Tag: vertical farming

During the year 1994, 5.314 hectares were eradicated .However, according to US estimates, poppy fields that year did not fall below 20,000 hectares, an estimate that was never denied by the Colombian authorities.The illegal heroin trade of the eighties and nineties appeared to follow a similar pattern to that of the marijuana business in the sixties and seventies. In the case of marijuana, the production triangle in this hemisphere had been made up of Mexico, Jamaica and Colombia. When repression took its toll in one country, especially due to the use of herbicides, the business moved to another, although it always returned to the spot where initially larger amounts had been planted. Something similar occurred with poppies between Mexico, Guatemala and Colombia. The original problem of illegal crops was never overcome, nor were the authorities able to dismount the equipment and infrastructure which enabled such plantations and laboratories to stay in business in the above-mentioned countries. By attacking temporarily, and in an isolated fashion an illegal crop, public anti-drug policy automatically attacks the weakest and least decisive link in the vast and complex chain of illegal drug dealing, and at the same time has the worst possible negative effect from a social viewpoint on small farmers and Indian populations, while affecting hardly at all the area of organized crime financed by the drug traffickers. The Gaviria administration had decided to deal with the drug problem by placing its emphasis on a policy of submission and making clear that it differentiated between drug trafficking and narco-terrorism. President Gaviria stated that “while narco-terrorism is our problem, drug trafficking is an international phenomenon.” Nonetheless, in the case of poppy growing, Gaviria did what former governments had done in their attacks on coca and marijuana fields.

The results of his efforts were insignificant and ephemeral, as were those of his predecessors. When a government acts on the basis of punishment alone and without offering incentives,indoor vertical farming believing that it is indulging in a technically-approved and non-harmful type of fumigation, it finishes up contributing to environmental damage and to a greater social breakdown in the zones where the plantations are grown.But discussions on the subject assumed an elitist, moral tone: on one side were the “good, hard-line, intelligent people” uncontaminated by drug traffic, and on the other “the softies, the badies, the dumb idiots” who were either mouthpieces of the traffickers or were unconsciously letting themselves be used by them. In February 1992, Colombia’s Justice Minister made a comment which illustrates this point: he claimed that “a cloak of complicity has been thrown over things by those who object to herbicide fumigation for environmental reasons, while all the time playing into the hands of the drug traffickers.”At no time was there a lobby sufficiently coherent, serious and affirmative to combat the government’s determination to keep on fumigating. The executive did not receive substantial criticism nor impediments to the actions it carried out through legislation and the judiciary. The government was therefore able to go ahead with its eradication policy with few internal restrictions. Even so, the result was not very positive; the rise of the poppy emporium in Colombia amply demonstrated the limits of the government’s public anti-narcotics policy and the dramatic consequences of unremitting prohibition on the part of the United States. The Colombian government did not attack drug trafficking or narco-terrorism on the financial front. In accordance with the logic of the so-called economic liberalization fomented by the government in the early nineties, it made no sense to place restrictions and greater controls on the free movement of capital. In 1993, a report by the Vienna-based United Nations International Board on Fiscal Control of Narcotics recommended that “Colombian legislation consider the laundering of capital resources to be a crime and that banking laws should become stricter in order to allow for multilateral cooperation….”The alleged financing of the presidential election campaign with drug money formed the backdrop to the anti-narcotics policies of President Ernesto Samper’s administration . As months went by, the coercive diplomacy which the United States had hitherto been exerting on Colombia became transformed into “blackmail diplomacy.”The president’s capacity for political survival led him to “North Americanize” the fight against drug trafficking in Colombia; that is to say, the president accepted and implemented a strategy virtually imposed by the United States. The Samper government undertook an all-out chemical eradication campaign far beyond anything seen in the two preceding decades, with massive use of glyphosate. Fumigators also employed imazapyr, a more powerful granulated herbicide, and were planning to use tebuthiouron, an even more devastating killer than the others. Ernesto Samper also became the president who most helped criminalize the drug trade, while in Colombia it became almost impossible to discuss the subject of legalizing drugs, something Samper himself had suggested in the late seventies, given the failure of repressive measures taken at that time by the Turbay administration and fomented by the United States.The Samper government even went beyond the demands of the United States executive and legislature. In 1995, months before the infamous “Frechette Memorandum”began to circulate — a document which suggested that Colombia should adopt legislation and take drastic measures in the anti-drug war — President Samper had launched his “integral plan” announcing, amongst other things, the creation of Operación Resplandor designed to put a definite end to all illegal crops which existed in Colombia in the space of two years.”An all-out eradication policy had been set in place. In 1994 , 4.094 hectares of coca were eradicated. In 1995, the Samper administration eradicated 25,402 hectares; and in 1996, 9,711 hectares. In 1994, the Gaviria and Samper administrations had eradicated 5,314 hectares of poppies. In 1995, the Samper government eradicated 5,074 hectares; and in 1996, 6,044 hectares Between the years 1995 and 1996, glyphosate was used on a massive scale to destroy illegal crops.Even so, the idea of putting an end once and for all to illegal crops proved again to be illusory. In 1996, the US government estimated that the number of hectares dedicated to the planting of coca in Colombia had reached 53,800 hectares, while independent estimates placed the figure at around 80,000 hectares.This meant that Colombia had surpassed Bolivia, a country which traditionally was second only to Peru as a coca producer in South America. The same official US source estimated that Colombia had 4.133 hectares of marijuana and that the country had produced 63 tons of heroin in 1996. However, Colombians had their greatest surprise of all in 1996 when small farmers from the south, especially from the Caquetá region, suddenly made their presence felt in mass demonstrations and protest marches. Nobody had expected this. It was as if the whole population had discovered overnight, and a little belatedly, that the country had ceased to be the processor of these stimulants and had transformed itself now into something else: a huge grower of illegal crops. People also came to realize that the state simply did not operate at all in a large and strategic portion of the country’s territory, and that power, at the local level, was in the hands of insurgent groups, especially in those of the FARC . Colombians came to realize as well that violent measures alone were not going to solve the profound and intricate social, political and economic problems which had been incubating for decades in the nation’s geographic wilderness.In sum, fumigating with herbicides in southern Colombia in 1996 turned out to be as useless for dismantling the illegal business of drug dealing as had similar efforts in previous years. The difference was that, in 1996, paramilitary detachments were multiplying at a frightening rate in the south. The political blindness of people in government, hydroponic vertical farming police officers and the military, together with the administration’s obsequious submission to United States policies, led to a repeat, in 1997, of the indiscriminate fumigation with herbicides — on a huge scale with glyphosate, to a lesser extent with imazapyr. In 1997, Colombia sprayed 41,847 hectares of coca and 6,962 hectares of marijuana. Twenty-two hectares of coca were eradicated manually, as well as twenty-five hectares of poppies and 261 hectares of marijuana. In just over three years, the government had fumigated more than 100,000 hectares of illegal crops. But paradoxically that only went to prove, as never before, just how mistaken, harmful and counter-productive the chemical destruction of such crops could be; in 1998, almost 110,000 hectares of the national territory were dedicated to plantations of coca, marijuana and poppies. In that year, the Samper administration , and that of Andrés Pastrana , fumigated 66,083 hectares of coca and 2,931 of poppies, and manually destroyed 3,126 hectares of coca, 181 of poppies and 18 of marijuana.From the mid-nineties up to the present time, Colombia has broken all historical records in the matter of fumigation. And yet the data on the eradication of illegal crops in Colombia has never been more negative. For example, according to US estimates, in 1990 heroin production in Colombia was hardly worth mentioning; there were 32,100 hectares of coca plantations, and marijuana was being grown in 1,500 hectares. In 1996, Colombia was producing 63 tons of heroin annually, while 32,100 hectares were planted in coca and 4,133 in marijuana.In 1998, Colombia produced 435 metric tons of cocaine, and in 1999 it was producing 520 metric tons, and in the year 2000 production had gone up to 580 tons.According to Colombia’s Anti-narcotic Police, the Pastrana government had destroyed approximately 50,000 hectares of coca plantations by 1999 , and the US State Department gives a total of 56,254 hectares eradicated by Colombia in the year 2000.50Nonetheless, according to the Central Intelligence Agency , the total area planted in coca in 1999 amounted to 120,000 hectares,and the US State Department declared that this had increased to 136,200 hectares in the year 2000. This means that in just four years, from 1996 to 2000, the surface planted in coca in Colombia has doubled; the total number of hectares went from 8,280 to 13,200. An increase in the fumigation of illegal crops has not resulted in a decrease in the area planted with illegal crops, nor to a decrease in the production of illegal drugs. To this evident failure one must add the fact that, on the US market, cocaine and heroin have become both cheaper and purer. It is worth noting, also, that something similar has occurred in Western Europe where, in 1999, a gram of cocaine was worth US$90, and a gram of heroin was fetching US$98. So, the rationale which attempts to justify a strong eradication policy in the centers of supply has proved to be way off the mark. It had been presumed that the massive destruction of illegal drugs where production and processing were taking place was going to lead to less availability of narcotics in the centers of demand, an increase in price for the ultimate consumer and a lowering of standards of purity in the stimulants themselves. Quite the opposite has happened; in the year 2000 one could procure in the United States more drugs of better quality than ever before, and at lower prices. Besides, in terms of illegal drug consumption and of drug-related crime, the United States record has not shown substantial improvement. In 1988 the number of occasional consumers of heroin was reckoned at 167,000; in 1995 it had reached 322,000; while the total number of heroin consumers worldwide went from 692,000 in 1992 to 810,000 in 1995. The overall demand for heroin was 1,800,000 grams per year in 1988, but by 1996 it had soared to 2,400,000.53 Despite certain laudable achievements in reducing drug consumption in the United States, it is evident that a strong demand still exists. In this context it is worth quoting Bruce Bagley: “Some 13 million US drug users spent approximately US$67 billion on illicit drugs in 1999, making the US market the most lucrative one in the world for Colombian traffickers.”Concomitantly, in 1990 the total number of arrests in the area of drug-related law infringements was 1,089,500, whereas in 1996 the figure had risen to 1,128,647. In 1990, 53 percent of prisoners in federal jails were serving sentences for narcotic-related crimes; in 1995 the statistic had risen to 59.9 percent.

Inspection services are provided by the Federal Grain Inspection Service, the Food Safety Inspection Service, and the Packers and Stockyards Administration. The state government also provided approximately $147 million for agricultural plant and animal health, pest prevention and food safety services. Outlays for the Foreign Agriculture Service, Agricultural Marketing Service, and Office of Transportation comprise the federal portion of processing and marketing assistance. For the 1999-2001 period, the average state outlays for California Department of Food and Agriculture marketing, commodities and agricultural services totaled around $60 million. For those commodities with relatively small amounts of total support, marketing assistance provides the bulk of the support. Assessments are subtracted from outlays to determine the contribution to the PSE. Finally, there are state and federal marketing order, board and commissions for many California commodities. These are generally financed by check-off systems that apply a kind of excise tax on the marketed commodity to support promotion or research .Infrastructure support includes federal soil conservation programs, which provide assistance in reducing soil erosion and degradation of resources. While the contribution of these programs to overall support of California agriculture is small, they are included as a separate category for consistency with the PSE calculation.There are various tax benefits for agriculture and foreign sales corporations that indirectly support the agricultural industry. Nelson, Simone and Valdes have compiled the total value of federal tax benefits to agribusiness and have also calculated the value of inland waterway construction and railroad interest rate subsidies. In general, plastic pots 30 liters the value of transportation subsidies is relatively small, usually around 2 percent of total support for each commodity. This is likely an over-estimate, however, because the California share in these benefits is likely smaller than the California share of agricultural output .

Tax breaks were a larger share of the support, but were not substantial by themselves. We did not include in our PSE calculations the value of state and local real estate tax benefits to agriculture. California, like many other states in the United States, provides for a special taxation rate on agricultural real estate. The state’s Williamson Act, introduced in 1965, provides a preferential assessment program for agricultural land. Williamson Act acreage currently represents almost half of California agricultural land. Under the Williamson Act, landowners sign a contract with the appropriate local government agency restricting urban use of that land for ten years. In return, property under Williamson Act protection is assessed for tax purposes according to its capitalized agricultural income. Capitalized income assessments are usually about half of the market value-based assessments for Williamson Act land; thus landowners receive approximately $120 million in tax benefits. Contracts may be terminated through non-renewal or cancellation. Non-renewal gradually phases in the market value-based assessment over nine years; at the end of the ten-year contract, the land is appraised at full market value. Cancellation of Williamson Act contracts must be approved by the local governing board after conducting public hearings. If the contract cancellation is approved, the landowner pays a penalty of 12.5 percent of the current market value of the land .Dairy policy is discussed in detail above. Here we note only that, in addition to trade protection and internal price policies, the dairy industry receives support from several smaller programs as well. In addition, the dairy industry receives indirect support in the form of subsidies to the grain industry and, especially, the alfalfa hay industry. Hay is important in dairy production, accounting for about 20 percent of total costs. The major subsidy for alfalfa is irrigation water; some have argued that the water subsidy to alfalfa is a major contributor to lower dairy production costs in California. Let’s examine this proposition. Total alfalfa support is about $34 million. Most of this, about $15 million is attributable to the irrigation water subsidy.

