and plated on a Costar 96-well black plate and fluorescence read at an excitation wavelength of 370 nm and an emission wavelength of 460 nm using a SpectraMax M2 Microplate Reader with an average control value of 11.2 nmol 7-hydroxycoumarin/mg protein.AOX is a potentially important factor in drug metabolism with many studies examining its in vitro inhibition and the proposed effects on xenobiotic action. There is a wide range of AOX activity between species with rabbits, monkeys and humans the highest, mice intermediate and rats and dogs having the lowest activity. This same species dependent relationship is also observed for in vitro IMI nitroreduction by liver cytosol. Tungsten and hydralazine treatments provide a way to reduce AOX activity in vivo in mammals to evaluate its relevance in xenobiotic metabolism. Tungsten replaces molybdenum at the active center of AOX, rendering it inactive , but the mechanism of AOX inactivation by hydralazine is unknown.The level of AOX inhibition by tungsten treatment in mice was less than that by hydralazine , a difference reflected in their effect on IMI metabolism. Hydralazine treatment resulted in significantly reduced IMI metabolism to IMI-NNO and IMI-NH,maceta redonda but tungsten treatment only significantly reduced IMI metabolism to IMI-NH. There are four AOX genes in mice with two of the variants being expressed in the liver, AOH1 and AOX1. DBA/2 mice are completely deficient in the expression of AOH1 and have low expression of AOX1 compared to CD-1 mice. Our data also establish that DBA/2 mice have significantly lower AOX activity in the liver and further show that the reduced AOX activity decreased IMI metabolism to IMI-NNO and IMI-NH, but not to IMI-5-OH or IMI-ole.

The AOX-generated IMI metabolites are not all detoxification products. IMI-NH is a likely contributor to the nicotinic effects of IMI. It is over 300 times more potent than IMI at the mammalian nAChR and the mouse ip toxicity is also increased several fold. IMI-NNO retains insecticidal activity and as an N-nitroso compound it was subjected to extensive toxicological tests and cleared of potential problems. Our study concludes that reduced AOX activity is tightly correlated with reduced IMI metabolism to IMI-NNO and IMI-NH indicating that these products are mostly from AOX, not CYPs. Based on the metabolic sequence and relevant correlations, IMI-NH is mostly formed via IMI-NNO rather than another pathway. This is the first report on the metabolism of CYC with an emphasis on in vivo metabolites in mice at 15 and 120 min post-treatment. Metabolites are not conclusively identified since synthetic standards were not available. However, based on calculated m/z values and characteristic chlorine isotope patterns, it is concluded that CYC is converted in part to NMI, but is mostly oxidized to multiple mono- and dihydroxylation products within 15 min and dissipate by 120 min in liver. There are many possible isomeric CYC oxidation products from hydroxylation on the 6-, 7-, 10- or 11- position in each case with two possible stereoisomers of which five are detected as distinct peaks by LC/MS. Minor products include 2-CYC and nitroreduction on the nitro group to NO-CYC and NH2-CYC. CYC is readily hydrolyzed to NMI. Therefore, extraction and analytical conditions were carefully chosen to limit degradation. However, minor amounts of NMI detected may be due to degradation of CYC rather than its in vivo metabolism. NMI per se was not extensively metabolized. Only minor amounts of one hydroxylation and one nitroso product were evident. Detection methods may limit observing the extent of metabolism since only 3 mg/kg was administered to mice. The findings reported here lay the background for future studies on characterization of metabolites and in vitro species comparisons. In vitro conditions will allow detection of more metabolites and the enzymes responsible for their formation can be determined. 

