Month: December 2022

California’s record high average yields of 30.75 tons per acre in 1991, the highest in the world, are due largely to sustained research efforts over a long period of time. These efforts, which included variety testing, culture, soil fumigation, disease-free plants, drip irrigation, mulching, and annual replanting, are documented in Alston, Pardey and Carter . California Strawberry Advisory Board grants accounted for 42.5 percent of all state funds for strawberry research during the 15-year period from 1978 to 1992. The distribution of the returns from production research is an issue that has been studied extensively by agricultural economists. Alston, Norton, and Pardey provide an excellent summary of this work. Depending upon the relative price elasticities of demand and supply, consumers may receive half or more of the short-run benefits from production research. Huang and Sexton demonstrated recently that market power can have an important effect on both the level and distribution of benefits. Processors with market power may be able to capture a large share of the benefits at the expense of both consumers and producers. To the extent that the benefits from producer-funded research accrue to consumers and processors, it diminishes the farm sector’s incentive to fund such research.As Table 5 shows, some commodity programs have been effective for a long period while others are of more recent origin. Many programs have been terminated as a result of changing economic and political relationships.

Despite the turnover, the number of government-mandated commodity programs has grown over time,chicken fodder system and the group approach to solving commodity marketing problems remains popular. The periodic renewal votes conducted for most programs reveal their popularity, with positive votes typically above 90 percent. A number of marketing programs have, however, encountered problems. As a group, the programs using quantity controls to practice price discrimination have lost governmental and legislative support, due to perceived adverse impacts on U.S. consumers. The programs with the strongest potential for increasing producer prices, including hops, lemons, Navel oranges, and Valencia oranges, have been terminated by the Secretary of Agriculture. Those orders with quantity controls nowadays use them infrequently.Informed observers agree that it will be very difficult to gain approval for a new marketing order with strong quantity controls. Programs compelling producer and handler support of commodity advertising programs have faced withering legal challenges in recent years based upon the argument that they represent an undue restriction on commercial free speech under the First Amendment to the U.S. Constitution. Two recent rulings on the issue by the U.S. Supreme Court have done little to clarify matters.6 Additional litigation is working its way through the court system. If the courts find ultimately that producers and handlers cannot be compelled to support an industry’s advertising program, it will likely fail due to free-rider problems. If the courts decide in favor of mandatory support, current programs will continue and new programs may emerge. There will, however, be increased monitoring of program costs and benefits to assure program supporters that their funds are being well-spent. Research funding pressures may require commodity groups to increase their support for research programs, if they want research to be done. The mandated programs provide a proven means for commodity-based research support, and they may take on an increased research role, as has been done by the California strawberry industry.This chapter surveys California’s agricultural trade environment and prospects.

We pay particular attention to the impacts of the 2002 United States Farm Bill, the Farm Security and Rural Investment Act on California’s trade in agricultural products and the prospects for California agriculture from further agricultural trade liberalization. We argue that foreign markets are extremely important to California agriculture, and that increased trade liberalization will be beneficial to most California producers since they competitively supply specialty products and continue to face barriers to trade in important markets. We also discuss the benefits of subsidies provided to agriculture in California and agricultural exports in particular. While a quantitative comparison of this support versus the potential benefits of increased trade liberalization is beyond the scope of this chapter, there is suggestive evidence that California agriculture would be better off with reduced subsidies to U.S. agriculture and concomitant increased access to markets abroad. Thus, to the extent that the political fallout from the Farm Bill results in less ambitious World Trade Organization negotiations, the 2002 Farm Bill is costly for the California agricultural sector.The remainder of this chapter is organized as follows: First, the chapter describes the main characteristics of California’s agricultural trade. Second, the international trading environment facing California agriculture is discussed. Third, we review and discuss elements of the Farm Bill that have important implications for California’s agricultural trade. These include the export programs, the highly controversial country-of-origin-labeling guidelines, and environmental programs. Fourth, we discuss how the 2002 Farm Bill affects the U.S.’s ability to meet its current WTO obligations and its potential effect on current liberalization talks from which California has much to gain.California agricultural producers rely on foreign markets for a significant portion of their revenues and export relatively more than producers in other states do. The value of California agricultural exports totaled about $6.5 billion in 2002, or about 20 percent of the value of agricultural commodities produced in California.

While it is not surprising that California’s export earnings exceed those of every other state since its farm cash receipts are the highest in the country, exports are relatively more important to California than to other states. While California accounts for 12 percent of national farm cash receipts , it accounts for an estimated 15 percent of total U.S. agricultural export revenue. To put these figures in an international context, the state of California exports more agricultural products than some leading agricultural countries do, including such countries as Chile and China. The annual value of Mexico’s agricultural exports is only slightly larger than California’s estimated value.As shown in table 1, the top six food product exports from California in 2002 were almonds, cotton, wine, table grapes, dairy, and oranges. The state is not a significant producer or exporter of grain crops such as corn, wheat, or soybeans. In fact, the state is a net importer of feed grains. Figure 1 highlights the diversity of California’s exports. The top five products account for just over one-third of California’s agricultural exports by value. Even when exports are aggregated into commodity groups, as opposed to individual products, the range of products exported by California is striking . According to UC Agricultural Issues Center statistics, fruit exports comprise 25 percent of the state’s agricultural exports, followed by field crops , tree nuts , vegetables , animal products and wine . This diversity of exports reflects California’s production diversity and differentiates the state from other important agricultural states in the U.S., which tend to produce only a few commodities. For instance, the agricultural sector in Iowa and Illinois is concentrated in just three commodities: corn, soybeans and hogs,fodder systems for cattle which account for 70-80 percent of those states’ farm cash receipts. Nebraska’s production of corn and cattle generates over 70 percent of that state’s farm receipts. Texas depends on the cattle sector, which produces 50 percent of its farm cash receipts . Of any other state in the U.S., the profile of Florida’s agriculture is perhaps most similar to California’s. While the value of agricultural production in Florida is about 25 percent of that in California, Florida’s agriculture is quite diversified and the state produces fruits, vegetables, and dairy products. However, Florida is not as dependent on foreign markets as California is; many of the state’s fruits and vegetables are sold domestically.

Not surprisingly, this means that Florida’s growers tend to be more protectionist than growers in California. As we explain a little later, California growers have a great deal to gain from breaking down foreign barriers to trade in fruits and vegetables; this is less true for Florida growers. California’s exports are destined for a diverse group of relatively high income countries, with the exception of the increasingly important Chinese market. The major foreign markets for almonds and wine are in Europe, while significant markets for the other top commodities are in Canada, Mexico, and Asia. Penetration of these desirable markets is all the more impressive because these countries remain quite protectionist with respect to agriculture, as discussed in the next section. It is estimated that about 40 percent of California agricultural exports is destined for Asia, 20 percent to Europe, and 30 percent to North America. California exports nearly twice as much of its agricultural output to the relatively wealthy European Union markets compared to the U.S. as a whole .California agriculture faces a complex international trading environment, characterized by import tariffs, non-tariff trade barriers, new competitors, and relatively little traditional federal assistance compared to other states. In this section, we review the market environment in which California’s agricultural producers compete. Increasing foreign competition and relatively closed markets have created demand within California for both increased government support for agriculture , and further trade liberalization in foreign markets . The internal contradictions between these positions have not been resolved. We argue later that California receives little benefit from the taxpayer dollars spent on foreign marketing; consequently, the California agricultural industry may wish to concentrate on achieving global trade liberalization even if this necessitates funding reductions for foreign marketing activities. In the last decade, the nominal value of total U.S. agricultural exports grew by about 30 percent. Exports of some commodities important to California grew more rapidly and some less rapidly than the national average. Over this time period, U.S. dairy exports increased by 265 percent and fresh vegetable exports increased by 73 percent. Figure 3 shows how the nominal values of some major California exports changed over the period 1995-2002. According to UC AIC and the Foreign Agricultural Service , the fortunes of California’s commodities have been mixed; almonds and wine have fared somewhat better than tomatoes and raisins. While the total nominal value of California’s agricultural exports has declined by about 5 percent since 1995, this figure masks widely divergent trends across commodities, so no general conclusions can be drawn.In the 1990s the most significant import growth in world markets was in high valued and processed food products like those grown in California. The share of high value and processed agricultural products in world agricultural trade has increased from less than 40 percent in the early 1980s to well over 50 percent by the end of the 1990s . At the same time, the share of fruits and vegetables in world agricultural trade remained at about 17 percent from 1990 to 2001, with a dollar value of $69.8 billion in 2001, up from $51 billion in 1990 . The fact that fruit and vegetable trade did not increase any faster than total agricultural trade is very surprising given the growing per capita demand in developed countries for fresh fruits and vegetables. The stagnant share of fruit and vegetable trade no doubt reflects the high level of protectionism around the world for these food categories. For instance, two-tiered tariffs known as tariff-rate quotas are commonly used to restrict imports of fruits and vegetables. Worldwide, there are more than 350 TRQs placed on trade in fruits and vegetables, and more than 25 percent of all agricultural TRQs are concentrated in the fruit and vegetable trade . This phenomenon critically affects California agriculture. As an exporter of high-value food commodities, California must contend with the fact that import tariffs in important markets such as in the EU are generally higher on processed agricultural products than on the primary commodities. This tariff wedge between a processed commodity and its corresponding primary commodity is referred to as tariff escalation, and this is a significant obstacle to California exports. Tariff escalation produces a trade bias against processed agricultural products and value added products. There is general evidence of tariff escalation in OECD countries , especially for fruits, vegetables, and nuts—major California exports. For many countries, bound tariffs tend to be higher for processed food products than for unprocessed products . Furthermore, recent tariff reductions on agricultural products exceeded tarrif reductions on processed food products in Australia, Canada, the European Union and Mexico . Government transfers to the agricultural industry have contributed to the sector’s profitability in California, particularly for those farmers not growing nuts, fruits and vegetables.