Some of the alfalfa and other hay grown in the state is consumed by other livestock. Approximately $12 million of the water subsidy to hay is ultimately of benefit to the dairy industry. If the $12 million were added to a subsidy of about one billion dollars, it would raise the overall dairy subsidy from 33.4 percent to 33.6 percent. In other words the effect of irrigation subsidy on dairy is very small, especially compared to the subsidy from other sources.Commodities in this category have little government intervention in their markets. The PSEs range from about 3 to 5 percent of the revenue. There are no significant trade barriers or direct payments for these commodities. The main portion of support comes from input assistance, marketing assistance, broad government infrastructure and economy-wide policies. While these commodities have no explicit export subsidies, they do benefit from foreign market development funding to some degree, especially almonds and strawberries . Crop insurance benefits and disaster payments are also a source of a small amount of support for this group . In the citrus industry, crop insurance and disaster payments comprise almost 30 percent of the support; large payments were made following the 1990 freeze that took a heavy toll on the California citrus industry . Most commodities in this group have some sort of marketing order, either federal, state, or both. The marketing order share of total support ranges from 3 percent to around 25 percent . The share of support from research is relatively high for these commodities, around 25 percent. Nevertheless, since these percentages equal very small PSEs for the horticultural commodities, the overall subsidy is quite small.The federal programs for these commodities were discussed in detail above. Direct government payments provide the lion’s share of support: 90 percent for rice, 74 percent for cotton, 86 percent for feed grains, and over 76 percent for wheat. Cotton, wheat, and rice have active marketing orders but compared to the value of the direct income supports, the marketing order budgets are relatively small. The magnitude of the direct payments and the export subsidies also make the value of the input assistance, marketing assistance, infrastructure, and economy-wide policies a small percentage of total support.

As noted above, the most important feature of support for alfalfa and other hay is the water input subsidy. Alfalfa production in California uses approximately 2.3 million acre-feet of CVP or SWP water per year. Like fruits, nuts, and vegetables, alfalfa production does not benefit from trade barriers or direct payments. Research accounts for about 15 percent of alfalfa support, while the input assistance , marketing assistance, infrastructure, and economy-wide policies provide about 35 percent. Excluding water, the alfalfa industry would have a PSE of 2.2 percent.One of the major problems in California is that the state’s water is concentrated in the north, but the majority of the state’s urban population and irrigated agriculture is located in the south. California contains 32 million acre-feet of developed water,round plastic pots of which 84 percent is used to irrigate 9.68 million acres of agricultural land. Because such a large proportion of water resources is used for irrigated agriculture, most water management conflicts involve the movement of water to or from irrigated agriculture. While most of the water is used to irrigate field and fodder crops, the high value vegetable and fruit crops generate the majority of agricultural revenues.From the 1950’s to 1970’s different government agencies at the State and Federal level implemented a massive water development program in California. This program was built upon the traditional supply augmentation approach to water development. Unfortunately this approach to water development is flawed. The main weakness of the traditional supply based method is that it assumes that the demand for water is perfectly inelastic and unchanging over time. An inelastic demand assumes that there is little quantitative response to changes in the price of water. Under this planning approach the quantity of water to be delivered by a water project is fixed, and the only question is how to minimize the costs of supplying it. Economic analysis is then performed to see if the total costs of the water project are less than the total benefits. Both the State Water Project and the Federal Central Valley Water Project were developed using the principles of the supply-based approach to water development. The SWP was originally projected to supply an average annual quantity of 4.2 million acre-feet of water in two stages. The first stage of 2.2 million acre-feet was built and put into service in the late 1960’s and early 1970’s.

However, subsequent attempts to build the remaining 2 million acre-feet capacity have met with effective opposition from environmental interests, who want to prevent any further water development, and current contractors, who know that the average cost of water delivered by the system will have to increase by up to 300 percent to finance the completion of the planned project. In 1994 the SWP project contractors and operators met to renegotiate the conditions for water sales among contractors and the allocation of cuts in water deliveries during drought periods. The resulting Monterey agreement also enabled contractors who overlie a state operated groundwater storage project to exchange the control of the project for surface water entitlements; these entitlements could then be transferred to urban contractors. Finally, the agreement sanctioned the permanent transfer of 130 thousand acre-feet of water from agricultural to urban users. The CVP parallels the SWP and delivers 4.6 million acre-feet of water to both urban and agricultural contractors. Urban contractors receive 10 percent of total water deliveries while the remaining 90 percent of water is diverted to agricultural contractors. The CVP was operational in 1965, but by 1992 there was considerable political pressure to modify the operation of the project to reduce environmental damage to different fish populations in the Sacramento River Delta. The resulting Central Valley Project Improvement Act reallocated water to environmental uses by cutting water deliveries by 1 million acre-feet in normal rainfall years and by 804 thousand acre-feet in critical rainfall years. The CVPIA mandated that 800 thousand acre-feet of water be reallocated to in stream uses to protect the salmon runs, while 400 thousand acre-feet of water be reallocated to wildlife refuges . Water markets in the CVP districts are limited to local sales among agricultural contractors. These sales are short in duration and are generated by differences in the water allocations between farm regions and years. Due to institutional constraints, CVP water is still largely used for agricultural irrigation despite a three-fold difference between the value of water in nearby urban sectors and agricultural sectors.In recent years, State and Federal law have mandated a set of modifications that affect both the state and federal water projects in California. In 1996 and 1997 California developed the 4.4 Plan that aims to reduce diversions from the Colorado River to 4.4 million acre-feet over a period of 15 years. Moreover, in 2000 the Environmental Water Account was implemented by the state and federal governments. The purpose of the EWA is to regenerate the fisheries of the San Francisco Bay-Delta system while simultaneously securing water supplies to both urban and agricultural users. Both these developments have encouraged water trading.This transfer, while historic, is more like an intergovernmental reallocation than a prototypical water market exchange. The QSA settled a large array of issues regarding use and conveyance of Colorado River water, many of which were unrelated to the transfer itself. There is also some question as to the willingness of IID to enter into the agreement. While it appears that many landowners and the IID itself will benefit substantially from the agreement, local opposition to the transfer remained strong until the Bureau of Reclamation found under a Section 517 proceeding that IID’s use of water exceeded “reasonable and beneficial” amounts. This finding raised the possibility that, unless transferred, IID stood to lose a significant share of its annual use with no compensation.Figure 3 plots both actual transfers and regression predictions of water transfers in California between 1985 and 2001. The regression fitted to water transfer data confirms that rainfall levels have a significant effect on annual water transfers . The data also confirms a positive correlation between the time trend and water transfers. When expressed as a percentage of the mean level of water transfers, the regression time trend shows an annual growth rate of 1.26 percent over the period.

Several California marketing order and commission commodity promotion and research programs have recently been involved in litigation as a small minority of unhappy producers and handlers have turned to the courts with requests to modify or terminate the programs. Recent court cases involving constitutional challenges include actions against the marketing orders for peaches and nectarines, kiwifruit, plums, apples, grape root stocks, cut flowers, almonds, milk, cling peaches, and table grapes.Marketing order quantity controls can be a powerful economic tool when the commodity group controls most of the production of the commodity and when there are different markets with different price elasticities of demand. Under these conditions, the commodity group can gain a measure of monopoly power and enhance returns through price discrimination. However, since they are unable to control entry, any short-run price enhancement will lead to a longer-run supply response. It is not surprising that quantity controls have been controversial—monopoly pricing practices reduce the welfare of some consumers and may distort resource allocation decisions, while producers face all of the problems of maintaining a cartel. Marketing orders for several California commodities include quantity control provisions , although the use of quantity controls has decreased over time as a result of problems noted above. The federal marketing orders for citrus, with their prorate provisions, were terminated at the end of the 1993-94 crop year after more than 50 years of almost continuous use. The citrus prorates set the amount of lemons and oranges that could be shipped to the domestic fresh market on a weekly basis.The demand facing packers in the fresh market is very inelastic relative to the demands in the processing and export market. Thus,stacking pots price discrimination against the fresh market, by restricting the flow of product to it, is both possible and profitable. Short-run producer price enhancement without any controls on entry led to an acreage response for both lemons and Navel oranges.

As new plantings reached bearing age, the Administrative Committees were forced to divert increasing proportions of the annual crop to exports and processing to maintain fresh market prices. Producer returns from all markets decreased over time, until new plantings were no longer profitable. However, when compared to a competitive solution, prorate resulted in increased acreage and production of citrus, as well as increased exports and processed products . Opponents of the citrus volume regulations, who had been sued in 1983 by the United States for violations of prorate, discovered evidence of over shipments by a large number of competing orange and lemon packing houses.Because of allegations of limitations violations of shipments under the order, a series of lawsuits, investigations, and proposals for penalties under AMAA forfeiture rules threatened to keep the industry in court for years and create economic hardships for many industry participants. To minimize long-term damage to the industry and “to end the divisiveness in the citrus industry caused by over ten years of acrimonious litigation,” the Secretary of Agriculture terminated the California-Arizona citrus marketing orders, effective July 31, 1994, and dismissed all litigation brought pursuant to the AMAA.Raisins provide another good illustration of volume controls in California. California is the largest volume raisin producer in the world, and this industry has operated under a federal marketing order program with volume controls since 1949. Under the raisin marketing order, annual production is divided between free tonnage and a reserve pool, and the Raisin Administrative Committee controls the reserve tonnage. Only free tonnage can be sold on the domestic market, but the RAC can allow packers to buy additional tonnage for free use from the reserve when the RAC determines that such actions are justified by supply and demand conditions. Until 1977, the majority of raisins in the reserve pool were exported at prices that were much lower than for raisins sold on the domestic market. Raisins from the reserve were also used for the school lunch program, government subsidized exports, other government programs, sales to wineries for distilling into alcohol, donations to charity, and cattle feed. Thus, the raisin industry working through the RAC successfully used the reserve pool to practice price discrimination in separate domestic and export markets.

Conditions and markets changed, however, and beginning in 1977, exports were considered free tonnage shipments, and the initial free tonnage was increased to serve favorable export markets. Since 1977, the RAC has often exported reserve pool raisins at prices competitive with world prices but below prices on the domestic market. Finally, the experience of the California almond industry illustrates how changing market conditions can alter the effectiveness of volume controls.The federal marketing order for California almonds includes provisions for market allocation and a reserve pool. At the beginning of each marketing season, the Almond Board of California recommends to the Secretary of Agriculture a maximum annual quantity to be sold in domestic and export markets and the quantity that cannot be sold . The reserve may be designated as either unallocated or allocated reserve. The unallocated reserve is essentially forced storage; nuts can be released from the unallocated reserve as the season progresses or carried over to the following season. The allocated reserve must be utilized in noncompetitive outlets such as almond butter, almond oil, airline samples, or cattle feed. The reserve provision of the almond marketing order was used to encourage export sales through 1972, while maintaining higher prices in the domestic market than in the export market. This price discrimination ended when export markets became an important outlet for California almonds , with price elasticities tending to equalize between domestic and export markets. Recent work indicates that the price elasticity of demand for almonds is now more elastic in the domestic market than in major export markets, leading to the result that short-run revenue maximization through price discrimination could involve restricting sales to export markets . Recent models of acreage response to changing returns indicate that U.S. and Spanish producers each increase production when returns appear favorable . Thus, if the Almond Board were to use the reserve to practice price discrimination and raise world almond prices, increased prices would stimulate production in Spain as well as the United States. As a consequence of these various considerations the almond industry has not implemented volume controls for the past several years. All existing federal marketing orders for California fruits, vegetables, and nuts include provisions for grades and minimum quality standards. However, only ten of the California State marketing programs include quality standards and inspection provisions, and just seven actively use the provisions. Given typical seasonal price relationships for fresh fruit, with high early-season prices, there are strong incentives to ship fruit as early as possible, even though it may not be fully matured. Most consumers are unable to judge the maturity of fruit from appearance and may find that fruit that “looks good” does not “taste good.” The result is an adverse selection problem.