TMX undergoes metabolic activation to CLO in insects, plants and mice. The neonicotinoids generally have favorable mammalian toxicology with the exception of TMX which is a hepatotoxicant and hepatocarcinogen in mice but not rats or dogs. The mechanism of this TMX- and mouse-specific hepatotoxicity/ hepatocarcinogenicity is of considerable interest relative to neonicotinoid risk assessment. Green et al.observed that liver microsomal metabolic rates are greater for mouse than rat or human in the production of TMX metabolites, dm-TMX, CLO and dm-CLO. They also found that mouse-specific adverse effects of TMX are due to dm-TMX exacerbated by dm-CLO which mimics the structure of L-NAME, a standard inhibitor of iNOS, an enzyme with a regulatory role in the development of hepatotoxicity. The structure-activity relationships of neonicotinoids as hepatotoxicants or hepatocarcinogens help focus mechanistic hypotheses on specific molecular substituents. TMX and dm-TMX are hepatotoxicants/ hepatocarcinogens and contain the oxadiazinane substituent uniquely among the neonicotinoids so this moiety is of particular interest. N-Methyl substituents on five of the neonicotinoids are not the hepatotoxic moiety because dm-TMX lacks this group. The Green et al.hypothesis is that dm-TMX is the hepatotoxicant exacerbated by dm-CLO as an iNOS inhibitor. The present study examines an alternative hypothesis that the unique aspect of the oxadiazinane moiety is its metabolic conversion to HCHO and N-methylol intermediates , the ultimate hepatotoxicants and hepatocarcinogens which may be synergized by dm-CLO as a NOS inhibitor.high-throughput oxymyoglobin assay by following the procedure of Dawson and Knowles. To determine nNOS inhibition, HEK293 cells overexpressing rat nNOS were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.4 mg/mL geneticin. For enzyme activity assays, cells were cultured in Costar 96-well black plates and treated with 5 µM A23187 ionophore to activate nNOS then with dm-CLO or L-NAME for 8 h. To measure nitric oxide production, cell media was removed and replaced with reaction buffer containing 1 mM L-arginine, 10 µM 4,5-diaminofluorescein diacetate , 1 mM NADPH and dm-CLO or L-NAME. After a 2-h incubation in the dark, fluorescence was read at an excitation wavelength of 490 nm and an emission wavelength of 520 nm using a SpectraMax M2 Microplate Reader.Mouse liver microsomes were prepared by homogenizing livers from male albino Swiss Webster mice in PBS followed by differential centrifugation of the supernatant. The microsomal 100,000g pellet was resuspended in PBS and protein concentration measured. Liver microsomes for species comparison studies were from BD Biosciences. Recombinant CYP3A4 was compared to recombinant CYP2C19 , the two isoforms previously shown to be responsible for TMX metabolism. Each neonicotinoid was incubated with microsomes from mouse, rat or human or rCYP isoform and 0 or 1 mM NADPH in PBS for 1 h at 37°C. Alachlor and hexamethylphosphoramide , compounds known to produce HCHO via N-methylol intermediates on activation by CYPs , were also incubated under the same conditions with mouse liver microsomes alone or with NADPH. Enzymatic reactions were terminated by addition of 25% ZnSO4 aqueous solution and saturated Ba2 aqueous solution. Samples were briefly vortexed and placed on ice for 5 min then centrifuged at 18,000g for 5 min. HCHO levels were analyzed after conversion to the 2,4-dinitrophenylhydrazine derivative. An aliquot of the supernatant was mixed with 7.2 mM DNPH and incubated at room temperature for 30 min followed by addition of carbon tetrachloride , vortexing for 30 seconds and a final incubation at room temperature for 30 min. The lower organic layer was evaporated to dryness under N2 at 25°C and resuspended in 80:20:0.1 ACN/water/HCO2H and filtered through 0.2 µm nylon for HPLC analysis. Samples were analyzed on a Waters Alliance 2695 HPLC equipped with an Agilent Zorbax SB-C18 column and Waters Alliance 2487 dual UV absorbance detector.

A constraint in one system could not only affect economic security in that system but could inhibit access in another. Therefore, the nexus provides a powerful means to improve synergies in food, energy, and water production , to identify how stressing food, energy and/or water systems creates resource vulnerabilities and/or resource scarcities in all three, to understand and quantify the production of ecosystem services, and to develop climate adaptation strategies. However, historically, food, energy, and water systems have been pigeonholed politically as well as broken up into small disjointed pieces that cross political boundaries and do not align with bio-regions or watersheds. This type of disjointed management leaves policy makers ill-equipped to provide resilient management strategies. Thus, the success of using the nexus concept to improve food, energy, and water systems will likely depend on how it incorporates issues surrounding resource governance , including how governance and the discourse of securitization become a way to legitimize political agendas. Many have argued that the nexus discourse of security places economic variables over access to resources for the world’s poor, an idea that can be traced back to Foucault’s theory of the linkages between security and the circulation of the global economy. In addition to this discursive ‘securitization’, resource governance outcomes are based on the larger political goals of the government or states involved in policy making. The connections between discourse, policy,diametro maceta 10 litros and land management, therefore, raise important questions regarding how policy is already impacting nexus outcomes and communities on the ground.