By sales value, California agriculture is comprised of a large number of small farms, while a small number of large farms represent most of the sales. The 16 percent of California farms with sales of more than $250,000 in 1997 also represented over 90 percent of total sales value. In 1997, almost 44 percent of California farms sold less than $10,000 of agricultural products. Retired or part-time farmers operate most of these farms.There appears to be a continuing trend toward fewer young people choosing farming as an occupation. Between 1987 and 2002 there were fewer farmers in the younger age categories and an increase in the oldest category. The percent of California farmers over 65 increased from 23 percent to almost 30 percent. Farming is likely a retirement occupation for an increasing number of individuals. Meanwhile, the share of the state population over 65 remained unchanged at about 10.5 percent between 1990 and 2000.Anecdotal information suggests that many family farms remain in the name of the oldest family members, even if they are less actively involved in farming than younger members. This trend may place an upward bias on age estimates since almost all of California’s farms are family owned and operated. In 1997, about 19 percent of U.S. farm operators described themselves as retired.Total pesticide use in California agriculture shows an upward trend, with total reported pounds applied fluctuating from year to year depending on pest problems, weather, and acreage and types of crop planted. Also,hydroponic dutch buckets the types and forms of the pesticides have changed to meet new pests and environmental demands. In 2000, more than 550,000 pounds of chemicals defined by the United States Environmental Protection Agency as “reduced risk” were applied by commercial agriculture in California.

This was equivalent to about one half of one percent of total pounds of pesticides applied to California crops. In 1990, California became the first state to require reporting of the agricultural use of all pesticides: insecticides, herbicides, rodenticides, fungicides, and sanitizers. In contrast, much of the non-agricultural uses such as chlorine for swimming pools and home and garden pesticides are not reported. About one-third of all California farms did not report using any chemicals or fertilizer in the 1997 Census of Agriculture. California has about 1,526 registered organic farmers, only a tiny portion of those farms that did not report using any chemicals or fertilizer. Therefore, care is needed in interpreting these Census of Agriculture figures. Many farmers may have failed to respond to this particular question or were small livestock growers or other operators whose farms used no chemicals or fertilizer without being defined explicitly as “organic.”California receives about 200 million acre-feet of precipitation in a normal non drought year. Roughly 65 percent of this is lost to evaporation or vegetation. The remaining 71 maf of average runoff, plus imported water, supplies the state’s water “budget,” traveling through California’s complex water distribution system to environmental, agricultural, and urban uses. Groundwater is an additional important source. In 1998 the California Department of Water Resources released a normalized water budget showing the state’s supply and use of applied water in an “average” non drought year. Figures in the “average” year budget were based on the distribution infrastructure in place in 1995. The 1.6 maf shortage is largely accounted for by groundwater overdraft that was not included in the budget. More than 70 percent of the average annual runoff occurs north of Sacramento, but about 75 percent of the state’s water demand is south of Sacramento. California uses a combination of federal, state, and local water projects to capture, store, transport, and import surface water to meet demand around the state. The largest water projects are the federal Central Valley Project and the State Water Project.

The amount of water per acre used by urban areas varies according to land use, population density and water use efficiency. In some areas agriculture may use less water per acre than nearby urban development while in other areas the opposite case may be true. Groundwater provides 30 percent of the supply used by agriculture and the urban sector in a normal non-drought year. Agriculture accounts for over 90 percent of the groundwater used in the San Joaquin, Tulare Lake, and Central Coast hydrologic regions. Only a portion of the applied water is actually used by the crop. The remainder percolates through the soil, flows downstream to other uses, or is irrecoverably lost due to other factors. Crop water use is measured as evapotranspiration of applied water . The ratio of ETAW to applied water is an indication of irrigation efficiency. The amount of water applied to a particular crop depends on many factors including plant evapotranspiration, soil properties, irrigation efficiency, and weather. Plant intake is the primary purpose of water application, but water is also applied to crops for cultural purposes such as frost control, facilitating cultivation and leaching of salts out of the crop root zone. There is a wide range in water application rates among crops and hydrologic regions. For example, depending on the hydrologic region, anywhere between 2 and 10-acre-feet/acre are applied to alfalfa annually. Hay production, including alfalfa, accounts for almost 15 percent of total irrigation water used in agriculture. Cotton accounts for about 12.5 percent. The top 12 commodities, those that represent 60 percent of the total value of California agriculture, account for about 48 percent of the water used for irrigation in the state. Agricultural surface water costs differ greatly by hydrologic region and source of supply. According to the Department of Water Resources, the 2003 Central Valley Project contract rates range from $2 per acre-foot in the Sacramento Valley to $27 in the county of Tulare and almost $30 in some areas of the Delta.

Almost one-third of California’s irrigated acreage used sprinkler, drip or trickle systems in 1998. The rest used gravity flow systems such as furrows. More than one method was used on some acreage.Technological innovation, fueled by research and entrepreneurship, has been a driving force in U.S. agriculture during the past century, leading to both higher yields and lower prices. In California, technological change has facilitated significant yield increases for many crops as well as other changes. Inputs have been used more efficiently to produce greater quantities of output. For instance, cash receipts per irrigated acre increased by 35 percent between 1960 and 1995. This can be attributed partially to the development and implementation of more efficient irrigation, such as drip systems, and partially to a change in the type of crops produced. The most recent analysis available finds that the productivity index for California agriculture doubled between 1949 and 1991. During the 1990s, particularly toward the end of the decade, computers were increasingly incorporated into farming operations. In only two years, between 1997and 1999, the number of California farms with Internet access doubled to 46 percent, and reached 51 percent in 2001. Overall, about 36 percent of California farms reported using computers in their business operations in 2001, compared to 29 percent for the United States as a whole, although there are several states with higher usage than California.In 2001, U.S. agricultural experiment stations collectively spent $2.3 billion on scientists’ agricultural research. The University of California Division of Agriculture and Natural Resources accounted for about 10 percent of those resources. The DANR includes scientists with the UC Berkeley College of Natural Resources, the UC Davis College of Agricultural and Environmental Sciences, the Division of Biological Sciences,bato bucket and the School of Veterinary Medicine; and the UC Riverside College of Natural and Agricultural Sciences. The DANR’s two major organizational units are the Agricultural Experimental Station and the Cooperative Extension . The AES is basically a multi-campus research organization, with a staff of near 700 academics distributed in more than 50 different departments. The CE constitutes the main outreach program, with about 400 specialists and advisors dispersed throughout the state. During the 1990s DANR aggregate funding stayed approximately constant at an average of $235 million per year. From 1999 to 2002, total funding increased in constant terms by 25 percent. The three campuses ,accounted for 72 percent of the 2002 annual DANR expenditures, while regionally based units accounted for 14 percent of the budget, and statewide academic programs and their support 12 percent. In 2002, about 80 percent of total funding came from government sources ; 13 percent came from private gifts, grants and contracts, and 7 percent from other sources, such as county government, endowments, sales, services, etc.The number of CE County Advisors decreased by about 18 percent between 1990 and 1999, from 326 to 265, and their distribution among program areas has changed. Agriculture Program Area now accounts for 60 percent of the UC Cooperative Extension County Advisors, up from 55 percent in 1990, while Human Resources decreased from 34 to 30 percent. Natural Resources program changed slightly from 11 to 10 percent of the CE County Advisors.

Each dollar earned within agriculture fuels a more vigorous economy by stimulating additional activity in the form of jobs, income and output. In general, the greater the interdependence in the economy, the greater the additional activity, or multiplier effects. These multipliers may be applied to the county, state and regional levels using the IMPLAN4 model. Multiplier effects can be represented by four measures that reflect the impact that agriculture has on the state. The first measure, sales impact, records how agricultural purchases influence total private sector sales. A second measure is the amount of personal income produced directly and indirectly by the economic output of agriculture and agricultural processing. The third measure calculates the total value-added linked to agriculture. “Value added” in this case is equal to the value of goods and services sold by a firm or sector of the economy, minus the cost of inputs and services used to produce those goods. A final measure is the number of jobs in agriculture, agricultural processing and other sectors of the economy related to agriculture in the state. These multiplier effects may be demonstrated by tracing the activity of an individual farm. A farm’s sales impact would include all the inputs used on that farm, such as machinery, fertilizer, electricity—anything farm dollars buy. The personal income from the farm would include the farm’s income and a portion of the income of those from whom the farm purchased inputs. The farm’s value added would be equal to the cash receipts from sales of farm products less the costs of inputs that went into producing those goods. The jobs related to the farm’s efforts would include labor on that farm as well as in input and output industries that rely on business from that farm. For example, agricultural machinery manufacturers, chemical manufacturers, processors, and people working in retail food trade have jobs that are related to agriculture. The economic impacts shown in Table 22 can be interpreted as an indication of how the state would be affected if agricultural production and processing were to cease, and the associated inputs were not reemployed in any other economic use. Multiplier effects differ by commodity since some commodities may be related to more input and processing industries than others. For example, dairy production is related to a relatively extensive processing sector, for which a wide range of inputs and specialized machinery has been developed. Hence, the dairy industry may have a greater effect on the economy in terms of multiplier effects than some other commodities. Multiplier effects may differ by region due to geographic dispersion of industries related to agriculture, aggregate size of agriculture and type of commodities produced in that region. Some industries have more local impacts, while others have impacts that are spread farther afield. For example, county or multi-county multiplier effects do not include input and processing industries located outside of that region, even if those industries are located elsewhere in the state. Similarly, state multiplier effects do not include input and processing industries located outside of the state. Thus, multiplier effects for commodity groups with geographically diffuse input and processing sectors may be underestimated. Through multiplier effects, agricultural production and processing account for about 6 percent or 7 percent of the state’s total income, value-added, and jobs.