Sellers are aware of the product’s characteristics, but buyers are unaware. In these settings, low-quality products can drive high-quality products from the marketplace. Indeed,fodder growing system representatives of many commodity groups believe that shipments of immature fruit have a negative impact on total sales, because consumers may delay repeat purchases after being dissatisfied with their original purchases. Maturity standards based on sugar content, firmness, and color are used by several marketing orders to determine when fruit is mature enough to be shipped. Minimum quality standards may: increase the retail demand for a product, resulting in higher prices and/or increased sales; reduce marketing margins, with benefits accruing to both producers and consumers; and reduce supply, which with inelastic demand can increase total revenue to producers. Any effective minimum quality standard will restrict the quantity of commodity marketed, but supply control is not the usual focus of such standards. Federal marketing order regulations on grade, size, quality or maturity also applies to imports of the same commodities from other countries during the period the marketing order is in effect. The use of some minimum quality standards has been controversial. Concerns include charges that quality standards are a hidden form of supply control, that quality standards waste edible fruit with the primary impact being on the poorest consumers, and that quality standards are sometimes not equitable because of regional variations in production conditions. While empirical analyses of the economic impact of minimum standards of grade, size, and maturity for California commodities are limited, those available indicate that it is probably relatively small .The purpose of commodity group expenditures on generic advertising and promotion is to increase the demand for the commodity so that more commodity can be sold for the same price, or the same amount can be sold for a higher price. The rationale for mandatory support by all producers is based on the distribution of documented program benefits and the “free-rider problem.” 5 Research completed and underway documents significant increases in product demand as a result of commodity advertising and promotion programs, with net monetary benefits to producers being much greater than costs. For example, Alston et al. estimated that the elasticity of demand with respect to promotion for California table grapes was 0.16.

Using this promotion coefficient, they estimated that the promotional activities of the Table Grape Commission had increased per capita consumption by about 1.5 pounds over that which would have existed in the absence of a promotional program. This increase was about one-third of recent total per capita consumption. The benefits to producers were very high in both the short- and long-run. The short-run marginal benefit-cost ratio was estimated at over 80:1—for every $1 spent on the program, the industry gained net benefits of $80. When producer supply response was factored into the analysis, the benefit-cost ratios decreased. Using a supply elasticity of 5, the average benefit-cost ratio was about 10:1 and the marginal benefit-cost ratio was about 5:1. Studies of the estimated returns from advertising and promotion programs for other California commodities include avocados , prunes , almonds , eggs , raisins , and walnuts . Each of these studies found that advertising and promotion increased the demand for the product and that program returns exceeded costs by a significant margin. The U.S. government has funded agricultural commodity groups, as well as private firms, to conduct promotional programs in export markets. The Market Access Program and its predecessor programs, the Market Promotion Program, and the Targeted Export Assistance Program, have provided matching funds for the promotion of a number of California commodities. Federal allocations of funds to 15 commodity boards, commissions, and other groups promoting only California fruits, nuts, and vegetables totaled $18.67 million in fiscal year 2002. These funds accounted for 18.7 percent of $100 million awarded to all organizations. Organizations that promote products produced in other states as well as California also received large allocations. California organizations receiving 2002 awards greater than $1 million included Blue Diamond Growers/Almond Board of California, California Prune Board, California Table Grape Commission, California Walnut Commission, Raisin Administrative Committee, Sunkist Growers, Inc., and Wine Institute.Research and development provisions are included in most of the California marketing programs. In 1992, there were 28 programs with research expenditures totaling almost$8.5 million . This increased to 38 California programs with a total 2002/03 or 2003/04 budget of $20.2 million. The largest research budgets were for citrus , rice , market milk , almonds , avocados , fresh strawberries , and walnuts . Overall, research expenditures increased from about 7.5 percent of total 1992 commodity group expenditures to just over 10 percent of 2003 expenditures. In terms of the total farm level value of production, research budgets averaged just over 0.1 percent of the 2001 value of covered commodities. Summary statistics on the economic impacts of commodity group research expenditures are limited, but those available indicate attractive rates of return. Most of the research funded by commodity groups operating under state marketing orders and commissions is done at the University of California. A study valuing California agricultural research concluded that the average annual internal rate of return for public investment in California agricultural research and extension for 1949-85 was about 20 percent . Consider, for example, the case of strawberries. California has become the world’s pre-eminent strawberry producer, now accounting for about 80 percent of U.S. fresh and processed production.

California’s agriculture is also a source of demand for both annual and perennial plants and trees, e.g., vegetable transplants, strawberry plants, seeds of all kinds, root stock for trees, and young nursery stock for new plantings and replacements of vines, tree fruits, and nuts. The types of firms producing nursery products vary widely, including extensive field operations, outdoor nurseries, and intensive greenhouse operations.Consumer demand for lettuce is relatively inelastic, and prices vary widely for this perishable commodity depending on acreage and weather-dependent supply conditions. Large grower-shippers operate in the several production areas in California and Arizona, moving with the seasons. The nation’s “salad bowl” is the Salinas Valley in Monterey County, where lettuce is harvested from April through early November. Other coastal areas produce during the same period. The Imperial Valley and other desert areas ship from early December until mid-March. Production on the west side of the San Joaquin Valley fills the market niches between the two major production areas. Field packing, vacuum cooling, and refrigerated transportation are key components requiring coordination for moving lettuce from the field to the consumer with minimal post-harvest loss in quality. Development of value-added pre-package salad greens has reduced shipments of “Iceberg” head lettuce and effectively increased the demand for other greens, including romaine and leaf lettuce.Almost all breeds of beef cattle are raised in California. The dairy sector also contributes a significant quantity of steers, culled cows,hydroponic nft and bulls as animals marketed for beef. Cattle and calves were California’s #1 agricultural commodity until 1980, when the number one position was taken by milk and cream. Later, grapes and nursery products moved ahead of cattle and calves.

Lettuce became the fourth most important crop in 2000. Cattle and calves are now California’s #5 agricultural commodity. More than two-thirds of the state’s land area is essentially non-tillable because of steep slopes or poor soils. These areas are typically used as rangeland for cattle. In addition, cattle are raised an irrigated pasture lands in the foothill areas and on marginal agricultural lands in the Central Valley. The cattle feeding industry is located primarily in the Imperial and San Joaquin Valleys. Cattle are also shipped out of state to feed lots closer to midwest feed supplies. Climate, topography, and overall conditions vary widely within the state, as do the sizes and types of cattle operations. Some are purely cow-calf operations, while others buy and sell animals as stockers, replacements, or feeders to fit the carrying capacity of owned and leased lands. All areas present separate and distinct challenges to cattle production in terms of rainfall, temperature patterns, topography, breeding and calving conditions, transportation, marketing, urban development, and cattle rustling and vandalism.Hay as a commodity category includes alfalfa hay, grain hay, green chop, sudan hay, and wild hay, but alfalfa is by far the most important component, contributing about 85 percent of the value of all hay production. Alfalfa hay acreage in California has averaged about a million acres, but is influenced by profitability of alternative annual crops , trees, and vines. The demand for alfalfa hay is determined to a large part by the size of the state’s dairy herd, which consumes about 70 percent of the supply. Horses consume about 20 percent. Alfalfa hay is grown in every climatic zone of the state. Climate determines the number of cuttings of hay. In the low desert there are as many as eight to ten cuttings per year; in the cool northern intermountain region, farmers harvest only two to four cuttings a year. Most of the crop is not used on the farm where it is produced, but is usually baled and shipped to end users. Pellets and cubes are other forms for equines and export markets. Alfalfa, a perennial crop with a three- to five-year economic life, does best when planted on well drained, deep, medium-textured soils.

Because it is a highly water intensive crop, its production cost will be directly affected by higher water prices and pumping costs, reducing the long-term profitability of the crop in the state’s crop mix.Flowers and greens are sold in cut and in potted forms. The major areas of production are the coastal counties where the typical mild climate permits outdoor production and lower-cost greenhouse operations. The major production areas of cut flowers are in the counties surrounding San Francisco Bay, extending southwest to Salinas, and in the coastal regions of San Diego and Santa Barbara Counties. The marketing of cut flowers in California is extremely intricate and complex. Although air shipments are used for transcontinental deliveries, most cut flowers are now precooled and shipped by refrigerated trucks. Increased imports, particularly from Columbia and Mexico, are a concern to California greenhouse growers of the three main cut flowers—roses, chrysanthemums, and carnations. The three have historically accounted for as much as two-thirds of the annual income from cut flowers and cut greens. Potted plants, including the seasonal items—poinsettias, lilies and hydrangeas are favored as consumers bring flowers and greenery into residences and offices. There are now more than 250 species and varieties of foliage plants being offered for sale in the trade.About three-quarters of the California strawberry crop is sold fresh; the remainder is sold for processing. Production of California strawberries runs from mid-February through mid-November and occurs in several growing areas along the southern and central coast. Even though strawberry plants are perennials, growers replant annually to obtain maximum yields and the best quality of fruit. Development of new varieties from an industry-supported fruit breeding program at the University of California has been important to the growth of the California strawberry industry.In 1950, California production of 2 million tons of processing tomatoes accounted for only 36 percent of U.S. production. The combination of favorable climate, good soils, ample water, an excellent highway system, applied technology, and research and development has fostered the growth of the processing industry in California which now produces 9 to 10 million tons annually, and as much as 12.2 million tons in 1999. Prices have fallen, as has acreage, while the industry is undergoing structural changes and reduced profitability. Tomato production is specialized and capital-intensive. Processing has changed from consumer products produced at multi-product plants to now include single product production at specialized “industrial plants” where tomato paste product is packaged in aseptic plastic containers in boxes and drums and shipped throughout the year to end users. Paste is simply a commodity bought and further processed into final consumer products—catsup, sauces, soups, etc.

Processing tomatoes are produced from the Mexican border to the northern Sacramento Valley. Harvest begins in the desert valleys in mid-June and continues northward in the Central Valley through September. A late harvest ends in the southern coastal counties in November. All processing tomatoes are harvested mechanically.California agriculture is large, diverse, complex and dynamic. This chapter documents the industry and its relationship to the rest of the economy. It also provides an overview of unifying forces and trends. Our aim is to supply a convenient compilation of facts and figures from a variety of sources, and to help the reader interpret the wide array of data presented.2 California agriculture is far larger, measured by sales, than that of any other state. California agriculture produces more value than most countries and is larger than, for example, such major agricultural producers as Canada or Australia.California is part of the national and international agricultural markets. Californians consume food that is produced in the state, as well as food that is imported from other states and countries. Agriculture in California is the largest among the states, and produces a variety of animals and animal products, fruit, tree-nuts, vegetables, field crops,hydroponic channel and nursery and floriculture products. The Central Valley accounts for more than half of the State’s gross value of agricultural production.About 93 percent of California’s 101.5 million acres is in rural uses. This rural area is divided evenly between federal and non-federal ownership. The federal land mostly includes national forests, national parks and wildlife areas, and “other land,” such as marshes, open swamps, and bare rock deserts. Roughly 11 percent of the federal rural land is grassland pasture and range used for agriculture. Of California’s 53 million acres of non-federal land, about 80 percent is grassland pasture and range, forest land, and cropland. About 5.5 million acres of California’s non-federal land are defined by the Natural Resources Conservation Service of the U.S. Department of Agriculture as “developed” for residential, industrial, and commercial use. However, the intensity of use varies widely, with much of this land relatively unpopulated. The California Department of Conservation Farmland Mapping and Monitoring Program defines 3.1 million acres of California’s non-federal land as “urban and built-up,” that is, land occupied by structures with a building density of at least one unit to one and one-half acres.

This suggests that roughly 2.4 million acres of “developed” land in the NRCS survey are still relatively rural, or not mapped by FMMP. In total, about 27.7 million acres, including 5 million acres of federal grazing land, are used for agriculture in California. More than half is pasture and range, about 39 percent is cropland, and the remainder is divided between woodland and other land.Conversion of agricultural land to urban uses continues to be a public policy issue in the United States and in California. In California between 1988 and 2000, according to the California Department of Conservation Farmland Mapping and Monitoring Program , about 549,000 acres were converted to urban and built-up uses. At these conversion rates, about 4.2 million acres would be converted in the next 100 years. Of the total acres converted from 1988-2000, 213,000 were formerly cropland and 100,000 were formerly grazing land. Another 235,000 acres were formerly “other land,” as classified by the FMMP. A significant portion of the “other land” was idled farmland previously removed from agricultural production in anticipation of development. This indicates that the figures for cropland and grazing land conversion may be understated. Farmland conversion is a topic of particular interest in the Central Valley, which has over half of the state’s agricultural land and 64 percent of the cropland. The Central Valley has had a lower proportion of its cropland and grazing land converted than the rest of the state. The Valley recorded 43 percent of statewide cropland conversion between 1988 and 2000. Similarly, the Central Valley grazing land, about 44 percent of the state total, contributed only 25 percent of the total grazing land conversions.Farmland conversion to urban uses is associated with population growth. California’s population increased by about 76 percent between 1970 and 2002, while the Central Valley’s population doubled. There is general agreement that state population growth will continue, but little consensus on precise projections of future growth rates. The Bureau of the Census estimates that the state population will be about 50 million by 2025.Nationwide, over the last half-century, the number of farms and the total land in farms have decreased, while the size of an average farm has increased. This trend has been less pronounced in California. While the average U.S. farm doubled in acreage between 1954 and 2002, the average California farm increased by about 13 percent. The official definition of a “farm” was changed in 1954, 1959, and 1974, to remove many of the smallest “farms” from census statistics. Each of these definitional changes decreased the reported number of farms and increased the average farm size. Since1974 a “farm” has been defined in the Census of Agriculture as a place that generates agricultural sales of at least $1,000 annually. Under the current Census of Agriculture definition, the average acreage of California farms decreased by 30 percent between 1974 and 2002. The 2002 Census introduced a new methodology for estimating total number of farms and operators’ demographics. The Census has been conducted via mail returns, and coverage has been always below 100 percent, especially among very small operations. The 2002 methodology accounts for all farms. In 2002, about 80 percent of California farms were less than 180 acres, yet the “average farm” size was 347 acres. These two statistics highlight the fact that a small percent of large farms account for a large percent of total acreage. These large farms include ranches that graze livestock and may generate relatively little total revenue.