Given the recent emergence of the FEW concept, few studies to date have explored the use of the nexus concept simultaneously with an analysis of governance structures. To address this gap, we present a quantitative application of the FEW nexus concept to study resource vulnerabilities and scarcities in the Lower Colorado River Basin in California, Arizona, and Nevada, U.S.A. We analyze the nexus within its sociopolitical, economic, and bio-regional context that determine what resources are available, used, produced, and traded. We take a case study approach, as case studies are best able to translate the on-the-ground nexus realities of a variety of institutions, bureaucracies, and stakeholders across space, time, and scale. Our goal was to understand how the governance structure of the Colorado River constrains the utility of the nexus approach to deal with future stresses. To do this, we first quantified the nexus by identifying the local and global linkages between food, energy, and water as well as the choices confronting water managers, and the Indian Reservations in the study area. We use these findings to look for the emergence of tipping points under two different scenarios: drought and increased demand for alfalfa. We then discuss how the very rigid water laws in the LCRB constrain the ability to improve resource management and respond to these tipping points using nexus thinking. While the main focus of this paper is on how economic and hydraulic factors influence FEW’s nexus governance, in the discussion we also examine the impact of institutional and political factors as well as geopolitics across the transnational boundary between the U.S. and Mexico.The Colorado River Basin has a semiarid to arid climate with an average of 40 cm annual precipitation that originates as snow pack in the Rocky Mountains and contributes to about 70% of the total stream flow in the basin. However, temperatures have been rising for the past century, with winter temperatures increasing more than summer temperatures on average by 2 °C. 

Temperatures are predicted to rise at least another 1.1 to 2.0 °C by 2050. These higher temperatures increase evaporation rates and have coincided with a reduction in snow pack and snow melt in the UCRB. In addition, the LCRB has been in a drought since 2002, though at the same time the UCRB has experienced less severe or no drought conditions, outside of extremely dry periods in 2003 to 2004, and 2012. Historically, paleo climatic records indicate pervasive and long-lasting periods of drought occur in the region. However, more recently, the area has received significantly more snowfall.Its water elevation has been decreasing since 2000, which can be seen visually in aerial imagery.The Colorado River is operated under the “Law of the River,” which is comprised of a variety of U.S. federal laws, agreements, court decisions, and regulatory guidelines. These laws apply to seven Western U.S. states: Arizona, California, Colorado, Nevada, New Mexico, Utah, and Wyoming, and Mexico that utilize water from the river. The Law of the River allocates to each basin 7.5 million acre-feet 1 of water per year in perpetuity, with some exceptions that account for water scarcity. High variability in precipitation in the Rocky Mountains results in 5 MAF to 25 MAF of flow in any given year with an average annual flow of less than 16.4 MAF/yr. In terms of usage, on average 91.4% of the allocated water is used by municipalities, for power, or agriculture. The UCRB Indian reservations receive 1 MAF a year, while the LCRB Native American reservations are allotted 0.9 MAF a year. While this water is split amongst the 20 reservations in the LCRB, only six reservations have had their water rights quantified. This has resulted in a disproportional amount of water allotted per person on these reservations compared with the rest of the basin. Specifically, 6.7 AC-FT per person per year is allotted to Lower Basin Indian reservations while 0.9 AC-FT per person per year is allotted to the rest of the LCRB. It should be noted that the water allotments of both basins , Mexico ,macetas cultivo and the Native American Reservations , adds up to 18.4 MAF, 2 MAF more than the yearly average stream flow of the Colorado River not accounting for climatic fluctuations.

The rigid allotments based on the Law of the River have also resulted in most of the agriculture production in the study area taking place on Indian reservations. Prior to the Law of the River, American Indians practiced flood farming practices for thousands of years based on characteristic seasonal rains , as well as dry farming. Today, however, practices are much more water-intensive on Indian Reservations.The Colorado river allows local economies to exist in a semi-arid environment with two-thirds of Arizona’s, California’s, and Nevada’s state gross products dependent on the Lower Colorado River. The river provides 657.5 billion dollars of direct, indirect, and induced GDP to California; 185 billion dollars to Arizona; and 115.4 billion dollars to Nevada. Focusing on food systems, agricultural production in the study area is largely situated on Indian reservations due to their high allotment of river water.For alfalfa, one of the most common crops in the region, the water that is used to flood the fields is sent back into the canals. The high-water availability along with the warm climate allows the production of twelve harvests of alfalfa each year. Most of the alfalfa is exported as fodder to China, followed by Japan, Mexico, South Korea, and the EU.A diversity of data sources drawn from the Food and Agriculture Organization of the United Nations , the United States Department of Agriculture , and the Energy Information Association were used to quantify the connections between food, energy and water.Energy was quantified in terms of net generation and cost for hydroelectric, natural gas, solar, and wind generated in the region for 2001 to 2016. Food production was quantified in terms of area of agricultural crops as well as the price that farmers spent on production for 2008 to 2015. Only crops that took up greater than 1% of total area were analyzed. To determine the total impact of one sector on another , we combined the total amount of water used in energy or food, and the total amount of energy used to pump water or produce food.Water-energy linkages were quantified in two different ways. First, the amount of water used in energy production 2001 to 2016 was calculated by multiplying the appropriate water consumption factors of electricity generated from natural gas from Spang et al., 2014 by the amount of energy produced monthly from 2001 to 2016. Water used in natural gas extraction for gas used in the study area was not included since there are no natural gas extraction operations within the basin. Hydroelectric projects have no net water consumption, but reservoirs make evaporation rates higher, especially with higher temperatures. Second, energy used to transport water in the Colorado River Aqueduct was quantified using data obtained from the Metropolitan Water District of Southern California for 2001 to 2016.