A series of droughts and floods in the 1860s devastated many herds, and when recovery occurred in the 1870s, sheep-raising had largely replaced cattle-ranching. Indeed, by 1889, the state became the nation’s leading wool producer, with almost 13 percent of national output.Many of the livestock ranches of the nineteenth century operated on extremely large scales. Examples of these operations include Miller-Lux, Tejon, Kern County Land Company, Flint-Bixby, Irvine, Stearns, and Hearst. With the intensification of crop production in California, livestock activities tended to grow slowly. Although the smaller family-sized farms began to replace the large bonanza grain farms and livestock ranches, “general” or “mixed” farms modeled on mid western prototypes remained rare. This is reflected in the relatively small role of swine production in Figure 3. Largely as a result, over the 20th century, livestock production was relatively less important in California than in the country as a whole. For example, over the 1930-97 period, the share of the market value of sales of livestock and livestock products in the combined market value of sales of crops, livestock, and livestock products has almost always exceeded one-half nationally whereas, in California it usually hovered around one-third.The chief exceptions to the generalized pattern of slow growth over the early 20th century were dairy and poultry raising.These activities steadily expanded, primarily to serve the state’s rapidly growing urban markets. In 1993, California replaced Wisconsin as the nation’s number one milk producer.Between 1900 and 1960,hydroponic gutter the number of milk cows grew at a rate of 1.5 percent per annum and the number of chickens at a 3.3 percent rate. Output growth was even faster as productivity per animal unit expanded enormously, especially in the post-1940 period.

From the 1920s, California was a leader in output per dairy cow. For example, in 1924 milk production per dairy cow in California was 5,870 lbs., while similar figures for Wisconsin and the U.S. were 5,280 and 4,167 lbs. respectively.A similar pattern is found more recently. In 2000, California dairy cows produced an average of 21,169 lbs. of milk. The U.S. average was 18,204 lbs., while Wisconsin lagged behind with an average of 17,306 lbs.The post-1940 period also witnessed a dramatic revival of the state’s cattle sector outside dairying. The number of non-milk cows in California increased from about 1.4 million head in 1940 to 3.8 million in 1969. This growth was associated with a significant structural change that was pioneered in California and Arizona—the introduction of large-scale commercial feed-lot operations.By 1953, large feedlots had emerged as an important feature of the California landscape, with over 92 percent of the cattle on feed in lots of a capacity of 1,000 or more head. Between 1953 and 1963, the number of cattle on feed in California and the capacity of the state’s feedlots tripled. At the same time the average size of the lots soared. By 1963, almost 70 percent of the cattle on feed were in mega-lots of 10,000 or more head. A comparison with other areas provides perspective. In 1963, there were 613 feed lots in California with an average of about 3,100 head per lot. By contrast, Iowa had 45,000 feedlots with an average of less than 63 head per lot; Texas had 1,753 feed lots with an average of 511 head per lot. More generally, by the 1960s the size of cattle herds in California far exceeded the national average.Employment of state-of-the-art feed lots and modern science and veterinary medicine along with favorable climatic conditions allowed ranchers in California and Arizona to achieve significant efficiencies in converting feed to cattle weight. In the 1960s, larger commercial feedlots started to become more prevalent in the Southwest and in the Corn Belt.Thus, as in other cases, technologies developed in California spread to reshape agricultural practices in other regions.

A hallmark of California agriculture since the wheat era has been its highly mechanized farms. Nineteenth-century observers watched in awe as cumbersome steam tractors and giant combines worked their way across vast fields. In the twentieth century, California farmers led the nation in the adoption of gasoline tractors,mechanical cotton pickers, sugar beet harvesters, tomato harvesters, electric pumps, and dozens of less well-known machines. The story of agricultural mechanization in California illustrates the cumulative and reinforcing character of the invention and diffusion processes. Mechanization of one activity set in motion strong economic and cultural forces that encouraged further mechanization of other, sometimes quite different, activities.Specialized crops and growing conditions created demands for new types of equipment. Protected by high transportation costs from competition with large firms located in the Midwest, a local farm implement industry flourished by providing Pacific Coast farmers with equipment especially suited to their requirements. In many instances the inventors designed and perfected prototypes that later captured national and international markets. Grain combines, track-laying tractors, giant land planes, tomato pickers, and sugar beet harvesters, to name but a few, emerged from California’s shops. Several factors contributed to mechanization. In general, California farmers were more educated and more prosperous than farmers in many areas of the country. These advantages gave them the insight and financial wherewithal to support their penchant for tinkering. Nowhere was this more evident than on the bonanza ranches, which often served as the design and testing grounds for harvester prototypes. The large scale of many California farms allowed growers to spread the fixed cost of expensive equipment. The scarcity of labor in California meant relatively high wage rates and periods of uncertain labor supply. The climate and terrain were also favorable.

Extensive dry seasons allowed machines to work long hours in near-ideal conditions, and the flat Central Valley offered few obstacles to wheeled equipment. In the cases of small grains and cotton, mechanization was delayed in other regions of the country because free-standing moisture damaged the crops. Such problems were minimal in California. All things considered, the state’s climatic and economic conditions were exceptionally conducive to mechanization. As an index of the level of mechanization, Figure 4 shows the real value of implements per farm in California and other major regions. Over the years 1870 to 1930 the average value of implements per California farm was about double the national average. The new generation of farm equipment of the nineteenth century relied increasingly on horses and mules for power. Horses on any one farm were essentially fixed assets. A stock of horses accumulated for a given task was potentially available at a relatively low variable cost to perform other tasks. Thus, once a farmer increased his pool of horses, he was more likely to adopt new power-intensive equipment. For these reasons, an examination of horses on California farms will yield important insights into the course of mechanization. In 1870 the average number of horses and mules on a California farm was almost three times the national average, and the number of horses and mules per male worker was more than twice the national average. Throughout the nineteenth century, California farmers were using an enormous amount of horsepower.California was a leader in the early adoption of tractors. By 1920, over 10 percent of California farms had tractors compared with 3.6 percent for the nation as a whole. In 1925, nearly one-fifth of California farms reported tractors, proportionally more than in Illinois or Iowa, and just behind the nation-leading Dakotas. These figures actually understate the power available in California, because the tractors adopted in the West were, on average, substantially larger than those found elsewhere.25 In particular, western farmers were the predominant users of large track-laying tractors,u planting gutter which were invented in California. The state’s farmers were also the nation’s pioneers in the utilization of electric power. The world’s first purported use of electricity for irrigation pumping took place in the Central Valley just before the turn of the century. Consistent data on rural electricity use are not available until 1929. At that time, over one-half of California farms purchased electric power compared with about one-tenth for the United States as a whole.26 One of the best proxies for electrification is the number of agricultural pumps. Over the period 1910 to 1940, the state accounted for roughly 70 percent of all of the nation’s agricultural pumps.The abundant supply of power on California farms encouraged local manufacturers to produce new types of equipment, and in turn, the development of new and larger implements often created the need for new sources of power. This process of responding to the opportunities and bottlenecks created by previous technological changes provided a continuing stimulation to innovation. Tracing the changes in wheat farming technology will illustrate how the cumulative technological changes led to a distinctly different path of mechanical development in the West as compared to that which occurred elsewhere.Almost immediately after wheat cultivation began in the state, its farmers developed a distinctive set of cultural practices. Plowing the fertile California soil was nothing like working the rocky soils in the East or the dense sod of the Midwest. In California, ranchers used two, four, and even eight-bottomed gang plows, cutting just a few inches deep.In the East, plowing one-and-one-half acres was a good day’s work in 1880. In most of the prairie regions, two-and-one-half acres were the norm.