The spread of irrigation broadly paralleled the intensification movement. Between 1869 and 1889, the share of California farmland receiving water through artificial means increased from less than one percent to five percent. Growth was relatively slow in the 1890s, but expansion resumed over the 1900s and 1910s. By 1929, irrigated land accounted for nearly 16 percent of the farmland.Data on the value and composition of crop output put California’s agricultural transformation into sharper relief. Between 1859 and 1929, the real value of the state’s crop output increased over 25 times. Growth was especially rapid during the grain boom of the 1860s and 1870s, associated primarily with the expansion of the state’s agricultural land base. Subsequent growth in crop production was mainly due to increasing output per acre and was closely tied to a dramatic shift in the state’s crop mix. After falling in the 1860s and 1870s, the share of intensive crops in the value of total output climbed from less than 4 percent in 1879 to over 20 percent in 1889. By 1909, the intensive share reached nearly one-half, and by 1929, it was almost four fifths of the total.3 Figure 1 provides further documentation of the transformation of California’s crop mix over the late 19th and early 20th centuries. The Figure shows how cropland harvested in California was distributed across selected major crops over the 1879-1997 period. The acreage data reveal that in 1879,nft hydroponic wheat and barley were grown on over 75 percent of the state’s cropland whereas the combined total for the intensive crops was around five percent. By 1929, the picture had changed dramatically. Wheat and barley then accounted for about 26 percent of the cropland harvested and the intensive crop share stood around 35 percent.

In absolute terms, the acreage in the intensive crops expanded over ten times over this half century while that for wheat and barley fell by more than one-third.Data on shipments of California fresh, dried, and canned fruits and nuts reveal the sector’s spectacular expansion over this period. During the 1870s and 1880s, growth rates exceeded 25 percent per year . Shipments continued to grow at robust rates of about eight percent per annum over the 1890s and 1900s. By 1919, California produced 57 percent of the oranges, 70 percent of the prunes and plums, over 80 percent of the grapes and figs, and virtually all of the apricots, almonds, walnuts, olives, and lemons grown in the United States. In addition, California produced significant quantities of apples, pears, cherries, peaches, and other lesser crops. The spectacular growth in California production of specialty crops had important international consequences as traditional Mediterranean exporters of many crops were first driven from the lucrative U.S. market and then faced stiff competition from the upstart Californians in their own backyard of northern Europe. California production significantly affected the markets and incomes of raisin growers in Málaga and Alicante, prune growers in Serbia and Bosnia, and citrus growers in Sicily.Explanations for the causes and timing of California’s structural transformation have long puzzled scholars. The traditional literature yields numerous causal factors, including: increases in demand for income-elastic fruit products in eastern urban markets; improvements in transportation, especially the completion of the transcontinental railroad; reductions in the profitability of wheat due to slumping world grain prices and falling local yields; the spread of irrigation and the accompanying breakup of large land holdings; the increased availability of “cheap” labor; and the accumulation of knowledge about California’s environment and suitable agricultural practices. Yet a careful investigation of the transformation yields a surprising result: much of the credit for the shift to intensive crops must be given to exogenous declines in real interest rates and to “biological” changes as farmers learned more about how to grow new crops in the California environment. Isolated from America’s financial markets, California farmers faced high, even astronomical, interest rates, which discouraged capital investments. Rates fell from well over 100 percent during the Gold Rush to about 30 percent circa 1860. The downward trend continued with real rural mortgage rates approaching 8 to 12 percent by 1890. The implications of falling interest rates for a long-term investment such as an orchard were enormous.

As one Bay Area observer noted in the mid-1880s, the conversion of grain fields to orchards “has naturally been retarded in a community where there is little capital, by the cost of getting land into orchard, and waiting several years for returns.”Calculations indicate that the break-even interest rate for the wheat-to orchard transition was about 10 to 13 percent . These estimates conform fairly closely to the interest rate levels prevailing in California when horticulture began its ascent. A second key supply-side force was the increase in horticultural productivity associated with biological learning. Yields for leading tree crops nearly doubled between 1889 and 1919. When the Gold Rush began, the American occupiers knew little about the region’s soils and climate. As settlement continued, would-be farmers learned to distinguish the better soils from poorer soils, the more amply watered land from the more arid, the areas with moderate climates from those suffering greater extremes. Occasionally overcoming deep-seated prejudices, farmers learned which soils were comparatively more productive for specific crops. California fruit growers engaged in a similar time-consuming process of experimentation to find the most appropriate plant stocks and cultural practices. Existing varieties were introduced from around the world, and new varieties were created. In the early 1870s, USDA plant specialists established the foundation for the state’s citrus industry with navel orange bud wood imported from Bahia, Brazil. Plums and prune trees were brought in from France and Japan; grape vines from France, Italy, Spain, and Germany; and figs from Greece and Turkey. Plant breeders also got in on the act. The legendary Luther Burbank, who settled in California in 1875, developed hundreds of new varieties of plums and other fruits over his long career.In part, the growth of horticultural knowledge occurred through the informal “folk process” highlighted in William Parker’s classic treatment of American agriculture. Over time, the process of research and diffusion became increasingly formalized and institutionalized.As an example, a series of major citrus expositions, held annually in Riverside from the late 1870s on, helped popularize the new Bahia orange variety. An emerging group of specialty farm journals, such as the Southern California Horticulturist, California Citrograph, and California Fruit Grower, supplemented the stalwart Pacific Rural Press to spread information about fruit growing.The California State Board of Horticulture, formed in 1881, provided an active forum for discussion of production and marketing practices, especially through its annual convention of fruit growers. The Agricultural College of the University of California, under the leadership of Eugene Hilgard and Edward Wickson, intensified its research efforts on horticultural and viticultural problems after the mid-1880s.

By the early 1900s, the USDA, the state agricultural research system, and local cooperatives formed an effective working arrangement to acquire and spread knowledge about fruit quality and the effects of packing, shipping, and marketing on spoilage and fruit appearance.These efforts led to the development of pre-cooling and other improved handling techniques, contributing to the emergence of California’s reputation for offering higher-quality horticultural products. This learning process eventually propelled California’s horticultural sector to a position of global leadership.More generally, the example of the state’s horticultural industry highlights the important,nft system if relatively neglected, contribution of biological learning to American agricultural development before the 1930s.A second major transformation took place in the early twentieth century with the increased cultivation of row crops including sugar beets, vegetables, and most notably cotton . These changes represented an intensification of farming with significant capital investments and often led to shifts onto what had been marginal or under-utilized lands. The advent of cotton, which by 1950 had become the state’s most valuable crop, offers another important case study in the continuing evolution of California agriculture.From Spanish times, visionaries attempted to introduce cotton into California on a commercial basis. A variety of factors, including the high cost of labor, the distance from markets and gins, and inadequate knowledge about appropriate varieties, soils, etc. doomed these early efforts. The real breakthrough came during World War I when high prices coupled with government research and promotional campaigns encouraged farmers in the Imperial, Coachella, and San Joaquin Valleys to adopt the crop. Figure2 illustrates acres harvested, bales produced, and yields per acre, from 1910 to 1964. The tremendous absolute increase in California’s cotton acreage since the 1920s contrasts with the absolute decline nationally. California’s acreage in cotton ranked 14th out of 15 cotton-producing states in 1919; by 1959 it ranked second.Several factors distinguished California’s cotton industry from other regions. First, cotton yields were typically more than double the national average. High yields resulted from the favorable climate, rich soils, controlled application of irrigation water, use of the best agricultural practices and fertilizer, adoption of high quality seeds, and relative freedom from pests. Second, the scale and structure of cotton farms was remarkably different in California. From the mid-1920s through the 1950s, the acreage of a California cotton farm were about five times that of farms in the Deep South. As an example of the structural differences between California and other important cotton states, in 1939 farms producing 50 or fewer bales grew to about 17 percent of the output in California, but in other leading cotton states, farms in this class produced at least 80 percent of all cotton output. One-half of the output in California was grown on farms producing more than 200 bales. For the nation as a whole, one-half of the output was raised on farms producing fewer than 13 bales.Thus,it is not surprising that California’s gross income per cotton farm was almost nine times the national average.Other distinctive features of California cotton farms were their more intensive use of power and their earlier mechanization of pre-harvest activities. In 1929, a California farm was almost 20 times more likely to have a tractor than a Mississippi farm.The Pacific Rural Press in 1927 offered a description of the highly mechanized state of many California cotton farms: “[M]en farm in sections…By the most efficient use of tractor power and tools, one outfit with a two-man daylight shift plants 100 acres per day, 6 rows at a time, and cultivates 70 acres 4-rows at a time.”The more rapid adoption of tractors created a setting favorable to further modernization. When picking machines became available, farmers already possessed the mechanical skills and aptitudes needed for machine-based production. The larger size of cotton operations in California and the more intensive use of tractors reflected a fundamentally different form of labor organization than that which dominated the South. By the 1940s, on the eve of cotton harvesting mechanization, most cotton in California was picked on a piece-rate basis by seasonal laborers under a contract system.Although conditions varied, a key ingredient was that a labor contractor recruited and supervised the workers, and dealt directly with the farmer, who might have had little or no personal contact with his laborers. This type of arrangement implied different class and social relationships from those that prevailed in much of the South. The California farm worker was more akin to an agricultural proletarian than to a rural peasant. The proverbial paternalism of southern planters toward their tenants had few parallels in California. As with many crops, California cotton growers also led the way in harvest mechanization. Many of the factors discussed above, including pre-harvest mechanization , relatively high wages, large-scale operations, high yields, a flat landscape, and a relative absence of rain during the harvest season all aided in the adoption of the mechanical harvester. Spindle picking machines first appeared on a commercial basis following World War II. In 1951, over 50 percent of the California crop was mechanically harvested compared to about 10 percent for the rest of the nation. At that time, about 50 percent of all the machines in operation in the United States were at work on California farms.16Similar forces—early adoption of large-scale operations and advanced technologies—characterized California’s livestock economy. The broad trends in livestock production in California since 1850 are reflected in Figure 3, which graphs the number of head of various types of livestock in the state as aggregated into a measure of animal units fed.The region emerged from the Mexican period primarily as a cattle producer.

Samples were collected at 0, 3, and 6 h following illumination with red light. The remainder of the culture was then placed under an IR LED for an additional 12 h, after which a final sample was collected. Upon collection, cells were immediately treated with 475 μL 100% ethanol and fixed for 1 h at room temperature. Cells were then pelleted, washed once with 50 mM sodium citrate , and then resuspended in 500 μL 50 mM sodium citrate to which RNaseA had been added. Following an incubation for 1 h at 37˚C, 50 μL proteinase K was added to a final concentration of 2 mg/mL and cells were incubated at 50˚C for an additional hour. SYTOX green was then added to each sample to a final concentration of 2 μM, and cells were incubated for 1 h at room temperature. Inparallel, control samples were prepared containing exponentially growing cultures of yeast treated with α-factor for for ~3 h prior to fixation and staining. Ploidy was determined by measuring the fluorescence intensity of SYTOX green staining by flow cytometry using a FACSAria III and normalizing to the α-factor-treated samples, which have a ploidy of 1N. For each time point for each independent experiment, 50,000 cells were measured. Analysis of flow cytometry data was performed using FlowJo and Flowing Software .Intact natural landscapes provide ecosystem functions that result in numerous ecosystem goods and services from which humans benefit, including carbon storage, flood protection, and maintenance of species life cycles . However, many of these services are diminished in landscapes that have been converted for agricultural purposes. The provisioning services of these food-production landscapes are clear, and there is increasing recognition that agricultural landscapes can continue to supply or maintain other vital ecosystem services if well managed. These include flood mitigation and carbon storage , pollination ,mobile grow rack and wildlife habitat . Maintaining ecosystem functions to optimize these multiple services in agricultural landscapes is particularly important, given that cropland and pasture currently occupy ~40% of the earth’s land surface, with increases predicted to support a growing global population .