The aqueduct conveys water from Lake Havasu over the Santa Ana Mountains and to cities in Southern California, including Los Angeles. In addition, energy used to transport water from Lake Havasu to Phoenix and Tucson via the Central Arizona Project was drawn from a previous study by Kleiman. A final water-energy linkage, the amount of energy need to purify wastewater for domestic consumption was not quantified due to lack of data. Generally, however, wastewater treatment accounts for ∼3% of energy used in the United States.First, information on specific crop water usage was estimated using evapotranspiration rates in ft/growing period and average area cultivated annually for six of the seven most common monocrops in the LCRB. All data came from FAO estimates except for lettuce. FAO evapotranspiration estimates are given in mm/growing period, however, we converted the units to ft/growing period so we could easily translate the measurement to volume of water in AC-FT. Second, data for 2007, 2012, and 2013 from the National Agricultural Statistics Service for the entire Lower Colorado River Basin was used to estimate yearly water used for irrigation. In order to calculate the data at the watershed subunit level, we used the United States Agriculture Service 2012 Census and Cropland Data Layer to estimate the percent of irrigated land out of the total acres of cropland. The proportion of cropland that was irrigated varied based on data year, in 2013, 67.74% of cropland was irrigated; in 2012, 75.82% of cropland was irrigated; and in 2007, 71.21% cropland was irrigated. We averaged the percent of cropland irrigated in the watershed subunit for 2007, 2012, 2013 to extrapolate to the remaining years from 2008 to 2015. The average irrigation estimate was used in the production scenarios to understand responsiveness of irrigation and crop production to changes in climate.The linkage between food and energy was quantified in two ways. First, we estimated the amount of direct usage of fossil fuels by calculating the amount of fuel farmers purchased to run farm machinery in 2012. Second, using the 2013 NASS estimation for energy expended in irrigation for the entire LCRB, estimated at $49.00 per acre, we estimated the cost for irrigation in the study area. Using the cost per amount of energy we converted $49.00 per acre to the amount of energy in MWH used for irrigation in the study area. Assuming that the price of energy needed to irrigate remained constant, we calculated the energy expense for all years using the estimated irrigated cropland and cost per amount of energy for those years.As expected for a temperate semi-arid climate, the amount of water consumed followed a seasonal pattern with increased use during the summer months and decreased use during winter. Across Arizona, California, and Nevada, the main consumptive water use was either irrigation or municipal use, depending on the year. In 2013, the biggest consumers in Arizona and California were large cities such as Phoenix, Tucson, and irrigation districts. Water to cities in Arizona was transferred through the Central Arizona Project with over 1.5 MAF consumed. The Imperial Irrigation District was California’s largest water consumer in 2013, consuming more than 2.5 MAF of water. The Metropolitan Water District that transfers water to Los Angeles consumed less than the Central Arizona Project with just over one MAF. In Nevada, there are no consumptive uses over 100,000ACFT. However, the City of Las Vegas receives its water right before it reaches Lake Mead, so this is not accounted for in our analysis.

In multiple studies, slow sand filtration has been useful to remove and eliminate propagules of plant pathogens: many species of phytophthora – P. cinnamomi , P. cryptogea , P. nicotianae , and species of Pythium , Fusarium , E. coli , and nematodes including Radopholus similis. These zoosporic fungi, viruses, and nematodes cause crop damage including severe root rot and if not treated in a recirculating nursery and greenhouse system, widespread epidemic may impact all the crops within the facilities. While SSF has been shown to remove plant pathogens, its efficacy to remove other compounds has yet to be explored in depth. Contaminants of emerging concern , also named “emerging contaminants”, are a group of contaminants that consist of pharmaceutically active compounds, endocrine disrupting compounds, personal care products, plasticizers, pesticides and herbicides, and flame retardants that are found in trace amounts in the environments, primarily discharged from wastewater treatment plants that insufficiently treat these contaminants through secondary and tertiary treatment. Non-point sources, such as overland flow during rainfall or land drainage in agricultural areas deliver veterinary medicines and pesticide runoff to surface water or groundwater. The existence of emerging contaminants is unknown in the environment due to the lack of monitoring and due to their low concentrations in surface waters. Many of these compounds vary due to their application and consumption from region to region and will depend on the efficiency of removal by wastewater treatment plants. Technologies already proven effective in removing contaminants are activated carbon ,maceta 25l ozonation and advanced oxidation processes , and membrane filtration. However, these processes are costly and require large amounts of resources to operate.