In California, it was common for one man with a gang plow and a team of eight horses to complete six to ten acres per day. The tendency of California’s farmers to use larger plows continued into the twentieth century. After tractors came on line, the state’s farmers were also noted for using both larger models and larger equipment. This pattern influenced subsequent manufacturing and farming decisions.The preference for large plows in California stimulated local investors and manufacturers who vied to capture the specialized market. As evidence of the different focus of their innovative activity, the U.S. Agricultural Commissioner noted that “patents granted on wheel plows in 1869 to residents of California and Oregon largely exceed in number those granted for inventions of a like character from all the other states of the Union.”Between 1859 and 1873 California accounted for one-quarter of the nation’s patenting activity for multi-bottom plows. By way of contrast, the state’s contribution to the development of small single-bottom plows was insignificant.The experience with large plows directly contributed to important developments in the perfection and use of listers, harrows, levelers, and earth-moving equipment. The adoption of distinctive labor-saving techniques carried over to grain sowing and harvest activities. An 1875 USDA survey showed that over one-half of mid western farmers used grain drills, but that virtually all California farmers sowed their grain.California farmers were sometimes accused of being slovenly for using sowing, a technique which was also common to the more backward American South. However, the use of broadcast sowers in California reflected a rational response to the state’s own factor price environment, and bore little resemblance to the hand-sowing techniques practiced in the South. Among the broadcasting equipment used in California were advanced high-capacity endgate seeders of local design. By the 1880s improved models were capable of seeding up to 60 acres in one day. By contrast, a standard drill could seed about 15 acres per day and a man broadcasting by hand could seed roughly 7 acres per day.The use of labor-saving techniques was most evident on the state’s bonanza wheat ranches, where some farmers attached a broadcast sower to the back of a gang plow and then attached a harrow behind the sower, thereby accomplishing the plowing, sowing, and harrowing with a single operation.California wheat growers also followed a different technological path in their harvest operations by relying primarily on headers instead of reapers. This practice would have important implications for the subsequent development of combines in California. The header cut only the top of the straw. The cut grain was then transported on a continuous apron to an accompanying wagon. Headers typically had larger cutting bars and, hence, greater capacity than reapers, but the most significant advantage was that headers eliminated the need for binding. The initial cost of the header was about 50 to 100 percent more than the reaper, but its real drawback was in humid areas where the grain was not dry enough to harvest unless it was dead ripe. This involved huge crop risks in the climate of the Midwest, risks that were virtually nonexistent in the dry California summers.

The stakeholders consisted primarily of the Preserve Partners—a consortium of federal, state, and local agencies—in addition to non-profits such as TNC and Ducks Unlimited. The management scenarios were developed with reference to the Preserve Management Plan , created by the Preserve Partners through a 2-year planning process . The management plan gathered information from the public , the Preserve Partners, local municipalities, and other groups. We used a time-frame of ~30 years to 2050 . We considered each of the scenarios in isolation; for example, landscape-wide restoration does not account for development, nor urbanization for set-asides for wildlife. For parcels not affected by the management scenario, we assumed a static landscape with no change of land use occurring, as much of the land has remained pastoral for ~150 years before present. The objective of this scenario was to maximize restoration of agricultural lands to natural riparian habitat, focusing on areas of specific soil type and proximity to the river, based on an analysis in the Management Plan. In addition, Preserve goals, as set forth in the Management Plan, support maximizing the restoration of riparian habitat in the Cosumnes corridor. We applied six decision rules that relate the location of each parcel to existing landscape features. A parcel received a high likelihood of being restored if: the parcel was currently within the Cosumnes River Preserve lands; was managed for other conservation purposes ; was within a historical riparian corridor; was within 1km of standing water; was within 1km of grassland, shrubland, or wetland; or was within 1km of riparian forest.

The 1-km distance threshold was set to be inclusive of remnant riparian forest within the Preserve,flood table coupled with the assumption that areas within this threshold are practical targets for restoration. If a parcel fell within any one of these six categories, it was given a score of one. Scores were summed for each of the six rules, and a composite score was given to each parcel. Using the composite score, the upper quartile of all parcels with a score greater than one, weighted by area, were designated to be restored. We tested for multicollinearity using the variance inflation factor , which did not indicate substantial multicollinearity of the six decision rule variables . We filtered the final layer of restorable parcels to exclude existing urban areas. The restoration of these parcels was to grassland or riparian forest, which was assigned based on the potential natural vegetation . We did not consider areas defined by Kuchler as subtidal marsh within the study area for future restoration because of the current lack of feasibility; consequently, parcels remained under current land cover . The objective of this scenario was to represent a realistic growth outcome for the area projected to 2050. We assigned parcels as urban in 2050 based on whether they were currently urban or projected to become urban using the Preferred Blueprint Scenario for 2050 . SACOG generated the blueprint to help guide local government in growth and transportation planning through 2050 throughout the six-county region. This Preferred Blueprint Scenario promotes compact, mixed-use development and more transit choices as an alternative to low-density development . We filtered the final layer of potentially urbanized parcels by extracting current protected areas and areas of riparian forest, which are unlikely to be developed because they are within the 100-year floodplain. This provided the land cover data necessary to make projections in ecosystem services and disservices for 2050. The objective of this scenario was to maximize high-quality foraging habitat for the Swainson’s Hawk, and was developed based upon the literature and experience of the authors. A parcel received a high likelihood of being Swainson’s Hawk-friendly agriculture if it was designated as or within 1.25km of alfalfa, grain, pasture, or row crop . We selected all non-protected parcels of natural vegetation, such as grasslands, within 1.25km of existing fields under these four agricultural types to identify parcels available for conversion to agricultural types favorable to Swainson’s Hawk.

For practical reasons, certain agricultural types were not considered for conversion. For example, vineyards, given their high economic value , are unlikely to be converted; vineyard expansion remains a dominant trend for the region . We filtered the final layer of potentially enhanced parcels to exclude riparian forest and existing urban areas. Using the composite score, we converted the upper quartile of all parcels for these rules from existing land use to the four agriculture types more favorable for Swainson’s Hawk foraging. These types were randomly allocated in proportion to the number of parcels in which they currently occur: alfalfa 5%, grain 20%, pasture 45%, and row crop 30%. As with the restoration scenario, we tested for multicollinearity using the VIF, which indicated the model did not have substantial multicollinearity of the variables.We quantified the amount of carbon stored within three different land-cover types based on readily available data and literature: agricultural crops ; natural, non-forest vegetation types ; and forest types . We did not include soil carbon storage in this analysis, nor do we account for carbon storage associated with natural habitat within urban areas. We assumed that these steady-state estimates apply to all locations, and that changes in land cover would increase or decrease carbon storage to a new steady state.Above- and below-ground carbon storage for standing agricultural crops was based on a study by Kroodsma and Field . We divided the yield by the harvest index for each crop, and then multiplied the result by 0.45 as the proportion of biomass assumed to be carbon to estimate Mg C ha-1. For row crops not included in Kroodsma and Field , we estimated it using the National Agricultural Statistics Service yield data from the year 2000 and the average harvest index for all row crops . We estimated carbon storage for orchards by assuming the mid-point of the crop’s lifespan, multiplying this by wood accumulated , and again multiplying by 0.45 to provide Mg C ha-1.

For perennial crops not listed in Kroodsma and Field , we used the mid-point of the average lifespan and the average wood-accumulation estimates for crops within the same category as defined by the CDWR. For our broad categories of grains, orchards and row crops, we calculated an average value of Mg C ha-1 based on data available for each crop type within the category. We multiplied the area of each of the seven agricultural classes in the study area by the estimated carbon to provide total Mg C ha-1 . Carbon storage for non-forest natural vegetation types used estimates from the literature: pasture, grassland, and shrubland , and freshwater emergent wetlands . We divided forested vegetation types into three main types of riparian forest: valley oak , Fremont cottonwood , and willow . We improved the estimates of carbon storage associated with these types of riparian forest with plot data collected on the Cosumnes Preserve. Plot data included the measurement of all trees >10cm diameter at breast height within a 0.04-ha plot , applied allometric equations to calculate the amount of above-ground carbon , and summed these amounts to report a total Mg C ha-1 for riparian forests . These are in line with other estimates of live biomass from riparian studies in California . Using this variety of techniques,rolling benches we assigneda coarse estimate of total Mg C ha-1 for each parcel in the study area . Since this compilation of varied data includes a mix of both above- and below-ground carbon estimates for different classes , our estimates need to be considered conservative. We assessed the effect of different landscape management scenarios on the Swainson’s Hawk and also on a suite of 15 focal bird species. First, we used Boosted Regression Trees modeling techniques to fit the baseline land-cover data to presence and absence points of Swainson’s Hawk nest locations. We used known nest locations, identified using comprehensive field surveys of the area , to generate presence points . We generated absence points by randomly placing pseudo-absence points within the study area. We used 75% of the points to train the landscape suitability model, and 25% to test the predictive ability of those points.