In the past, agricultural production was characterized by growing one or more crops in the same place. Crop rotation would provide inputs of nitrogen, and suppress insects and weeds by breaking their life cycles, yielding modest but stable agricultural inputs . However, this link between ecology and agriculture has become strained over the past few decades a a result of mechanization, new crop varieties, development of agrochemicals, as well as political and economic forces associated with regional agriculture’s supplying international markets . This has led to concerns about the long-term sustainability of food-production systems, and the influence of these practices on the ecosystem services provided to people. An increasing number of studies show how creating diverse agricultural landscapes, through patches of remnant or revegetated native habitat on farmland, even on a small scale, can provide important habitat for native flora and fauna, as well as benefit farm productivity in unexpected ways . For example, fields with uncultivated margins have higher plant and moth diversity as well as more diverse soil macrofauna . Hedgerows have been associated with higher bird and moth diversity, provide movement corridors for fauna and host natural enemies that control agricultural pests . In addition, remnant areas close to agricultural areas improve pollination services with positive consequences for crop yields . Furthermore, agricultural lands store carbon through remnant native vegetation and from the crops cultivated, particularly annual row crops because of their dense planting . Similarly below-ground biomass could be increased by introducing cover crops with deeper roots to increase below-ground biomass while food is still produced . Other ecosystem services from certain types of agriculture include aesthetic landscapes , farm tourism , and the preservation of rural lifestyles . Agriculture can also be the source of ecosystem disservices such as habitat loss and pesticide poisoning of non-targeted species , while soil and nutrient runoff result in losses of soil carbon . The effect of these disservices ranges from the local scale to the regional scale , to the global scale . For example, 20% of the N fertilizer applied in agricultural systems globally moves to aquatic ecosystems . Agricultural production practices in California, which produces roughly half of the fruits, nuts, and vegetables for the U.S, has resulted in widespread nitrate contamination of groundwater aquifers . An emerging body of literature focuses on spatially quantifying ecosystem services and comparing these with patterns of biodiversity across the landscape and agricultural land returns . For example, Nelson et al. 

Assessed these three components under alternative land-use trajectories in the Willamette Basin, Oregon. The study found that a conservation scenario which resulted in high scores for ecosystem services also had high scores for biodiversity, while a development scenario had higher returns to land-owners but lower levels of biodiversity conservation and ecosystem services. Polasky et al. in an assessment of land-use alternatives over a 10-year period in Minnesota found a lack of concordance—the scenarios that created the greatest annual net returns to land-owners also had the lowest social benefits. Agricultural expansion was found to reduce stored carbon, negatively affect water quality, and reduce habitat quality for biodiversity and forest songbirds. The present study has elements similar to Nelson et al. and Polasky et al. . It is conducted at the parcel spatial scale, it is forward looking to 2050, and it addresses ecosystem services and disservices along with biodiversity and financial returns from agriculture. In this study, we examine the assumption that land use change in agriculturally dominated areas provides positive benefits for land-owners and financial agricultural returns at the expense of biodiversity and other ecosystem services, such as carbon storage. We do this by quantifying carbon storage, landscape suitability for birds, ecosystem disservices, and financial returns from agriculture within an area of the Central Valley of California. We ask how these change by 2050 under three alternative scenarios: restoration, urbanization, and enhanced agriculture tailored to the needs of a key species of conservation concern, the Swainson’s Hawk. The study area spans a 72,188-ha area in the Central Valley that includes the Cosumnes River Preserve and surrounding lands up to 50m in elevation, encompassing lands owned privately, by state and federal government, or by non-profit organizations including The Nature Conservancy . Historically, this area was dominated by native grassland, valley oak woodland and savanna, and riparian forest along the once-perennial Cosumnes River. However, conversion to agriculture has resulted in a landscape where only small patches of natural habitat remain. Many of these remnant natural areas are currently experiencing conversion to urban land use from the rapidly growing adjacent cities. Although some of these natural areas are habitat for state- and federally-listed threatened and endangered species and, consequently, development of these lands has resulted in mitigation funding to compensate for habitat losses of imperiled species. Currently, natural vegetation covers 44% of the study area,ebb and flow table based on a vegetation map of the Delta developed by the California Department of Fish and Wildlife in addition to project-based vegetation mapping.

The urban and developed footprint, which currently covers 9% of the study area, has increased by 35% over the last decade . Urban expansion associated with cities at the northern and southern edges of the study area continues to exert development pressure on remaining natural and agricultural lands. Today, the river channel is lined with agricultural levees, and adjacent floodplains are used largely for crops , with agriculture covering 46% of the study area. Agricultural activity in the study area, compared to large-scale agriculture in many other parts of California, has a high diversity of crops across many small parcels. Our agricultural land-use scenario represents an enhanced agriculture which favors compatible crops that are commonly the targets for mitigating habitat loss for the imperiled Swainson’s Hawk through mitigation funding. This scenario is reasonable, given that the region is highly suitable for Swainson’s Hawk nesting and foraging, supporting one of the highest concentrations of birds in the Pacific Flyway. Because B. swainsoni is a species of concern, priority has been placed on managing the landscape for its persistence. Both governmental and nongovernmental land managers currently engage in a number of practices intended to boost populations of avian species of concern, for example, paying farmers to flood fallow fields to provide habitat for migrating waterfowl. Given their conservation priority status, it is possible that management agencies might also pay farmers to enhance habitat for improved outcomes for the Swainson’s Hawk. The widespread use of conservation easements within the study area provides the mechanism and the means to accomplish an enhanced agricultural scenario. The Yolo County Habitat Conservation Plan/Natural Community Conservation Plan is one such policy that points toward management for this species. The Swainson’s Hawk can also be a valuable focal species because of its dependence on tree canopy nesting sites and nearby open-country foraging habitat. Many other raptors, riparian species, and migratory birds also depend on these ecosystem traits. The Swainson’s Hawk is able to forage in specific types of agriculture . In particular, alfalfa is a valuable resource because it is a perennial crop that continually supports high populations of prey whose availability peaks during monthly irrigation and harvesting events. Other crops, such as beet and tomato fields, are also hunted regularly during harvest, though crops such as rice or vineyards are not significantly utilized . The same crop associations are true for a number of other species. Alfalfa, in particular, is considered important habitat for other migratory birds, including shorebirds like Long-Billed Curlew and White-Faced Ibis , both of which are species of conservation concern. The Swainson’s Hawk responds well to protection and restoration of riparian forest habitats , within an agricultural landscape that is used for foraging . Thus, a varying matrix of natural riparian habitat and different forms of agriculture can reasonably be expected to significantly affect the value of the landscape for this species.

The potential for multi-species benefits from single species mitigation or management is rarely evaluated; therefore, we include an assessment of the ramifications of these land-use scenarios for 15 other focal bird species identified by the California Partners in Flight program . These are a suite of species whose requirements define different spatial attributes, habitat characteristics, and management regimes, and represent healthy habitats within our focal landscape . We conducted this study at the parcel scale , which is meaningful because land management decisions for agricultural practices are made at this scale and only a few ecosystem services quantification studies have been conducted at this scale . The average size of parcels in the study area that grow alfalfa, grain, orchard, rice, row crop, or vineyard was 3.88ha . To assess the changes over time among carbon storage, landscape suitability for birds, ecosystem disservices, and returns from agriculture with each of our management scenarios, we first developed a snapshot of the current land cover in the study area. We mapped urban areas using information from the Sacramento Area Council of Governments data from 2005, with a minimum mapping unit of five acres for urban areas and ten acres for rural areas. We defined agricultural land cover data from the CDFW’s Delta vegetation map and from the California Department of Water Resources , with a minimum mapping unit of 10 acres .These were considered sufficiently distinct from each other in terms of foraging value for Swainson’s Hawk. In addition to these agricultural classes, we used four natural vegetation classes along with developed/ urban and water . These land-cover types were intersected with land-owner parcels for the study area . In cases where a parcel contained more than one land use type, the parcel was split into the respective classes to retain this detail. These resultant land- cover data represent the contemporary baseline condition from which we measured changes resulting from the landscape management scenarios. We conducted spatial analysis in ESRI ArcMap version 10.3, in Universal Transverse Mercator projection with North American Datum 1983.We consulted with management stakeholders at the Cosumnes River Preserve to develop three management scenarios to simulate potential changes in the current landscape: extensive restoration, urbanization, and enhanced agriculture designed to benefit the Swainson’s Hawk.

In Arabidopsis, AtTic20-I and AtTic110 are expressed to a lower extent in roots than in leaves, similar to pea . These results seemingly contradict those of Hirabayashi et al., who concluded a comparable expression level of Tic20-I in shoots and roots. However, they used a non-quantifiable approach in contrast to our quantitative analysis. Furthermore, in our experiments the overall expression of AtTic20-I and AtTic110 differs notably from that in pea, AtTic110 RNA being about 3.5 and 6 times more abundant than AtTic20-I in leaves and roots, respectively. We also designed specific primers for the second Tic20 homolog in Arabidopsis, AtTic20-IV, and our quantitative method was sufficiently sensitive to precisely define its RNA levels in Arabidopsis leaves and roots, allowing direct comparison with the expression of AtTic20-I and AtTic110 . Transcription of AtTic20-IV had also been investigated in parallel to AtTic110 by Teng et al., who observed a differential ratio of expression using two different methods, of which one was not even sensitive enough to detect AtTic20-IV. A very recent study also investigated the expression of AtTic20-IV, however, without any quantification of their data. Our data show that AtTic20-IV is present in leaves and roots with transcript levels similar to AtTic20-I, but less abundant than AtTic110. Interestingly, and in accordance with the data presented by Hirabayashi et al., transcript levels of AtTic20-IV in roots are higher than those of AtTic20-I, while the opposite is true in leaf tissue. It can be speculated that the observed expression pattern reflects tissue-specific differentiation of both genes. AtTic20-IV may still partially complement for the function of AtTic20-I, as becomes evident from the viability of attic20-I knockout plants and the yellowish phenotype of attic20-I mutants over expressing AtTic20- IV. However, the severe phenotype of attic20-I plants, in conjunction with the observed differential expression pattern, clearly indicates specific functions of the two homologs. Furthermore, hydroponic nft a higher AtTic110 expression rate as observed in antisense attic20-I lines might indicate another possible compensatory effect .

The expression pattern of the three investigated genes was found to be similar in Arabidopsis growing hydroponically with or without sucrose or on soil . However, gene expression was generally higher in plants growing without sucrose.Semi-quantitative analysis of Tic20 and Tic110 on protein level was performed using immunoblots of envelope membranes isolated from two-week-old pea and four week-old Arabidopsis plants. In parallel, calibration curves were generated using a series of known concentrations of over expressed and purified proteins . After quantification of immunoblots from envelopes, amounts of PsTic20, PsTic110, AtTic20 and AtTic110 were determined using the corresponding calibration curve. The amount of PsTic110 in IE was found to be almost eight times higher than that of PsTic20 , which differs strikingly from the similar transcript levels of the two genes detected in leaves , indicating profound differences in post translational processes such as translation rate and protein turnover. In Arabidopsis, the absolute amount of AtTic110 is nearly the same as in pea , however, Arabidopsis envelopes represent a mixture, containing both outer and IE vesicles. Thus, the relative amount of AtTic110 is possibly higher than in pea. Surprisingly, the amount of AtTic20 is more than 100 times lower than that of AtTic110, showing an even greater difference in comparison to the observed RNA expression levels . Taking the different molecular size of Tic110 and Tic20 into account , we still observe 20 times more AtTic110 than AtTic20 protein. In pea, we found 1.4 times more Tic110 RNA than Tic20, whereas in Arabidopsis the ratio of Tic110 to Tic20 is 20.3. The number of channel forming units must even be more different, since Tic110 was shown to form dimers, whereas Tic20 builds very large complexes between 700 kDa and 1 MDa. Thus, two Tic110 molecules would be necessary to form a channel in contrast to Tic20, which would require many more molecules to form the pore. Though we cannot exclude that Tic20 might be subject to degradation by an unknown protease in vivo, protease treatments with thermolysin of right-side out IE vesicles in vitro clearly shows that Tic20 is very protease resistant,even in the presence of detergent. In contrast, Tic110 is easily degraded already without addition of detergent .

This argues against more rapid degradation of Tic20 compared to Tic110 during preparation of IE. The difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis may be due to the different age of the plants or the different needs under the given growth conditions, and suggests that there is no strict stoichiometry between the two proteins. Moreover, the low abundance of Tic20 in comparison to Tic110 in envelopes clearly demonstrates that Tic20 cannot be the main channel of the Tic translocon as previously suggested, since it cannot possibly support the required import rates of some highly abundant preproteins that are needed in the chloroplast.Experimental data suggested a common complex between Tic110 and Tic20 in chloroplast envelope membranes using a cross-linking approach. However, the interaction was not visible in the absence of Toc components, making a stable association unlikely. Furthermore, no evidence for a common complex was found by Kikuchi et al. using solubilized chloroplasts of pea and Arabidopsis for two-dimensional blue native/SDS-PAGE analysis. Likewise, the difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis indicates that a common complex, in which both proteins cooperate in translocation channel formation in a reasonable stoichiometry, is improbable. To clarify this issue, we addressed these partly conflicting results by using IE vesicles, which should minimize the possible influence of the interaction with Toc components on complex formation. Pea IE vesicles were solubilized in 5% digitonin and subjected to 2D BN/ SDS-PAGE. Immunoblots revealed that both Tic20 and Tic110 are present in distinct high molecular weight complexes : Tic110-containing complexes migrate at a size of ~ 200-300 kDa, whereas Tic20 displays a much slower mobility in BN-PAGE and is present in complexes exceeding 700 kDa, in line with the results from Kikuchi et al.. However, at a similar molecular weight of 250 kDa on BN-PAGE not only Tic110 but also Hsp93, Tic62 and Tic55 were described. The molecular weight of a complex containing all of these components would be much higher. Therefore, components of the Tic complex might associate with Tic110 very dynamically resulting in different compositions under different conditions, or alternatively, there are different complexes present at the same molecular weight.Tic22, the only Tic component located in the intermembrane space, is a potential candidate, since both proteins were identified together in cross-linking experiments.