In turn, bio-filtration systems are simple to operate, relatively low in cost and maintenance, removes both turbidity and propagules of pathogens, and overall improves the quality of the water. Thus, bio-filtration like SSF may offer a low cost alternative for the treatment of contaminants in wastewater.The most commonly researched bio-filtration systems to treat emerging contaminants are managed aquifer recharge processes. Managed aquifer recharge processes are robust and cost-effective systems and include a variety of applications such as aquifer storage and recovery, infiltration ponds, percolation tanks, soil aquifer treatment, and sand dams. MAR systems like bank filtration and artificial recharge are adopted by wastewater treatment plants to reduce the cost of using more costly advanced treatment systems like nanofiltration and reverse osmosis. MAR systems are typically adopted if surface water quality is inadequate or if the amount of raw water like in groundwater is not sufficient ; MAR systems can replenish these natural systems. However, in comparison to SSF, biodegradation in MAR systems are very important mechanisms, much more than sorption as sorption sites can become exhausted or desportion can occur. This practice may not be as easy to maintain like slow sand filters, as sand can be back washed and the supernatant of the disturbed schmutzdecke can be drained. Also, it is suggested that MAR such as bank filtration may not be optimal for smaller operating facilities that have less land space. There have been many studies that focus on biofilm reactors such as bank filtration and underground dams that can remove organic micropollutants but these are not usually designed, only grown in natural conditions. Therefore these systems are very limited and may need to overcome obstacles of releasing WWTP effluent water directly to natural waters. However, given substantial research on MAR systems, pharmaceuticals and endocrine disrupting compounds, which are usually main contaminants of interest due to their potentially adverse effects on human health and aquatic life even at low concentrations , have shown positive results for their removal.

Most of these studies were laboratory scaled and field studies. Some contaminants researched in MAR systems are endocrine disrupting compounds , antiseptics , pharmaceuticals , and disinfection by-products. Baumgarten et al. investigated the removal of poorly degradable antibiotic sulfamethoxazole in laboratory columns for its removal in bank filtration. Results showed that 60% of sulfamethoxazole was removed within 14 days of column passage in aerobic conditions while no removal occurred under anoxic conditions in a 2 year long system operation. The degradation of poorly degradable compounds in aerobic conditions may give bank filtration its benefits in the top layers of sand, but shows complications with anoxic conditions, which can be representative of some MAR and SSF. Adaptation of the system may require long operation time as would be realistic in an actual bank filtration site. Thus, more biofilter systems with MAR and SSF should be studied for their processes that provide both oxic and anoxic conditions. Typically, varying redox conditions are effective for removing redox-sensitive organic micropollutants during MAR. Maeng et al. summarized the literature in a review regarding removal efficiencies of CECs using bank filtration and aquifer recharge column and full scale studies. To briefly summarize that work, there are CECs that are promising for removal by filtration and others which are more recalcitrant such as carbamazepine are ineffective. Antibiotics in a study by Heberer et al. investigated 19 targeted antibiotics at a lake bank filtration site in Berlin, Germany for 2.5 years. They detected 7 out of 19 target antibiotics. All antibiotics were completely removed after 2-4 months of travel time except for sulfamethoxazole, which in previous studies was discovered to be redox-dependent that degrades more effectively in anoxic conditions. Depending on the residence time, removal of antibiotics can increase. It can be concluded MAR is an effective treatment step for removing antibiotics, giving way for other filtration studies to filter veterinary antibiotics that may potentially be found in runoff from dairy farms. For non-steroidal anti-inflammatory drugs and analgesics, many have been removed at rates greater than 50% during bank filtration and aquifer recharge systems.