We generated models by calculating the proportion of each land-use type contained within a 25-ha square that surrounded each presence and absence point. This threshold utilized research which found that 50% of Swainson’s Hawk foraging occurs within 25 to 86 ha of nest sites . We also tested model sensitivity using a 100-ha core area, and noted no significant changes n model results. Once we fitted the current land-cover type to the Swainson’s Hawk nest presence and absence data, we used the BRT to spatially project the probability of landscape suitability onto each of the three future scenarios. We assessed model performance using area under curve of the receiver operating characteristic curve scores . We converted model results to raster grids and assigned each parcel a landscape suitability score based on the average score of all grid cells contained within a parcel . Second, we assessed the effects of the three management scenarios relative to baseline on a suite of 15 other focal bird species identified as indicator species for natural habitats in the Central Valley . In contrast to the Swainson’s Hawk approach, we used existing suitability models developed for each of these focal species in the Central Valley, with suitability scores ranging from zero to one . We assigned suitability values to our baseline and three alternative scenario parcels using two steps. First, we estimated the average suitability for each bird species within each of our land-cover types by overlaying the spatial suitability surfaces onto our land-cover data and calculating the area weighted average suitability of each land-cover type for each bird species . Second, we assigned an area-weighted suitability value for each of the 15 species to each parcel in our baseline and future management scenarios, according to the parcel’s land-cover type. Based on these scores, we calculated the average suitability score for each landscape scenario across all 15 focal bird species, using a 5% increase or decrease as the threshold for meaningful change.We calculated nitrous oxide emissions for the agricultural land-use types in a manner consistent with International Panel on Climate Change Tier-1 guidelines . The key input parameter was nitrogen fertilizer use. We acquired estimates of nitrogen fertilizer application rates from a compilation of California data and summarized these by our seven agricultural types . For grain, orchard, pasture, and row crops, which contain multiple types of crops, we averaged emission rates of these individual crops to provide a single figure for the class. We used IPCC emissions factors to convert nitrogen fertilizer application to nitrous oxide emissions. This was 1% of nitrogen fertilizer applied for all crop groups except rice, for which we used an emissions factor of 0.3% . We excluded estimates for alfalfa since this crop rarely receives inorganic nitrogen fertilizer application and only accounts for a small proportion of the study area . For each parcel, we estimated the amount of nitrous oxide emissions per year under baseline and the three alternative management scenarios based on the land use type within each parcel. We calculated the amount of nitrate–nitrogen leaching for the agricultural land types based on the difference between nutrient inputs and nutrient losses. We compiled nutrient inputs from crop specific fertilization rates and based nutrient losses on the amount of nitrogen harvested in crops . We assumed atmospheric losses to be 10% of the fertilization rate, which is a conservative estimate developed to reflect the total N gaseous emissions . We assumed all surplus nitrogen was leached from soil into the groundwater in the form of nitrate , and for crops where the nitrogen harvested exceeds the nitrogen inputs, we assumed leaching loss was zero. As with emissions, we estimated the amount of nitrogen currently leached per year for each parcel and for the three alternative scenarios.Natural vegetation increased slightly in the restoration scenario from a baseline of 44% to 46% of the study area, and decreased in the urban and enhanced agriculture scenarios to 40% and 21%, respectively . Under baseline conditions, natural vegetation consisted primarily of grassland in the eastern portion of the study area , and riparian forest along the Cosumnes River accounts for 4%. Cover of riparian forest increased to 12% under the restoration scenario .

To investigate whether Tic20 can indeed form an ion channel, Tic20-proteoliposomes were subjected to swelling assays . Changes in the size of liposomes in the presence of high salt concentrations, as revealed by changes in the optical density, can be used to detect the presence of a poreforming protein. After addition of 300 mM KCl to liposomes and Tic20-proteoliposomes, their optical densities dropped initially, due to shrinkage caused by the increased salt concentration. However, the optical density of protein-free liposomes remained at this low level, showing no change in their size; whereas in the case of Tic20-proteoliposomes the optical density increased constantly with time. The increase in optical density strongly supports the presence of a channel in Tic20-proteoliposomes that is permeable for ions, thereby creating an equilibrium between the inner compartment of the proteoliposomes and the surrounding buffer. To exclude the possible effects of contaminating channel-forming proteins derived from the bacterial membrane and a protein inserted into the liposomes , a further negative control was set up: Tic110 containing only the first three transmembrane helices was purified similarly to Tic20 and reconstituted into liposomes. We chose this construct, since NtTic110 inserts into the membrane during in vitro protein import experiments. Furthermore, as the full length and N-terminally truncated Tic110 possess very similar channel activities, it is unlikely that the N-terminal part alone forms a channel. The insertion of NtTic110 into liposomes was confirmed by incubation under different buffer conditions followed by flotation experiments, similarly to Tic20 . However,hydroponic dutch buckets these NtTic110-proteoliposomes behaved similarly to the empty liposomes during swelling assays: after addition of salt, the optical density decreased, and except for a small initial increase, it remained at a constant level.

This makes it unlikely that a contamination from E. coli or simply the insertion of a protein into the liposomes caused the observed effect in the optical density of Tic20- proteoliposomes. To further characterize the channel activity of Tic20, electrophysiological measurements were performed. After the fusion of Tic20-proteoliposomes with a lipid bilayer, ion channel activity was observed . The total conductance under symmetrical buffer conditions , 250 mM KCl was dependent on the direction of the applied potential: 1260 pS and 1010 pS under negative and positive voltage values, respectively. The channel was mostly in the completely open state, however, individual single gating events were also frequently observed, varying in a broad range between 25 pS to 600 pS. All detected gating events were depicted in two histograms.Two conductance classes were defined both at negative and positive voltage values with thresholds of 220 pS and 180 pS, respectively.The observed pore seems to be asymmetric, since higher conductance classes notably differ under positive and negative voltages. This is probably due to interactions of the permeating ions with the channel, which presumably exhibits an asymmetric potential profile along the pore. Since small and large opening events were simultaneously observed in all experiments, it is very unlikely that they belong to two different pores. The selectivity of Tic20 was investigated under asymmetric salt conditions , 250/20 mM KCl. Similarly to the conductance values, the channel is intrinsically rectifying ,supporting asymmetric channel properties. The obtained reverse potential is 37.0 ± 1.4 mV . According to the Goldman-Hodgkin-Katz approach, this corresponds to a selectivity of 6.5:1 for K+ :Cl- -ions, thus indicating cation selectivity similar to Tic110.

To determine the channel’s orientation within the bilayer, two side-specific characteristics were taken into account: the highest total conductance under symmetrical buffer conditions was measured under negative voltage values, and the channel rectifies in the same direction under asymmetrical buffer conditions . Therefore, it seems that the protein is randomly inserted into the bilayer. The pore size was roughly estimated according to Hille et al.. Considering the highest conductance class , a channel length of 1-5 nm and a resistivity of 247.5 Ω cm for a solution containing 250 mM KCl, taking into account that the conductivity of the electrolyte solution within the pore is ~5 times lower than in the bulk solution, the pore size was estimated to vary between 7.8-14.1 Å. This is in good agreement with the size of protein translocation channels such as Toc75 in the outer envelope membrane and Tic110 in the IE. Thus, the size of the Tic20 pore would be sufficient for the translocation of precursor proteins through the membrane. NtTic110, as a negative control, did not show any channel activity during electrophysiological measurements, indicating that the measured channel is not the result of a possible bacterial contamination . Considering our data presented here and those published in previous studies, we can conclude that the Tic translocon consists of distinct translocation channels: On the one hand, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins; on the other hand, Tic20 might facilitate the translocation of a subset of proteins. This scenario would match the one found in the inner mitochondrial membrane, where specific translocases exist for defined groups of precursor proteins: the import pathway of mitochondrial carrier proteins being clearly separated from that of matrix targeted preproteins. The situation in chloroplasts does not seem as clear-cut, but an analogous separation determined by the final destination and/or intrinsic properties of translocated proteins is feasible. The severe phenotype of attic20-I mutants prompts us to hypothesize that Tic20 might be specifically required for the translocation of some essential proteins. According to cross-linking results, Tic20 is connected to Toc translocon components. Therefore, after entering the intermembrane space via the Toc complex, some preproteins might be transported through the IE via Tic20.

On the contrary, Kikuchi et al. presented that Tic20 migrates on BN-PAGE at the same molecular weight as the imported precursor of the small subunit of Rubisco and that tic20-I mutants display a reduced rate of the artificial precursor protein RbcS-nt: GFP. The authors interpreted these results in a way that Tic20 might function at an intermediate step between the Toc translocon and the channel of Tic110. However,bato bucket being a substantial part of the general import pathway seems unlikely due to the very low abundance of Tic20. It is feasible to speculate that such abundant proteins as pSSU, which are imported at a very high rate, may interact incidentally with nearby proteins or indifferently use all available import channels. To clarify this question, substrate proteins and interaction partners of Tic20 should be a matter of further investigation. Additionally, a very recent study suggested AtTic20-IV as an import channel working side by side with AtTic20-I. However, detailed characterization of the protein and experimental evidence for channel activity are still missing.Cerium oxide nanoparticles are widely used in applications such as catalyst automotive industry, glass mirrors, plate glass, and ophthalmic lenses . These NPs are among the 13 engineered nanomaterials in the list of priority for immediate testing by the Organization for Economic Cooperation and Development . However, the environmental release of CeO2 NPs from factories or applications, and their behavior and effects in the environment are not well known yet . Previous studies have shown that CeO2 NPs are stable in soil at pH values of 7 to 9 . This suggests CeO2 NPs will remain in soil for a long time. In addition, reports from recent investigations have shown a wide variety of plant responses after exposure toCeO2 NPs. For instance, Schwabe found that CeO2 NP treatments did not reduced the growth in pumpkin and wheat. However, Ma et al. reported that, at 2000 mg/L, nano-CeO2 reduced root elongation in lettuce . Van Hoecke et al. found that CeO2 NPs, at concentrations as low as 2.6 and 5.4 mg/L, produced chronic toxicity to the unicellular alga Pseudokirchneriella subcapitata. Previous results from our research group have shown that CeO2 NPs at 2000 mg/L reduced corn and tomato germination by 30% and cucumber germination by 20% . In a more recent study, we demonstrated that CeO2 NPs are taken up and stored without change in maize roots . This previous study also revealed that the uptake of CeO2 NPs by corn plants was affected by soil organic matter content and alginate surface coating . Alginates are naturally occurring polysaccharides that have been used to stabilize NPs for several applications . This suggests that excess of alginate can be released into the environment together with NPs, with unknown consequences for edible plants. Thus, more studies are needed to better understand the impact of CeO2 NPs in plants, in environments where excess alginates could be present. On the other hand, studies have shown that carbon-based nanoparticles such as single walled carbon nanotubes triggered reactive oxygen species generation in Arabidopsis and rice .