However, only minor amounts of Tic20 and Tic22 were shown to co-localize after gel filtration of solubilized envelope membranes. A second candidate for common complex formation is PIC1/Tic21: Kikuchi et al.demonstrated that a one-megadalton complex of Tic20 contains PIC1/Tic21 as a minor subunit. PIC1/ Tic21 was proposed to form a protein translocation channel in the Tic complex, mainly based on protein import defects of knockout mutants and on structural similarities to amino acid transporters and sugar permeases. An independent study by Duy et al. favours the hypothesis that PIC1/Tic21 forms a metal permease in the IE of chloroplasts,hydroponic channel rendering the import-related role questionable. This discrepancy will have to be addressed in the future. To test the complex formation of Tic20 in vitro without the involvement of other proteins, we used Tic20- proteoliposomes for 2D BN/SDS-PAGE analysis, similarly to IE vesicles . The migration behaviour of the protein resembles that observed in IE: the majority of the protein localizes in high molecular weight range, however, the signal appears more widespread and a portion is also detected at lower molecular weights, possibly as monomers. This observation reveals that Tic20 has the inherent ability to homo-oligomerize in the presence of a lipid bilayer. The less distinct signal could be due to different solubilization of Tic20 by digitonin in IE vesicles vs. liposomes, or could be an indication that additional subunits stabilize the endogenous Tic20 complexes, which are not present after the reconstitution. However, we interpret these observations as support for the hypothesis that the major component of the one megadalton complex in IE are homo-oligomers composed of Tic20.In silico analysis of Tic20 predicts the presence of four hydrophobic transmembrane helices positioning both N- and C-termini to one side of the membrane . According to these predictions, three cysteins in PsTic20 face the same side, while the fourth would be located in the plane of the membrane. We used pea IE vesicles prepared in a right-side-out orientation to determine the topology of Tic20 employing a Cys-labelling technique. To this end, the IE vesicles were incubated with a membrane-impermeable, Cys-reactive agent that adds a molecular weight of 5,000 Da to the target protein for each reactive Cys residue. In our experiments PEG-Mal did not strongly label any Cys residues of Tic20 under the conditions applied , indicating the absence of accessible Cys residues on the outside of the membrane. Only one faint additional band of higher molecular weight was detectable , possibly due to a partially accessible Cys located within the membrane. In the presence of 1% SDS, however, all four Cys residues present in PsTic20 are rapidly PEGylated, as demonstrated by the appearance of four intense additional bands after only five minutes of incubation. The observed gain in molecular weight per modification is bigger than the expected 5 kDa for each Cys, but this can be attributed to an aberrant mobility of the modified protein in the Bis-Tris/ SDS-PAGE used in the assay. Our results support a four transmembrane helix topology in which both the C- and N-termini are facing the stromal side of the membrane , with no Cys residues oriented towards the intermembrane space. Cys108 is most likely located in helix one, Cys227 and Cys230 are oriented to the stromal side of helix four and Cys243 is located in the stroma.

This topology is also in line with green fluorescent protein-labelling studies by van Dooren et al.indicating that the N- and C-termini also of the Toxoplasma gondii homolog of Tic20 face the stromal side of the inner apicoplast membrane.Tic20 was identified more than a decade ago but since then no heterologous expression and purification procedure has been reported, which could successfully synthesize folded full-length Tic20. Here, we report two efficient Escherichia coli based systems for Tic20 expression and purification from both pea and Arabidopsis: codon optimized PsTic20 was over expressed in a S12 cell lysate in presence of detergents, and AtTic20 over expression was successfully accomplished by adaptation of a special induction system. Following these steps, both pea and Arabidopsis proteins could be purified to homogeneity by metal affinity purification . Using the purified protein, we performed structural characterization studies of Tic20 by subjecting it to circular dichroism spectroscopy . The recorded spectra of PsTic20, displaying two minima at 210 and 222 nm and a large peak of positive ellipticity centered at 193 nm, are highly characteristic of a-helical proteins, and thus demonstrate that the protein exists in a folded state after purification in the presence of detergent. The secondary structure of Tic20 was estimated by fitting spectra to reference data sets resulting in an a-helical content of approximately 78%, confirming in silico predictions.To better characterize Tic20 in a membrane-mimicking environment, heterologously expressed and purified AtTic20 was reconstituted into liposomes in vitro. Initially, flotation experiments were performed to verify a stable insertion. In the presence or absence of liposomes, Tic20 was placed at the bottom of a gradient ranging from 1.6 M to 0.1 M sucrose. In the presence of liposomes, Tic20 migrated to the middle of the gradient, indicating a change in its density caused by interaction with liposomes. In contrast, the protein alone remained at the bottom of the gradient . Proteoliposomes were also treated with various buffers before flotation , to test whether the protein is firmly inserted into the liposomalmembrane or just loosely bound to the vesicle surface. None of the applied conditions changed the migration behaviour of Tic20 in the gradient , indicating that Tic20 was deeply inserted into the liposomal membrane. Thus, proteoliposomes represent a suitable in vitro system for the analysis of Tic20 channel activity.Even though Tic20 has long been suggested to form a channel in the IE membrane, this notion was solely based on structural analogy to other four-transmembrane helix proteins, and no experimental evidence has been provided so far.

Despite successfully expressing thaumatin in yeast , bacteria, fungi, and transgenic and transfected plants, biotechnological large-scale production facilities have yet to be established. Molecular farming, the production of recombinant proteins in plants, offers several advantages over bioreactor-based systems. In this application, plants are thought of as nature’s single use bioreactors, offering many benefits such as reduced upstream production complexity and costs , linear scalability, and their inability to replicate human viruses. specifically, open-field growth of plants has the potential to meet the market’s need for a large-scale, continuous demand of a commodity product at a competitive upstream cost. It has been marked suitable for this operation as plants can be easily adapted on an agricultural scale to yield several metric tons of the purified protein per year. Here, we present a feasibility study for a protein production level of tens of metric tons per year. The success of a new product in the biotechnology process industry depends on well-integrated planning that involves market analysis, product development, process development, and addressing regulatory issues simultaneously, which requires some decisions to be made with limited information. This generates demand for a platform to help fill in those gaps and facilitate making more informed process and technology decisions. Process simulation models can be used in several stages of the product life cycle including idea generation, process development, facility design, and manufacturing. For instance, based on preliminary economic evaluations of new projects, they are used to eliminate unfeasible ideas early on. During the development phase of the product, as the process undergoes frequent changes, such models can easily evaluate the impact of these changes and identify cost-sensitive areas.Additionally, PSMs are widely used in designing new manufacturing facilities mainly as a tool for sizing process equipment and supporting utilities,ebb and flow tray as well as for estimating the required capital investment and cost of goods.

This ultimately helps companies decide on building a new facility versus outsourcing to contact manufacturers. There are currently few published data-driven simulations of techno-economic models for plant-based manufacturing of proteins for pharmaceutical, biofuel, commercial enzyme, and food safety applications. However, to the best of our knowledge, no studies have proposed or assessed the feasibility of plant-based protein bio-production platforms on the commodity scale in tens of metric tons per year. The feasibility of production at this scale is critical for the emergence of thaumatin as a sugar substitute. Here, we present a preliminary process design, process simulation, and economic analysis for the large-scale manufacturing of thaumatin II variant by several different molecular farming production platforms.The base case scenario assumes an annual production capacity of 50 MT thaumatin. To achieve this level of production in a consistent manner, manufacturing is divided into 157 annual batches. Upstream production is attainable through open-field, staggered plantation of Nicotiana tabacum plants. Each batch has a duration of 45 days and a recipe cycle time of 2 days. A full list of process assumptions can be found in Table S1. The proposed design achieves the expression of thaumatin in N. tabacum leaves using magnICON® v.3. This technology developed by Icon Genetics GmbH allows for the separation of the “growth” and the “expression” phases in a manufacturing process. Moreover, this process obviates the need to use agroinfiltration, which requires more capital and operational costs for inoculum preparation and implementation of expensive units for the infiltration process, containment of the genetically engineered agrobacteria, and elimination of bacteria-derived endotoxins. In this design, transgenic N. tabacum or N. benthamiana plants carry a double-inducible viral vector that has been deconstructed into its two components, the replicon and the cell-to-cell movement protein. Background expression of recombinant proteins prior to induction remains minimal; however, inducible release of viral RNA replicons—from stably integrated DNA proreplicons—is triggered upon spraying the leaves and/or drenching the roots with a 4% ethanol solution resulting in expression levels as high as 4.3 g/kg fresh weight in Nicotiana benthamiana.

Nonetheless, Nicotiana tabacumhas several advantages that make it more suitable for large-scale open field production such as field hardiness, high biomass yields, well-established infrastructure for large-scale processing, plentiful seed production, while attaining expression levels up to 2 g/kg FW. Furthermore, it is unlikely that transgenic tobacco material would mix with material destined for the human food or animal feed chain, unless it is grown in rotation with a food crop, but further development of strict Good Agricultural Practice for transgenic plants should overcome these issues.An alternative upstream facility design scenario was developed to evaluate the process economics of a more controlled supply of thaumatin by growing the plant host in a 10-layer vertical farming indoor environment. Nicotiana benthamiana is chosen as a host because it is known to be a model for protein expression for both Agrobacterium and virus-based systems, but its low biomass yield and difficulties regarding adaptation in the field hinder its application for open outdoor growth. However, this species grows very well in indoor, controlled environments and has high recombinant protein production. This upstream production facility uses the same method of expression and follows the same schedule as the base case upstream facility.Transient expression in plants is a method of recombinantly producing proteins without stable integration of genes in the nuclear or chloroplast genome. The main advantages of using this method are reducing the extensive amount of time needed to develop a stable transgenic line and overcoming bio-safety concerns with growing transgenic food crops in the field expressing heterologous proteins. Transient expression is attainable through several systems including biolistic delivery of naked DNA, agrobacteria, and infection with viral vectors. Notably, the use of viral vectors has been marked suitable for application on a field-scale due to the flexibility of production, and the quick accumulation of target proteins while achieving high yields. A new report has shown efficacy in delivering RNA viral particles using a 1–3 bar pressure, 1–4 mm atomizer nozzles spray devices in the presence of an abrasive to cause mechanical wounding of plant cell wall. GRAS notices GRN 738 and GRN 910 describe production of thaumatin in edible plant species and N. benthamiana, respectively. The expression of thaumatin in leaf tissue of the food crops Beta vulgaris , Spinacia oleracea , or Lactuca sativa is generally lower than in N. benthamiana.

However, despite having lower expression levels, the absence of pyridine alkaloids that are present in Nicotiana species is a major advantage for production in food crops because of the significant downstream resources needed to remove alkaloids in Nicotiana-based products. The ultimate solution may be a high-expressing engineered Nicotiana host devoid of alkaloid biosynthesis, but that option was not modeled in this study. The transient production facility is designed to produce 50 MT of purified thaumatin in spinach, annually, over 153 batches due to longer turnaround time required for S. oleracea compared to N. tabacum crops. Each batch has a duration of 67.8 days and a recipe cycle time of 1.94 days. The proposed base case upstream field production facility,4×8 flood tray displayed in Figure 1, consists of a 540 acre block of land divided into 22 plots, each of which is suitable for growing 318,000 kg FW of N. tabacum, carrying 477 kg of thaumatin, accounting for downstream recovery of 66.8%. It is assumed that the facility is located in a suitable climate where the growth of N. tabacum is attainable throughout the year, ignoring variations in production between batches . Each batch starts with direct seeding of transgenic N. tabacum plants in the field . The seeds are left to germinate for two weeks followed by vegetative growth for 3 more weeks post germination . A fertigation stream is applied to deliver the necessary nutrients for optimal plant growth. After a total of 35 days post seeding, a tractor sprayer applies 4900 L of a 4% ethanol solution to the plot’s crop, triggering the synthesis and accumulation of thaumatin in plant biomass. The plants are incubated for 7 more days, during which time they continue to uptake nutrients and express thaumatin. After 42 days from seeding, the batch is harvested through two mechanical harvesters and four hopper trucks at a rate of 17,000 kg/h and transported to downstream processing facility using a conveyer belt . The plot undergoes a turnaround period of three days for which the labor and equipment cost is included. No pesticides, fungicides, or herbicides costs are added due to the assumption that not enough growing degree days are accumulated during the batch cycle duration , for disease-causing organisms to be a concern. Transgenic Nicotiana benthamiana seeds are germinated in soilless plant substrate at a density of 94 plants per tray . Seedlings are then grown hydroponically, under LEDs, until reaching manufacturing maturity after 35 days. Induction occurs in a separate hydroponic reservoir, where plants are root drenched and sprayed with 0.01 L of 4% ethanol per kg FW plant tissue. The plants are left to grow for 7 additional days during the incubation period. N. benthamiana biomass is mechanically harvested at a rate of 54,000 kg/h and transported to downstream processing facility using a conveyer belt . The indoor upstream facility flowsheet can be found in Figure S1. The total facility footprint was calculated to be 83,000 m2 . The base case downstream processing facility is designed to purify and formulate 318.5 kg/batch of thaumatin with 98% purity.