Field and laboratory scale studies have shown significant removals of diclofenac, ibuprofen, naproxen, and phenazone during soil passage. Diclofenac, ibuprofen, and naproxen have moderately high octanol-water partition coefficients , suggesting sorption would be the main mechanism of removal. Phenazone, however,frambuesas en macetas is more redox-dependent and can be removed under oxic conditions than anoxic conditions. Therefore, it is necessary to monitor the pH during soil passage as these NSAIDs may remain as ionic species in the aquatic environment, with more potential to be sorbed. Anticonvulsant pharmaceuticals have been shown numerous times their persistency in degradation in multiple treatment methods. Carbamazepine is one of the notorious poorly degradable compounds and has low removal in wastewater treatment plants. Drewes et al. showed no change in carbamazepine and primidone concentrations in soil aquifer treatment for estimated travel times up to six years. The extended research on this contaminant concluded bank filtration and aquifer recharge are not effective for anticonvulsant removal. Antidepressants removal has still yet to be studied. A study by Snyder et al. investigated three antidepressants and their fate during a pilot scale bank filtration. Fluoxetine was removed significantly at 99% and meprobamate was only 66%. There could be more research developed in understanding the fate of more antidepressants since they are commonly used drugs in the pharmaceutical industry. Lipid regulators are similar to NSAIDs, where they remain in ionic species. Thus, pH plays an important role in the removal mechanism. One of these lipid regulators, clofibric acid, is a common metabolite of clofibrate, and is detected frequently in the aquatic environment. Interestingly, research suggests clofibric acid concentrations increased at bank filtration sites in Germany due to the high consumption of liquid regulators during the 1990s. They discovered clofibric acid present in deeper layers of the aquifer. Lipid regulators and NSAIDs can be included in a joint research to test pH conditions for their removal. Steroid hormones are also a very particular group of CECs because they can produce potentially adverse effect on human health and aquatic life even at very low concentrations. However, laboratory scale and field studies using bank filtration in Berlin, Germany showed positive results. 17β-estradiol and 17α-ethinylestradiol were not detected in surface water from Berlin and estrone was removed greater than 80%. Snyder et al. used batch experiments and field studies with bank filtration and demonstrated estrone, 17β-estradiol, and 17α-ethinylestradiol were removed by biodegradation and sorption.Given conditions of certain MAR, steroid hormones and potentially a wide range of endocrine disrupting compounds can be reliably treated. For pesticides/herbicides, most studies have been focused on atrazine.Atrazine is resistant in the environment and penetrates through the surface and subsurface due to its high mobility, persistence, low vapor pressure, and massive application since it has been in use since 1959. Ho et al. and Zhang et al. showed triazine herbicides like atrazine were poorly removed by biofilters, but some studies have shown that atrazine can be readily biodegradable in aquatic environments, with reported removal rates ranging from weeks to years. 

Based on the previous studies, it is apparent that emerging contaminants can be removed and have shown adequate removals with a variety of conditions; aerobic and anaerobic conditions, biological substrate feeds, bacterial community development, filter mediums, variety of physicochemical properties of contaminants, and type of leachate water filtered using a SSF system. However, despite the variance in studies, most research agrees that the general contaminant removal increases over time as filter matures. Now that available freshwater resources are continuously limited and increases in world population have raised the pressure on natural resources , water resource management have turned to water reclamation and reuse to sustain agricultural activities. Reclaimed water use is not only limited to agriculture but widely used in other purposes such as irrigating landscapes, nurseries and greenhouses, flushing toilets, and replenishing groundwater aquifers. In 2006, an estimated 9.8×106 m3 d -1 of treated municipal wastewater was used in the United States. Studies have documented the presence of many micro-contaminants such as pharmaceuticals and personal care products, pesticides, phenolicestrogens, surfactants, biocides, and disinfection by-products in reclaimed water. These contaminants may accumulate in the area irrigated by reclaimed water and may result in contamination of the soil and plants. Along with the combined use of pesticides in greenhouses, it is possible for receiving waters to receive a wide range of pollutants. This is especially true in rural areas where contaminated runoff from farmland contributes a significant proportion of the pesticide load. The overall objective of this study was to promote slow sand filtration columns as a cost effective engineered solution to treat emerging contaminants. Specifically we simulated a greenhouse irrigation system that contains emerging contaminant concentrations of 400 ng L -1 based on the range of literature values for concentrations of emerging contaminants found in reclaimed nonpotable wastewater , a concentration higher than the average to be able to adequately detect the compounds in our study. This pilot scale study is a preliminary study to see how viable SSF columns can be to remove emerging contaminants. Results of the present study can be combined with previous studies of using SSF columns to remove both pathogens and contaminants provided by reclaimed water and pesticide use. The removal efficiencies of 14 selected pharmaceuticals and personal care products , 7 pesticides, 3 plasticizers, and 2 detergents/emulsifers, and the trends after the project’s initial start and declogging maintenance removal rates thereafter were examined. Chemical residues have been ubiquitous in the environment as they are found in many environmental matrices, from sewage water, effluent water from wastewater treatment plants, river water, to even drinking water. These compounds can come from sources such as households, nurseries, wastewater treatment plants, factories, hospitals, and any other facility dispensing chemical waste such as pharmaceuticals, personal care products, antibiotics, plasticizers, pesticides, etc. This group of compounds, known collectively as “emerging compounds” or “compounds of emerging concern” , is notorious for their occurrence in the environment and their complexity existing in particular environmental matrices. Even at their low concentrations they can affect human health and environmental health. Analytical techniques using Gas Chromatography-Mass Spectrometry or Liquid Chromatography-Mass Spectrometry have paved the way for determining the concentrations of CECs even at their trace amount. GC-MS was first used to determine pharmaceuticals and personal care products in the environment in 1976. Advances to the quantification of CECs in environmental samples have increased detection sensitivity and reliability. CECs come in a wide variability in their concentrations, polarities, and thermal labilities. 