In addition, multi-wall carbon nanotubes have been found to induce gene expression of heat shock protein 90 in tomato leaves and roots . However, there are no reports on the effect of CeO2 NPs on heat shock protein expression in plants. A few studies have described the physiological impacts of rare earth elements in plants. For example, at concentration higher than 89 µmol/L, cerium affected the foliar chlorophyll content, nitrate reductase activity, shoot root length and relative yield in cowpea plants . The authors suggested the effects could be produced by the substitution of Mg2+ by Ce in chlorophyll synthesis. It has also been suggested that, due to their similar chemical characteristics, Eu, a REE, may compete with Ca for organic ligands . These studies suggest that REE elements can have serious impacts on the uptake of nutritional elements in food crops. However, to the authors’ knowledge the impact of REE NPs on the uptake of nutritional elements by plants has yet to be reported. The purposes of this work were to determine the effects of alginate on: the transport of Ce within corn plants treated with CeO2 NPs, the uptake and transport of micro and macro nutrients, the chlorophyll content, and the expression of stress related heat shock protein 70. Maize was selected for this study because it is a crop widely cultivated throughout the world for direct and indirect consumption. In addition, 40% of the corn world’s harvest is produced in the United States . In this study, corn plants were grown in soil spiked with CeO2 NPs with various alginate concentrations for one month. After harvest, the concentration of Ce and many nutrient elements were determined by ICP-OES in the root and shoots tissues. This suggests that in an eventual release of CeO2 NPs, the higher risk of food contamination would occur in organic matter enriched soil. The mechanism involved in the increase of Ce uptake and translocation by alginate is still unknown. However, our previous work showed that alginate surface coating increased the Ce translocation to shoots in corn plants grown in a soil with low organic matter content and treated with 400 mg/kg CeO2 NPs. Sodium alginate has been associated with seed germination, shoot elongation, root growth, and flower production, among others in Foeniculum vulgare Mill . However, the mechanisms of these effects are still unknown. The presence of CeO2 NPs with/without alginate did not alter the uptake of macro-nutrients Mg, K, Ca, S, and P in one-month old corn roots. However, the uptake of Al and the micro-nutrients Fe, Mn, and Zn was increased . Compared to control , the concentrations of Fe and Al were significantly higher in all NP treatments. For Al, the difference was significant at p ≤ 0.023, but for Fe, the significance was only at p ≤ 0.09. The accumulation of both Fe and Al in roots was similar in all treatments. Moreover, compared to NPs alone and NPs-low alginate, the concentrations of both Fe an Al were significantly higher at medium and high alginate concentrations. It is very likely that the CeO2 NPs were bound with Fe and Al oxides, which are widespread soil colloids. Previous results showed that Fe and Al are co-released from the soil column with ZnO NPs . Manganese accumulation pattern was different. The addition of CeO2 NPs without alginate increased Mn accumulation in roots by 34% compared to control ; but NPs-low alginate and NPs-medium alginate treatments increased the accumulation of Mn by 92% and 90% respect to NPs without alginate and 158% and 155% respect to control. These differences were significant at p ≤ 0.005.

Since thaumatin is a 22 kDa protein, a membrane with MWCO of 5 kDa is used per working process knowledge. Assuming a conservative flux of 30 L/, the inlet stream is concentrated using a concentration factor of 5, diafiltered 10 times against reverse osmosis water, then re-concentrated using a CF of 5 over 20.6 h, resulting in a 75% pure thaumatin and nicotine content of 1.08 mg/kg thaumatin. A retention coefficient of 0.9993 was assumed for thaumatin, resulting in 5.8% thaumatin loss in UF/DF . The retentate is then sent to five CEX chromatography columns operating in parallel which was modeled based on unpublished data from Nomad Bioscience GmbH . GE Healthcare Capto S resin with an assumed binding capacity of 150 g/L was used in this analysis. Table S2 shows the downstream losses breakdown per unit operation. Spray drying is used as a final formulation step over other means of industrial drying due to the heat sensitivity of thaumatin. The simulated facility consists of three sections—Virion production laboratory , spinach field growth, and DSP. A list of base case design parameters and assumptions is shown in Table S3. The VPL process is adopted from a recent article entailing the production of RNA viral particles from agrobacteria carrying a PVX construct. The laboratory is sized to produce 7900 L of spray solution per batch for application in the field. Nicotiana benthamiana plants are used as the host to produce the viral particles to inoculate spinach. N. benthamiana seeds are germinated in soilless plant substrate at a density of 94 plants per tray.

Seedlings are grown hydroponically , under LEDs, until reaching manufacturing maturity at day 35. Agrobacterium tumefaciens is grown for 24 h, before being left in a 4 L flask overnight, and the A. tumefaciens suspension is added to MES buffer in V-101. N. benthamiana infiltration takes place in a vacuum agroinfiltration chamber for 24 h followed by incubation for 7 days in . N. benthamiana biomass production,ebb flow tray agrobacterium growth, agroinfiltration, and incubation parameters are adapted from. After the incubation period, 41.5 kg of N. benthamiana fresh weight are ground and mixed with PBS buffer in a 5:1 buffer:biomass ratio. The extract is then sent to a decanter centrifuge to separate plant dry matter from the liquid phase which is clarified by dead-end filtration , followed by mixing the permeate with 35.9 kg of diatomaceous earth and 7780 L of water to reach a final concentration of 1014 viral particles/L and 4.55 g diatomaceous earth/L. Diatomaceous earth is used as an abrasive to mechanically wound plant cell walls allowing the virions to enter the cytoplasm of the cell. The final spray is stored in for 13 h before field application. Field operation starts at the beginning of each batch with the direct seeding of 28.3 million Spinacia oleracea seeds over 22.6 acres. Spinach is planted over 80-inch beds with an assumed 3 ft spacing between beds, resulting in 14,520 linear bed feet per acre. Seeds are germinated and grown in the field for 44.5 days, during which time a drip irrigation system delivers irrigation water and soluble fertilizer to the soil.A tractor on which multiple high-pressure spray devices are mounted is used to deliver the viral particle solution at a rate of 2 acres/h. This method of delivery has shown high effectiveness. Spinach plants are incubated in the field for 15 days post-infection. During that period, thaumatin starts to accumulate in the crop at an average expression level of 1 g/kg FW after 15 days post-spraying. At day 60, two mechanical harvests collect a total of 344 MT spinach biomass, carrying 344 kg thaumatin, with the aid of four hopper trucks, which is transferred to a 500-m-long conveyor belt that extends from the field collection site to the DSP section of the facility. Harvesting occurs at an average rate of 17,000 kg FW/h, which is estimated based on a harvester speed of 5 km/h and 14,520 linear bed feet per acre.

A more simplified downstream processing, enabled by the use of spinach as a host, starts with mixing plant material with 65 ◦C water before extracting the green juice through a screw press . The resulting GJ is heated for 1 h at 65 ◦C in ten jacketed tanks , then concentrated by evaporation to reduce product stream volume for further purification steps. Since thaumatin is not stable at temperatures above 70 ◦C at neutral pH, evaporation is performed at a low temperature of 40 ◦C and 0.074 bar vacuum pressure. Thermally degraded host cell proteins and impurities are eliminated in a P&F filtration unit designed to include 10 filter sheets with decreasing particle retention size from 25 to 0.1 µm. Smaller impurities are removed using a diafiltration unit with 5 kDa molecular weight cut off cassettes in a similar process as described in Section 3.3, the retentate is spray dried in to obtain a final product which has 5% water content, and 348 kg of solid material containing 94% pure thaumatin and 6% spinach impurities. These impurities are expected to be water soluble, heat stable molecules in the range of 5–100 kDa, according to the theoretical design of the filtration scheme. As shown in Figure 3a, field labor is the highest contributor to the upstream field facility followed by consumables. Detailed labor requirement and cost estimation calculations can be found in Tables S7 and S8. Consumables include mechanical harvester and tractor’s fuel, lubrication, and repair costs and other field equipment repair costs. Upstream indoor facility AOC breakdown elucidates a high cost of consumables due to the cost of soilless plant substrate, followed by high energy consumption from the LED lighting system used for plant growth. The labor category does not appear clearly on the chart because of the low need for labor hours since the indoor facility is highly automated. In both DSP scenarios, facility-dependent costs have the highest cost impact. Insurance, local taxes, and other overhead expenses are estimated to be 1%, 2%, and 5% of the section’s DFC, respectively. Maintenance costs are also included in this category and estimated to be 10% of equipment purchase prices. Facility dependent cost estimation parameters are shown in Tables S9 and S10. Consumables account for 38% of the DSP facility with chromatography due to the high cost of Capto S resin that is changed every 100 cycles. The effect of varying resin binding capacity to the product on the DSP AOC and COGS is shown in Figure 3d.