A DSP batch starts with shredding plant biomass using two industrial shredders , each processing 40,000 kg of plant biomass/h. This step is designed to homogenize the leaves and stems to facilitate the extraction process. Shredded plant material is then mixed with an acetate buffer in a 0.8 L of buffer to 1 kg of biomass ratio. This step leverages stability of thaumatin at low pH to precipitate host plant proteins that aren’t stable under acidic conditions. The extraction buffer consists of 50 mM acetic acid and 150 mM sodium chloride mixture at a pH of 4.0. The resulting plant slurry is then fed into a screw press to separate most of the dry plant material. A screw press is recommended for this step because it minimizes the amount of extraction buffer needed by forcing out more plant sap with the increasing pressure inside the chamber. The crude extract stream obtained from the screw press unit is sent to three parallel P&F filtration units for initial clarification, each having a membrane area of 190 m2 . Furthermore, the model assumes the use of food-grade filtermembranes designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. The acetate buffer is applied once again as cake wash with a 0.2 L buffer to 1 L extract ratio. Diatomaceous earth is added to this step as a filter aid in a 6:100. The stability of thaumatin at low pH and high temperatures facilitates the precipitation of more host cell proteins as well as other undesired plant-derived compounds. Using seven heating tanks , the plant extract is then heated to 60 ◦C for 60 min. Following heat incubation, the stream is sent to a P&F filtration unit to capture the heat-precipitated proteins. It is assumed that a 90% reduction of N. tabacum total soluble proteins is attainable following the heat incubation and precipitation steps. Concentrating the thaumatin stream prior to the ultrafiltration/diafiltration step is necessary to avoid processing large liquid volumes ~573,000 L further downstream. It has been reported that thaumatin experiences a loss in sweetness when heated above 70 ◦C at a pH of 7.0; therefore, the product stream undergoes concentration by evaporation prior to neutralizing the solution since the protein can sustain higher temperatures at a low pH.The triple effect evaporation unit is designed to evaporate 90% of the water content in the stream at 109 ◦C, 77 ◦C, and 40 ◦C in the first, second, and third effect, respectively, over 4 h. The exiting stream is then neutralized with 1:1 molar ratio and mixed in V-101 for 30 min and sent to the P&F filtration unit to remove any precipitated materials. An additional 1.5% loss of thaumatin during this step is assumed. Because soluble impurities such as nicotine and other pyridine alkaloids are abundant in N. tabacum plants, a UF/DF step is necessary to eliminate small molecules. The UF/DF unit consists of 4 stacked cassette holders, each containing twenty 3.5 m2 cassettes.

The potential for a Mars mission in the early 2030s underscores the urgency of developing a road map for advantageous space bio-technologies.The replicative capacity of biology reduces mission launch cost by producing goods on-demand using in situ resources , recycling waste products , and interacting with other biological processes for stable ecosystem function . This trait not only lowers initial launch costs, but also minimizes the quantity and frequency of resupply missions that would otherwise be required due to limited food and pharmaceutical shelf-life on deep space missions. Biological systems also provide robust utility via genetic engineering, which can provide solutions to unforeseen problems and lower inherent risk . For example, organisms can be engineered on-site to produce a pharmaceutical to treat an unexpected medical condition when rapid supply from Earth would be infeasible . A so-called “bio-manufactory” for deep space missions based on in situ resource utilization and composed of integrated biologically-driven subunits capable of producing food, pharmaceuticals, and biomaterials will greatly reduce launch and resupply cost, and is therefore critical to the future of human based space exploration .The standard specifications for Mars exploration from 2009 to 2019 are not biomanufacturing-driven due to the novelty of space bioengineering. Here, we outline biotechnological support to produce food, medicine, and specialized construction materials on a long-term mission with six crew-members and surface operations for ∼ 500 sols flanked by two interplanetary transits of ∼ 210 days . We further assume predeployment cargo that includes in situ resource utilization hardware for Mars-ascent propellant production , which is to be launched from Earth to a mission site. Additional supplies such as habitat assemblies , photovoltaics , experimental equipment, and other non-living consumables will be included. The proposed bio-manufactory would augment processes for air generation and water and waste recycling and purification—typically associated with Environmental Control and Life Support Systems —since its needs overlap but are broader,grow hydroponic and drive a wider development of an array of ISRU, in situ manufacturing , food and pharmaceutical synthesis , and loop closure technologies .

Food, medicine, and gas exchange to sustain humans imposes important ECLSS feasibility constraints . These arise from a crewmember physiological profile, with an upper-bound metabolic rate of ∼ 11–13 MJ/CM-sol that can be satisfied through prepackaged meals and potable water intake of 2.5 kg/CM-sol . Sustaining a CM also entails providing oxygen at 0.8 kg/CM-sol and recycling the 1.04 kg/CM-sol of CO2, 0.11 kg of fecal and urine solid, and 3.6 kg of water waste within a habitat kept at ∼ 294 K and ∼ 70 kPa. Proposed short duration missions lean heavily on chemical processes for life support with consumables sent from Earth . As the length of a mission increases, demands on the quantity and quality of consumables increase dramatically. As missions become more complex with longer surface operations, biotechnology offers methods for consumable production in the form of edible crops and waste recycling through microbial digestion . Advancements in bio-manufacturing for deep space exploration will ensure a transition from short term missions such as those on the ISS that are reliant on single use-single-supply resources to long-term missions that are sustainable.Efficiency gains in a bio-manufactory come in part from the interconnection and modularity of various unit operations . However, different mission stage requirements for assembly, operation, timing, and productivity can lead to different optimal biomanufactory system configurations. A challenge therefore exists for technology choice and process optimization to address the high flexibility, scalability, and infrastructure minimization needs of an integrated biomanufactory. Current frameworks for biomanufacturing optimization do not dwell on these aspects. A series of new innovations in modeling processes and developing performance metrics specific to ECLSS biotechnology is called for, innovations that can suitably capture risk, modularity, autonomy, and recyclability. Concomitant invention in engineering infrastructure will also be required.An estimated ∼ 10,000 kg of food mass is required for a crew of six on a ∼ 900 days mission to Mars . Food production for longer missions reduces this mission overhead and increases food store flexibility, bolsters astronaut mental health, revitalizes air, and recycles wastewater through transpiration and condensation capture . Pharmaceutical life support must address challenges of accelerated instability [ ∼ 75% of solid formulation pharmaceuticals are projected to expire mid-mission at 880 days ], the need for a wide range of pharmaceuticals to mitigate a myriad of low probability medical risks, and the mismatch between the long re-supply times to Mars and often short therapeutic time windows for pharmaceutical treatment.

Pharmaceutical production for longer missions can mitigate the impact of this anticipated instability and accelerate response time to unanticipated medical threats. In early missions, FPS may boost crew morale and supplement labile nutrients . As mission scale increases, FPS may meet important food and pharmaceutical needs . A biomanufactory that focuses on oxygenic photoautotrophs, namely plants, algae and cyanobacteria, enhances simplicity, versatility, and synergy with intersecting life support systems and a Martian atmosphere has been shown to support such biological systems . While plant-based food has been the main staple considered for extended missions , the advent of cultured and 3D printed meat-like products from animal, plant and fungal cells may ultimately provide a scalable and efficient alternative to cropping systems . FPS organisms for Mars use must be optimized for growth and yields of biomass, nutrient, and pharmaceutical accumulation. Providing adequate and appropriate lighting will be a challenge of photo autotrophic-centric FPS on Mars . Developing plants and algae with reduced chloroplast light-harvesting antenna size has the potential to improve whole-organism quantum yield by increasing light penetration deeper into the canopy, which will reduce the fraction of light that is wastefully dissipated as heat and allow higher planting density . Developing FPS organisms for pharmaceutical production is especially complicated, given the breadth of production modalities and pharmaceutical need . Limited resource pharmaceutical purification is also a critically important consideration that has not been rigorously addressed. Promising biologically-derived purification technologies should be considered for processing drugs that require very high purity . Developing FPS growth systems for Mars requires synergistic biotic and abiotic optimization, as indicated by lighting systems and plant microbiomes. For lighting, consider that recent advancements in LED efficiency now make LEDs optimal for crop growth in extraterrestrial systems.The ideal spectra from tunable LEDs will likely be one with a high fraction of red photons for maximum production efficiency, but increasing the fraction of shorter wavelength blue photons could increase crop quality . Similarly, higher photon intensities increase production rates but decrease production efficiency. Understanding the associated volume and power/cooling requirement trade offs will be paramount to increasing overall system efficiency. For microbiomes, consider that ISS open-air plant cultivation results in rapid and widespread colonization by atypicaly low diversity bacterial and fungal microbiomes that often lead to plant disease and decreased plant productivity .

Synthetic microbial communities may provide stability and resilience to the plant microbiome and simultaneously improve the phenotype of host plants via the genes carried by community members. A subset of naturally occurring microbes are well known to promote growth of their plant hosts , accelerate wastewater remediation and nutrient recycling , and shield plant hosts from both abiotic and biotic stresses , including opportunistic pathogens . While SynCom design is challenging, the inclusion of SynComs in life support systems represents a critical risk-mitigation strategy to protect vital food and pharma resources. The application of SynComs to Mars-based agriculture motivates additional discussions in tradeoffs between customized hydroponics versus regolith-based farming,growing lettuce hydroponically both of which will require distinct technology platforms and applied SynComs.Our biomanufactory FPS module has three submodules: crops, pharmaceuticals, and functional foods . The inputs to all three submodules are nearly identical in needing H2O as an electron donor, CO2 as a carbon source, and light as an energy source, with the required nitrogen source being organism-dependent . H2O, CO2, and light are directly available from the Martian environment. Fixed nitrogen comes from the biomanufactory ISRU module. The submodules output O2, biomass, and waste products. However, the crop submodule chiefly outputs edible biomass for bulk food consumption, the pharmaceutical submodule synthesizes medicines, and the functional foods submodule augments the nutritional requirements of the crop submodule with microbially-produced vitamins . These outputs will be consumed directly by crew-members, with waste products entering the LC module for recycling. All submodules will have increased risk, modularity, and recyclability relative to traditional technological approaches. Increased risk is associated with biomass loss due to lowerthan-expected yields, contamination, and possible growth system failure. Increased modularity over shipping known pharmaceuticals to Mars derives from the programmability of biology, and the rapid response time of molecular pharming in crops for as-needed production of biologics. Increased recyclability stems from the lack of packaging required for shipping food and pharmaceuticals from Earth, as well as the ability to recycle plant waste using anaerobic digestion. At a systems integration level, FPS organism care will increase the crew time requirements for setup, maintenance, and harvesting compared to advance food and pharmaceutical shipments. However, overall cost impacts require careful scrutiny: crop growth likely saves on shipping costs, whereas pharmaceutical or functional food production on Mars may increase costs relative to shipping drugs and vitamins from Earth.Maintaining FPS systems requires cultivation vessels/chambers, support structures, plumbing, and tools. Such physical objects represent elements of an inventory that, for short missions, will likely be a combination of predeployment cargo and supplies from the crewed transit vehicle . As mission duration increases, so does the quantity, composition diversity, and construction complexity of these objects. The extent of ISM for initial exploration missions is not currently specified . Nevertheless, recent developments imply that ISM will be critical for the generation of commodities and consumables made of plastics , metals , composite-ceramics , and electronics as mission objects, with uses ranging from functional tools to physical components of the life-supporting habitat . Plastics will make up the majority of high-turnover items with sizes on the order of small parts to bench-top equipment, and will also account for contingencies . Biotechnology—specifically synthetic biology—in combination with additive manufacturing has been proposed an a critical element towards the establishment of off world manufacturing and can produce such polymeric constructs from basic feed stocks in a more compact and integrated way than chemical synthesis, because microbial bioreactors operate much closer to ambient conditions than chemical processes .