Reconfiguration is used extensively in Corfu, to replace failed drives, to add capacity to the system, and to add, remove, or relocate replicas. This makes reconfiguration latency a crucial metric for our system. Recall that reconfiguration latency has two components: sealing the current con- figuration, which contacts a subset of the cluster, and writing the new configuration to the auxiliary. In our experiments, we conservatively seal all drives, to provide an upper bound on reconfiguration time; in practice, only a subset needs to be sealed. Our auxiliary is implemented as a networked file share.We now demonstrate the performance of Corfu-Store for atomic multi-key operations. Figure 3.19 shows the performance of multi-put operations in Corfu-Store. On the x-axis, we vary the number of keys updated atomically in each multi-put operation. The bars in the graph plot the number of multi-puts executed per second. The line plots the total number of Corfu log appends as a result of the multi-put operations; a multi-put involving k keys generates k +1 log appends, one for each updated key and a final append for the commit record. A client appending to a materialized stream first obtains the current layout and makes a request to the sequencer with a stream id. The sequencer returns both alog token, which is a pointer to the next address in the global log, and a stream token, which is a pointer to the next address in the stream. Using these tokens and the layout, the client determines the set of replicas to write to. In contrast to traditional designs, replica sets in vCorfu are dynamically arranged during appends. For fault tolerance, each entry is replicated on two replica types: the first indexed by the address in the log ,best vertical garden system and the second by the combination of the stream id and the stream address. To perform a write, the client writes to the log replica first, then to the stream replica.

If a replica previously accepted a write to a given address, the write is rejected and the client must retry with a new log token. Once the client writes to both replicas, it commits the write by broadcasting a commit message to each replica it accessed. Replicas will only serve reads for committed data. The write path of a client, which takes four round trips in normal operation is shown in Figure 4.5. A server-driven variant where the log replica writes to the stream replica takes 6 messages; we leave implementation of this variant for future work. The primary benefit of materialized streams is that they provide an abstraction of independent logs while maintaining a total global order over all appends. This enables vCorfu to support atomic writes across streams, which form the basic building block for supporting transactions. To append to multiple streams atomically, the client obtains a log token and stream tokens for each materialized stream it wishes to append to. The client first writes to the log replica using the log token. Then, the client writes to the stream replica of each stream. The client then sends a commit message to each participating replica. The resulting write is ordered in the log by a single log token, but multiple stream tokens. Materialized strreams are a first class abstraction in vCorfu, unlike streams in Tango which are merely tags within a shared log. Materialized streams strike a balance that combines the global consistency advantages of shared logs with the locality advantages of distributed data platforms. Specifically, the following properties enable vCorfu materialized streams to effectively support state machine replication at scale: The global log is a single source of scalability, consistency, durability and history. One may wonder, why have log replicas at all, if all we care to read from are materialized streams? First, the global log provides a convenient, scalable mechanism to obtain a consistent snapshot of the entire system. This can be used to execute long running read-only transactions, a key part of many analytics workloads, or a backup utility could constantly scan the log and move it to cold storage. Second, the log provides us with a unique level of fault tolerance – even if all the stream replicas were to fail, vCorfu can fall back to using the log replicas only, continuing to service requests.

Materialized streams are true virtual logs, unlike streams. Tango streams enable clients to selectively consume a set of updates in a shared log. Clients read sequentially from streams using a read Next call, which returns the next entry in the stream. Tango clients cannot randomly read from anywhere in stream because streams are implemented using a technique called backpointers: each entry in a stream points to the previous entry, inducing a requirement for sequential traveral. Materializing the stream removes this restriction: since clients have access to a replica which contains all the updates for a given stream, clients can perform all the functions they would call on a log, including a random read given a stream address, or a bulk read of an entire stream. This support is essential if clients randomly read from different streams, as back pointers would require reading each stream from the tail in order. vCorfu avoids backpointers, which pose performance, concurrency and recovery issues. Back pointers can result in performance degradation when concurrent clients are writing to the log and a timeout occurs,vertical farming equipment causing a hole filling protocol to be invoked. Since holes have no back pointers, timeouts force a linear scan of the log, with a cost proportional to the number of streams in the log. Tango mitigates this problem by keeping the number of streams low and storing multiple back pointers, which has significant overhead because both the log and the sequencer must store these back pointers. Furthermore, back pointers significantly complicate recovery: if the sequencer fails, the entire log must be read to determine the most recent writes to each stream. vCorfu instead relies on stream replicas, which contain a complete copy of updates for each stream, resorting to a single back pointer only when stream replicas fail. Sequencer recovery is fast, since stream replicas can be queried for the most recent update. Stream replicas may handle playback and directly serve requests. In most shared log designs, clients must consume updates, which are distributed and sharded for performance. The log itself cannot directly serve requests because no single storage unit for the log contains all the updates necessary to service a request.