Transgenic production models were resized based on scenario design requirement for production levels ranging from 10–150 MT and expression levels ranging from 0.5–2.5 g/kg, while keeping the scheduling parameters the same from base case models. The significant impact of expression level on CAPEX and COGS is elucidated in Figure 4a–c. Production level shows a very small decline in COGS for indoor upstream facility and a linear increase in CAPEX with increasing production level. On the other hand, the field upstream facility showed a significant increase in COGS at lower production levels due to the minimum ownership costs of field equipment regardless of the small acreage size. DSP followed the expected behavior that economy of scale dictates,flood and drain tray with sharp decrease in COGS at lower production levels and diminishing returns at higher production levels. The deviation from linear trend at 150 MT/year in field upstream and DSP is likely due to the model’s specified equipment maximum rating, which allows for the inclusion of a new equipment in parallel beyond this rating. As shown in Table 2, the DSP section of the facility accounts for 79% of the project’s CAPEX and 63% of AOC. This is justified by the high equipment purchase prices, piping, instrumentation, buildings, engineering, and construction costs for a plant of this size. Figure 5a shows field labor as the highest cost contributor to the spinach field growth section due to the high direct demand of 48,800 labor-h/year, followed by the cost of spinach seeds, which is estimated to be $23.68/kg for the leafy Bloomsdale variety. Mechanical harvester and tractor’s fuel, lubrication, and repair costs are in included as consumables as well as other field machinery repair costs. Due to the small-scale scope of the VPL, labor is the highest contributor of the section’s operating cost.The impact of varying the highest cost drivers in each of the facility’s category by 25% on COGS is portrayed as a tornado diagram in Figure 5c. Field labor was the most sensitive cost variable, having the highest impact on the COGS, followed by the ultrafiltration membrane, which is replaced every 30 cycles. In this model, we assume a relatively high downstream recovery of the protein from harvest to formulation. The reason for this assumption is that spinach, being edible crop, allows for a lower target product purity and a consequently fewer DSP steps. It is particularly important to focus resources on maximizing downstream recovery during process development because it ultimately affects plant biomass and spray volume requirement upstream to appropriately compensate for these losses, which in turn affects equipment sizing in DSP based on the amount of plant material to be processed. The unit operations were resized according to the scenario design requirement for downstream recovery ranging from 50 to 95% while scheduling parameters were left unchanged. This effect of downstream recovery on the facility’s AOC and COGS is shown in Figure 5d and shows a 1.5× increase in AOC and COGS as downstream recovery decreases from 95% to 50%. Although our analysis indicates a relatively high COGS range for a sugar substitute, there are unrealized costs savings from thaumatin use due to its unique sweetness intensity. Thaumatin’s use in extremely small quantities is essentially why it is considered a noncaloric sweetener, as it provides only 4 calories per gram. Sensory evaluation studies have found that a sample with 5% sucrose +4.6 ppm thaumatin II had similar sweetness as a 10% sucrose control with minimal lingering aftertaste, suggesting that up to one-half of the sugar could be replaced by thaumatin II . SSBs including sodas, fruit drinks, and sport drinks account for 50% of the total added sugar in Western diets, and therefore provide an attractive avenue for thaumatin emergence as a sugar substitute.

The incorporation of thaumatin by the industry not only offers a tool to help decelerate the obesity epidemic caused by increased childhood sugar intake decades ago, but also provides itself with a more economically viable solution. Firstly, as sugar taxations emerge, sugar reduction becomes a financial incentive. Secondly, the reduction of sugar and the addition of thaumatin to retain the same level of sweetness has the potential to save millions of dollars per day on the cost of sweetening beverages. Assuming that the average “standard” sucrose concentration in SSBs is 35.5 g per 12 floz. drink ~10%, and a $0.30/kg sugar price, Figure 6 shows the potential savings from using thaumatin to reduce sugar content by 20%, 30%, and 50%, while maintaining the same sweetness as the standard for a range of thaumatin purchase prices. The amount of thaumatin needed to obtain the same sweetness as a 10% solution in each sugar reduction scenario was calculated using the sensory regression analysis included in a published GRAS notice . Table 3 shows the daily and annual amount of thaumatin needed for each sugar reductions scenario, assuming that one billion 12 fl oz drinks are to be sweetened per day. Successful implementation of thaumatin in this avenue can liberate R&D resources to improve expression levels and increase production volumes, both of which have a substantial impact on COGS reduction, as we have demonstrated.Our preliminary engineering facility design indicates the feasibility of thaumatin manufacturing by various molecular farming platforms. The most economic method is the field grown ethanol-inducible, transgenic N. tabacum, assuming a downstream facility without chromatography . It remains unclear whether heat incubation is sufficient to achieve the desired purity for a safe product without the inclusion of chromatography on a large-scale. In a previous plant-made food safety product techno-economic analysis, a chromatography unit was included for protein purification from N. benthamiana; however, heat precipitation of host cell proteins was not included as a purification step.

Relying on Halomonas spp. in combination with acetate as substrate may allow very rapid production of the required bio-plastic, but substrate availability constraints are higher than for CH4 or CO2/H2. A terminal electron acceptor is required in all cases, which will almost certainly be O2. Supplying O2 safely without risking explosive gas mixtures, or wasting the precious resource, is again a question of reactor design and operation. Certain purple non-sulfur alphaproteobacteria and Rhodopseudomonas palustris also feature remarkable substrate flexibility and can produce PHAs . Bioplastic recovery and purification is a major challenge. To release the intracellular compound, an osmolysis process may be employed with the halophile . However, the transfer of cells into purified water and separation of the polyesters from the cell debris, potentially through several washing steps, may require substantial amounts of water. An alternative and/or complement to the common process for extraction of PHAs with halogenated organic solvents, is to use acetate or methanol as solvents . This is applicable independent of the organism and the inputs can be provided from other bio-manufactory modules. The high crystallinity of pure PHB makes it brittle and causes it to have a narrow melting range, resulting in warp during extrusion and 3D-printing. Such behavior places operational constraints on processing and hampers applications to precision manufacturing . Workarounds may be through additives, bio-composite synthesis, and copolymerization. However, this ultimately depends on what biology can provide . There is a need to advance space bio-platforms to produce more diverse PHAs through synthetic biology. ISM of bio-materials can reduce the mission cost, increase modularity,growing hydroponically and improve system recyclability compared to abiotic approaches. In an abiotic approach, plastics will be included in the payload, thereby penalizing up-mass at launch.

As with elements of FPS and ISRU, ISM increases flexibility and can create contingencies during surface operations, therefore reducing mission risk. The high modularity of independent plastic production, filament formation, and 3D-printing allows for a versatile process, at the cost of greater resources required for systems operations. Overall, this maximizes resource use and recyclability, by utilizing mission waste streams and byproducts for circular resource management.Biomanufacturing on Mars can be supported by flexible biocatalysts that extract resources from the environment and transform them into the complex products needed to sustain human life. The Martian atmosphere contains CO2 and N2 .It is very likely that the expensive and energy-intensive Sabatier plants for CH4 production will be available per Design Reference Architecture . While a HaberBosch plant could be set up for ammonia production, this is neither part of the current DRA nor exceptionally efficient. Thus, for a biomanufactory, we must have carbon fixation reactors to fix CO2 into feed stocks for nonmethanotrophs, and have nitrogen fixation reactors to fifix N2 to fulfill nitrogen requirements for non-diazotrophs. Trace elements and small-usage compounds can be transported from Earth, or in some cases extracted from the Martian regolith. In the case where power is provided from photocollection or photovoltaics, light energy will vary with location and season, and may be critical to power our bioreactors. Although photosynthetic organisms are attractive for FPS, a higher demand for carbon-rich feed stocks and other chemicals necessitates a more rapid and efficient CO2 fixation strategy. Physicochemical conversion is inefficient due to high temperature and pressure requirements. Microbial electrosynthesis , whereby reducing power is passed from abiotic electrodes to microbes to power CO2 reduction, can offer rapid and efficient CO2 fixation at ambient temperature and pressure . MES can produce a variety of chemicals including acetate , isobutanol , PHB , and sucrose , and therefore represents a flexible and highly promising ISRU platform technology . Biological N2-fifixation offers power- and resource-efficient ammonium production. Although photoautotrophic N2 fixation with, for example, purple non-sulfur bacteria, is possible, slow growth rates due to the high energetic demand of nitrogenase limit throughput .