The versatility of microbial metabolisms allows direct use of CO2 from Mars’ atmosphere, methane from abiotic Sabatier processes , and/or biologically synthesized C2 compounds such as acetate, as well as waste biomass. A class of bio-plastics that can be directly obtained from microorganisms are polyhydroxyalkanoates . While the dominant natural PHA is poly , microbes can produce various copolymers with an expansive range of physical properties . This is commonly accomplished through co-feeding with fatty acids or hydroxyalkanoates, which get incorporated in the polyester. These co-substrates can be sourced from additional process inputs or generated in situ. For example the PHA poly-lactic acid can be produced by engineered Escherichia coli , albeit to much lower weight percent than is observed in organisms producing PHAs naturally. PHA composition can be modulated in other organisms . The rapid development of synthetic biology tools for non-model organisms opens an opportunity to tune PHA production in high PHB producers and derive a range of high-performance materials. Before downstream processing , the intracellularly accumulating bio-plastics need to be purified. The required degree of purity determines the approach and required secondary resources. Fused filament fabrication 3Dprinting, which works well in microgravity , has been applied for PLA processing and may be extendable to other bio-polyesters. Ideally, additive manufacturing will be integrated in-line with bio-plastics production and filament extrusion.Figure 4 depicts the use of three organism candidates from genera Cupriavidus, Methylocystis, and Halomonas that can meet bio-plastic production. This requires a different set of parameters to optimize their deployment, which strongly affects reactor design and operation. These microbes are capable of using a variety of carbon sources for bio-plastic production, each with a trade-off. For example, leveraging C2 feed stocks as the primary source will allow versatility in the microbe selection, but may be less efficient and autonomous than engineering a single organism like Cupriavidus necator to use CO2 directly from the atmosphere. Alternatively, in the event that CH4 is produced abiotically for ascent propellant , a marginal fraction of total CH4 will be sufficient for producing enough plastic without additional hardware costs associated with ISRU C2 production.

The product of NO2 ! was measured using the UV-Vis spectrophotometer at 540 nm. The content of H2O2 was determined after extraction by homogenizing plant tissues with 2 mL cold acetone . After centrifugation at 5000g at 4 ” C for 10 min, a 1.0 mL aliquot of the supernatant was mixed with 0.1 mL of 5% TiSO4 and 0.1 mL ammonia. After centrifugation, the titanium-peroxide complex pellet was resuspended in 3.0 mL of 2 M H2SO4, and absorbance was determined at 415 nm with a standard curve generated with known concentrations of H2O2.Plasma membrane integrity was evaluated by staining roots with Evans blue solution as in Yamamoto et al. with minor modifications. After the PPCP treatment, roots were stained with Evans blue solution for 15 min, and the stained roots were washed thoroughly with deionized water. The trapped Evans blue was released by extracting the roots in 5.0 mL of N,N-dimethylformamide. Absorbance of the supernatant was determined spectrophotometrically at 600 nm. The level of lipid peroxidation in roots and shoots of cucumber was measured in terms of malondialdehyde, which was determined according to the reaction with thiobarbituric acid as described in Yamamoto et al. . Historical staining for lipid peroxidation was conducted with Schiff’s reagent .For the measurement of reduced glutathione and oxidized glutathione , plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice. The homogenate was centrifuged at 12,000 g for 15 min at 4 ” C, and the supernatant was used for analysis of GSH and GSSG . A 0.5-mL aliquot of the supernatant was added to a reaction mixture containing 100 mM PBS , 0.2 mM NADPH and 1 mM 50 50 – dithiobis-2-nitrobenzonic acid . The reaction was started by the addition of 3 U of glutathione reductase, and absorbance at 412 nm was measured after 5 min. For GSSG, 2- vinylpyridine was added to the neutralized supernatant to mask GSH. Simultaneously,grow hydroponic the same volume of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG from the total GSH.Fresh plant tissue samples were frozen in liquid nitrogen, and homogenized in 50 mM PBS containing 1 mM EDTA and 1% PVP, with the addition of 1 mM ascorbate for ascorbate peroxidase.

The homogenate was centrifuged at 12,000 g for 20 min at 4 ” C, and the supernatant was used for the following enzyme assays . Protein content in enzyme extracts was determined by Coomassie brilliant blue G-250 with a standard curve using bovine serum albumin as the standard. Measurement of superoxide dismutase activity was carried out by inhibiting photochemical reduction of nitro blue tetrazolium . The assay mixture contained 50 mM PBS , 13 mM methionine, 75 mM NBT and 2 mM riboflavin. After addition of 100 mL of enzyme extract, the glass tubes were placed under light for 15 min, and then read at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT. To determine ascorbate peroxidase activity, a 200 mL aliquot of enzyme extract was added to the reaction mixture of 1 mM ascorbate and 0.3 mM H2O2 in 50 mM PBS. The absorbance changes were monitored at 290 nm for 3 min as ascorbate was oxidized, the enzyme activity was calculated using the extinction coefficient of 2.8 mM! 1 cm! 1 for ascorbate. Peroxidase activity was monitored by oxidation of 0.2% guaiacol using 0.3% H2O2 after addition of 50 mL enzyme extract. The enzyme activity was calculated using an extinction coefficient of 26.6 mM! 1 cm! 1 . Glutathione S-transferase activity was determined in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM 1-chloro-2,4-dinitrobenzene , 5 mM GSH and 100 mL enzyme extract. The GST activity was measured spectrophotometrically at 340 nm based on GSH-CDNB adduct synthesis using extinction coefficient 9.6 mM! 1 cm! 1 for GSH-CDNB.To evaluate the sensitivity of cucumber plants to PPCPs, an initial dose-response experiment was carried out. Statistical analysis showed no significant difference in the biomass of plants grown in 0.5, 5 ng L! 1 PPCP-spiked and control solutions . However, treatment with PPCPs at 50 mg L! 1 progressively caused an increase of leaf necrosis . The level of chlorophyll a and chlorophyll b decreased with increasing PPCP treatment rates . Meanwhile, root activity decreased by 15.4% and 28.2% after exposure to 5 and 50 mg L! 1 PPCPs, respectively, as compared with the control .Previous studies showed that reactive oxygen species were produced when plants were exposed to xenobiotics .

However, little is known if exposure to PPCPs would induce intracellular ROS production in higher plants. It was found that even at 0.5 mg L! 1 , PPCPs induced H2O2 accumulation in both shoots and roots, and the maximum elevation occurred at the highest concentration , where H2O2 levels were about 3.0- and 3.2-fold higher in shoots and roots, respectively, than those from the control treatment . Additionally, O2 .- did not increase significantly after exposure to PPCPs at 0.5 mg L! 1 , which could be due to the fast conversion of O2 .- to H2O2.Analysis of Evans blue uptake and malondialdehyde content showed that PPCPs caused oxidative damage to the plasma membrane and lipid fraction in plant seedlings; however the damage was less pronounced in the leaves. A significant increase in Evans blue uptake was found at 50 mg L! 1 PPCPs in the root , and histochemical staining clearly indicated that cell death occurred in the root tip that is the most active and sensitive region of the root. Lipid peroxidation measured as MDA increased in all of the stressed plants, and its content was higher in roots than in shoots in all of the PPCP treatments . These results were further confirmed by histochemical analysis using Schiff’s reagent to detect lipid peroxidation in plants .After 7 d of cultivation, SOD showed the maximal activity in roots after exposure to PPCPs at 5 mg L! 1 and decreased thereafter . Ascorbate peroxidase showed a dose-dependent response, increasing about 2.0- and 1.1-fold in roots and shoots, respectively, after exposure to PPCPs at 50 mg L! 1 . Peroxidase is among the enzymes with a potential role in the detoxification of a variety of xenobiotics, and GSTs often detoxify exogenous compounds by conjugation with GSH. The total POD and GST activities both increased appreciably after exposure to PPCPs. The GSH content increased in leaves after exposure to PPCPs, while the root showed a maximal GSH content at 0.5 mg L! 1 PPCPs, followed by decreases thereafter . The decreases of GSH in roots may be due to GSH serving as an antioxidant for preventing oxidative damage, and also acting to detoxify PPCPs by conjugation. The GSSG content displayed little change when PPCP concentrations were low . However, when the PPCP concentration was increased to 50 mg L! 1 , there was a significant increase in GSSG content .Results from the present study illustrated the physiological, biochemical and molecular mechanisms involved in the detoxification of PPCPs in plants by considering especially homeostasis of ROS and anti-oxidant metabolism.

The results clearly showed that PPCP-induced morphological indicators changed at elevated PPCP concentrations , and the impact was more pronounced in roots than shoots. The enhanced sensitivity of roots to PPCP toxicity may be due to the greater accumulation of PPCPs in the root . Similar observations were previously reported in alfalfa, lettuce,and pepper after PPCP exposure . In addition, Christou et al. suggested that PPCPs in a mixture displayed a different uptake pattern compared with that when exposed individually. The present study also showed that roots consistently accumulated PPCPs to a higher level than shoots when exposed to mixed PPCPs . In shoots, growing lettuce hydroponically the relative accumulation of individual PPCPs differed somewhat from that in Wu et al. , where carbamazepine and diazepam were found at the highest levels, followed by meprobamate and trimethoprim. The discrepancy between the studies may be attributed to the different growth conditions, plant cultivars, and sampling intervals. Acetaminophen, however, was not detected in the plant tissues , likely owing to its rapid metabolism after uptake . Recent studies showed that contact with PPCPs was capable of inducing a complex set of physiological responses in higher plants . Generally, contents of leaf pigments, including chlorophyll and carotenoids, provide valuable information about the physiological status of a plant. Here, a clear leaf necrosis and reduction in contents of chlorophyll and carotenoids were observed at higher PPCP concentrations . Findings from this and previous studies together suggest that PPCPs may significantly affect plant growth. Prior to the induction of whole plant morphological effects, stress may also lead to physiological, biochemical and molecular changes within the plant. However, little information is available about the toxic effects in plants from a mechanistic perspective. A direct result of stress-induced cellular changes is the enhanced ROS accumulation, consequently imposing oxidative stress to bio-molecules . Although additional research is needed to establish oxidative stress as the primary mechanism of PPCP toxicity to higher plants, it is clear that oxidative stress is involved in the development of PPCP-induced toxic symptoms. In this study, changes in ROS levels were observed in comparison to the control after exposure to PPCPs , and the response occurred at much lower concentrations than that for morphological effects. Overproduction of ROS can cause cell damage and is the final consequence of oxidative stress. In the current study, the increase in ROS production coincided with the increase in membrane damage and lipid peroxidation in the cucumber plants, indicating the presence of oxidative stress. In a previous study, exposure to 10 mg L! 1 of diclofenac, sulfamethoxazole, trimethoprim or 17a-ethinylestradiol did not induce significant lipid peroxidation in alfalfa leaves . Exposure to mixed PPCPs was found to exacerbate cytotoxicity to a rainbow trout gonadal cell line as compared to exposure to individual compounds . These results indicated that studies using individual PPCPs might underestimate the actual environmental impacts of trace organic contaminants that usually occur as mixtures. During the period of time in which cucumber plants were exposed to the PPCP mixture, an overall induction of enzymatic and non-enzymatic antioxidant systems was observed. Superoxide dismutase constitutes the first line of defense against ROS, which can dismutate O2 .- into the more stable H2O2 . In this study, the root experienced more significant oxidative damage as a result of greater ROS accumulation than shoots, and elevated activities of SOD were observed in treatments below 5 mg L! 1 of PPCPs .

Above the 5 mg L! 1 treatment rate, SOD activity in roots was significantly inhibited. In a previous study,increased SOD activity was detected after exposure to paracetamol , while decreased SOD activity was found under triclosan and galaxolide stress in wheat seedlings. A possible explanation for the decreased SOD activity may be that the oxidative stress exceeded the capability of the enzymatic machinery. It must be noted that there exist three forms of SOD isoenzymes: copper/zinc containing SOD, manganese containing SOD and iron containing SOD, which are all localized in different cellular compartments. Further research should take into account the specific roles of different SOD isoenzymes from different cellular compartments after exposure to trace xenobiotics. Using ascorbate as a reductant, H2O2 was reduced to water by ascorbate peroxidase , the major component of the ascorbate-glutathione cycle . Thus the increase in APX activity in the PPCP treated cucumber plants may be related to the functioning of the ascorbate-glutathione cycle that detoxifies H2O2 thereby preventing further damage. This was consistent with previous observations in Brassica juncea with acetaminophen treatment . It has been shown that APX may be responsible for the fine modulation of ROS for signaling, whereas CAT might be responsible for the removal of excess ROS during stress. The significantly increased catalase activity with increasing PPCP concentrations reaffirmed that the cucumber plants experienced serious oxidative stress when in contact with the PPCP mixture. Beside anti-oxidation, another key role of POD and GSTs is their ability to inactivate toxic compounds . For example, Agostini et al. demonstrated the capacity of peroxidases to degrade the pesticide 2,4-dichlorophenol in a cell culture of Brassica napus. Xia et al. suggested that the induced activity of GST by chlorpyrifos indicated formation of glutathione S-conjugates to detoxify the insecticide in plants.