Stream replicas in vCorfu, however, contain all the updates for a particular stream, so a stream replica can playback updates locally and directly service requests to clients, a departure from the traditional client-driven shared log paradigm. This removes the burden of playback from clients and avoids the playback bottleneck of previous shared log designs. Garbage collection is greatly simplified. In Tango, clients cannot trim streams directly. Instead, they must read the stream to determine which log addresses should be released, and issue trim calls for each log address, which can be a costly operation if many entries are to be released. In vCorfu, clients issue trim commands to stream replicas, which release storage locally and issue trim commands to the global log.While the Corfu log provides a scalable fabric for consistency, programmers need more than just a shared log abstraction to write reliable distributed programs. This chapter introduces the rich, object-oriented data services provided by Corfu. Instead of forcing programmers to produce and consume entries on the log, the distributed objects provided by Corfu enables applications to use the log by interacting with in-memory objects which are similar to common in-memory data structures used today. In addition to supporting in-memory objects, Corfu also supports transactions, which enable applications to access and modify multiple objects consistently. Finally, materialized streams enable local playback of the log, which enable thousands of clients to consume the log simultaneously.A Corfu application is typically a service running in a cloud environment as a part of a larger distributed system, managing metadata such as indices, name spaces, membership, locks, or resource lists. Application code executes on clients and manipulates data stored in Corfu objects, typically in response to networked requests from machines belonging to other services and subsystems. The local view of the object on each client interacts with a Corfu runtime, which in turn provides persistence and consistency by issuing appends and reads to an underlying shared log. Importantly, Corfu run times on different machines do not communicate with each other directly through message-passing; all interaction occurs via the shared log. Applications can use a standard set of objects provided by Corfu, providing interfaces similar to the Java Collections library or the C++ STL; alternatively, application developers can roll their own Corfu objects.The code for the Corfu object itself has three main components. First, it contains the view, which is an in-memory representation of the object in some form, such as a list or a map; in the example of a Corfu Register shown in Figure 5.1, this state is a single integer. Second, it implements the mandatory apply upcall which changes the view when the Corfu runtime calls it with new entries from the log.

The view must be modified only by the Corfu runtime via this apply upcall, and not by application threads executing arbitrary methods of the object. Finally, each object exposes an external interface consisting of object-specific mutator and accessor methods; for example, a CorfuMap might expose get/put methods, while the Corfu Register in Figure 5.1 exposes read/write methods. The object’s mutators do not directly change the in-memory state of the object, nor do the accessors immediately read its state. Instead, each mutator coalesces its parameters into an opaque buffer – an update record – and calls the update_helper function of the Corfu runtime, which appends it to the shared log. Each accessor first calls query_helper before returning an arbitrary function over the state of the object; within the Corfu runtime, query_helper plays new update records in the shared log until its current tail and applies them to the object via the apply upcall before returning.Based on our description thus far, a Corfu object is indistinguishable from a conventional SMR object. As in SMR, different views of the object derive consistency by funneling all updates through a total ordering engine. As in SMR, strongly consistent accessors are implemented by first placing a marker at the current position in the total order and then ensuring that the view has seen all updates until that marker. In conventional SMR this is usually done by injecting a read operation into the total order, or by directing the read request through the leader; in our case we leverage the check function of the log. Accordingly, a Corfu object with multiple views on different machines provides linearizable semantics for invocations of its mutators and accessors. A Corfu object is trivially persistent; the state of the object can be reconstructed by simply creating a new instance and calling query_helper on Corfu. A more subtle point is that the in-memory data structure of the object can contain pointers to values stored in the shared log, effectively turning the data structure into an index over log-structured storage. To facilitate this, each Corfu object is given direct, read-only access to its underlying shared log, and the apply upcall optionally provides the offset in the log of the new update. For example, a CorfuMap can update its internal hash-map with the offset rather than the value on each apply upcall; on a subsequent get, it can consult the hash-map to locate the offset and then directly issue a random read to the shared log. Since all updates are stored in the shared log, the state of the object can be rolled back to any point in its history simply by creating a new instance and syncing with the appropriate prefix of the log. To enable this, the Corfu query_helper interface takes an optional parameter that specifies the offset at which to stop syncing.