Therefore, heterotrophic production with similar bacteria using acetate or sucrose as a feed stock sourced from electromicrobial CO2-fifixation represents the most promising production scheme, and additionally benefits from a high degree of process redundancy with heterotrophic bioplastic production. Regolith provides a significant inventory for trace elements and, when mixed with the substantial cellulosic biomass waste from FPS processes, can facilitate recycling organic matter into fertilizer to support crop growth. However, regolith use is hampered by widespread perchlorate , indicating that decontamination is necessary prior to enrichment or use. Dechlorination can be achieved via biological perchlorate reduction using one of many dissimilatory perchlorate reducing organisms . Efforts to reduce perchlorate biologically have been explored independently and in combination with a more wholistic biological platform . Such efforts to integrate synthetic biology into human exploration missions suggest that a number of approaches should be considered within a surface biomanufactory.A biomanufactory must be able to produce and utilize feed stocks along three axes as depicted in Figure 5: CO2-fifixation to supply a carbon and energy source for downstream heterotrophic organisms or to generate commodity chemicals directly, N2-fifixation to provide ammonium and nitrate for plants and non-diazotrophic microbes, and regolith decontamination and enrichment for soil-based agriculture and trace nutrient provision. ISRU inputs are sub-module and organism dependent, with all sub-modules requiring water and power. For the carbon fixation sub-module , CO2 is supplied as the carbon source, and electrons are supplied as H2 or directly via a cathode. Our proposed biocatalysts are the lithoautotrophic Cupriavidus necator for longer-chain carbon production [e.g., sucrose ] and the acetogen Sporomusa ovata for acetate production. C. necator is a promising chassis for metabolic engineering and scale-up , with S. ovata having one of the highest current consumptions for acetogens characterized to date . The fixed-carbon outputs of this sub-module are then used as inputs for the other ISRU sub-modules in addition to the ISM module . The inputs to the nitrogen fixation sub-module include fixed carbon feedstocks, N2, and light. The diazotrophic purple-non sulfur bacterium Rhodopseudomonas palustris is the proposed biocatalyst, as this bacterium is capable of anaerobic, light-driven N2 fixation utilizing acetate as the carbon source, and has a robust genetic system allowing for rapid manipulation . The output product is fixed nitrogen in the form of ammonium, which is used as a feed stock for the carbon-fixation sub-module of ISRU along with the FPS and ISM modules.

The inputs for the regolith enrichment sub-module include regolith, fixed carbon feedstocks, and N2. Azospira suillum is a possible biocatalyst of choice due to its dual use in perchlorate reduction and nitrogen fixation . Regolith enrichment outputs include soil for the FPS module , H2 that can be fed back into the carbon fixation sub-module and the ISM module, chlorine gas from perchlorate reduction, and waste products. Replicate ISRU bioreactors operating continuously in parallel with back-up operations lines can ensure a constant supply of the chemical feed stocks, commodity chemicals, and biomass for downstream processing in ISM and FPS operations. Integration of ISRU technologies with other biomanufactory elements, especially anaerobic digestion reactors,grow strawberries hydroponically may enable complete recyclability of raw materials, minimizing resource consumption and impact on the Martian environment .Waste stream processing to recycle essential elements will reduce material requirements in the biomanufactory. Typical feed stocks include inedible crop mass, human excreta, and other mission wastes. Space mission waste management traditionally focuses on water recovery and efficient waste storage through warm air drying and lyophilization . Mission trash can be incinerated to produce CO2, CO, and H2O . Pyrolysis, another abiotic technique, yields CO and H2 alongside CH4 . The Sabatier process converts CO2 and CO to CH4 by reacting with H2. An alternate thermal degradation reactor , operating under varying conditions that promote pyrolysis, gasification, or incineration, yields various liquid and gaseous products. The fact remains however, that abiotic carbon recycling is inefficient with respect to desired product CH4, and is highly energy-intensive. Microbes that recover resources from mission wastes are a viable option to facilitate loop closure. Aerobic composting produces CO2 and a nutrient-rich extract for plant and microbial growth . However, this process requires O2, which will likely be a limited resource. Hence, anaerobic digestion, a multi-step microbial process that can produce a suite of end products at lower temperature than abiotic techniques , is the most promising approach for a Mars biomanufactory to recycle streams for the ISM and FPS processes. Digestion products CH4 and volatile fatty acids can be substrates for polymer-producing microbes . Digestate, with nutrients of N, P, and K, can be ideal for plant and microbial growth , as shown in Figure 6. Additionally, a CH4 and CO2 mixture serves as a biogas energy source, and byproduct H2 is also an energy source . Because additional infrastructure and utilities are necessary for waste processing, the extent of loop closure that is obtainable from a treatment route must be analyzed to balance yield with its infrastructure and logistic costs. Anaerobic digestion performance is a function of the composition and pretreatment of input waste streams , as well as reaction strategies like batch or continuous, number of stages, and operation conditions such as organic loading rate, solids retention time, operating temperature, pH, toxic levels of inhibitors and trace metal requirements . Many of these process parameters exhibit trade-offs between product yield and necessary resources. For example, a higher waste loading reduces water demand, albeit at the cost of process efficiency. There is also a potential for multiple co-benefits of anaerobic digestion within the biomanufactory. Anaerobic biodegradation of nitrogen-rich protein feed stocks, for example, releases free NH3 by ammonification. While NH3 is toxic to anaerobic digestion and must thus be managed , it reacts with carbonic acid to produce bicarbonate buffer and ammonium, decreasing CO2 levels in the biogas and buffering against low pH.

The resulting digestate ammonium can serve as a fertilizer for crops and nutrient for microbial cultures.FPS and ISM waste as well as human waste are inputs for an anaerobic digester, with output recycled products supplementing the ISRU unit. Depending on the configuration of the waste streams from the biomanufactory and other mission elements, the operating conditions of the process can be varied to alter the efficiency and output profile. Open problems include the design and optimization of waste processing configurations and operations, and the identification of optimal end-product distributions based on a loop closure metric against mission production profiles, mission horizon, biomanufacturing feed stock needs, and the possible use of leftover products by other mission elements beyond the biomanufactory. A comparison with abiotic waste treatment strategies is also needed, checking power demand, risk, autonomy, and modularity benefits.Biomanufactory development must be done in concert with planned NASA missions that can provide critical opportunities to test subsystems and models necessary to evaluate efficacy and technology readiness levels . Figure 7 is our attempt to place critical elements of a biomanufactory road map into this context. We label critical mission stages using Reference Mission Architecture -S and RMA-L, which refer to Mars surface missions with short and long durations, respectively. Reliance on biotechnology can increase the risk of forward biological contamination . Beyond contamination, there are ethical issues that concern both the act of colonizing a new land and justifying the cost and benefits of a mission given needs of the many here on earth. Our road map begins with the call for an extensive and ongoing discussion of ethics . Planetary protection policies can provide answers or frameworks to address extant ethical questions surrounding deep-space exploration, especially on Mars . Critically, scientists and engineers developing these technologies cannot be separate or immune to such policy development.The upcoming lunar exploration missions, Artemis and Gateway , provide additional opportunities for integration with Earth-based biomanufactory development. Early support missions will provide valuable experience in cargo predeployment for crewed operations, and is likely to help shape logistics development for short-term as well as long-term Mars exploration missions when a biomanufactory can be deployed. Here we present a subset of Artemis efforts as they relate to mission elements with opportunities for testing and maturing biomanufacturing technology. Although ISRU technologies for the Moon and Mars will be sufficiently distinct due to different resource availabilities, crewed Artemis missions provide a testing ground for crewed Mars bio-process infrastructure. Later Artemis missions also provide a suitable environment to test modular, interlocked, scalable reactor design, as well as the design of compact molecular biology labs for DNA synthesis and transformation.

In comparison with pesticides, only sporadic research has shown detoxification of PPCPs by POD and GST in plants. In the present study, activities of POD and GST increased in a dose-dependent manner in both roots and leaves after exposure to PPCPs . This observation was consistent with Bartha et al. and Huber et al. , who observed oxidation of diclofenac by plant peroxidases, and also glutathione conjugation in Typha latifolia. These mechanistic studies, together with our results, clearly show that the POD and GST enzyme families may play an important role in transformation and conjugation of PPCPs in plants. Glutathione is one of the major soluble low molecular weight antioxidants, and also the major non-protein thiol in plant cells , contributing to maintain the cellular redox homeostasis and signaling. Moreover, conjugation with xenobiotics by GSH may be a common pathway for plant metabolism of various man-made chemicals . Glutathione conjugation with diclofenac , 8:2 fluorotelomer alcohol and chlortetracycline have been previously observed in plants. It is well known that these processes require extensive utilization of reduced GSH as an electron donor and subsequently produce oxidized glutathione . The changes in the cellular glutathione pool, specially the associated ratio of reduced to oxidized glutathione, play a central role in plant defense responses . Glutathione homeostasis after exposure to trace levels of PPCPs, however,growing hydroponically has not been well documented so far. In this study, the glutathione content increased at low PPCP doses, while decreased to normal levels at the highest PPCP treatment level . The GSSG content was unchanged when the PPCP concentrations were low , but showed a significant increase when the PPCP concentration was increased to 50 mg L! 1 . The depletion observed in cellular GSH could contribute to the PPCP-induced oxidative stress and detoxification of xenobiotics.

Meanwhile, the different responses of GSH in root and leaves indicated that roots may be the main site to express PPCP toxicity and induce PPCP detoxification. Given the dominance of GSH conjugates as observed for pesticides, it is possible that trace contaminants such as PPCPs may be removed similarly by GSH conjugation. It is therefore imperative to conduct further research to explore the mechanisms and pathways of PPCP phytotransformation by GSTs after uptake by plants.On the whole, the present study provided evidence that some PPCPs may be translocated systemically, and ultimately posed toxicity effects in higher plants. Oxidative stress response may reflect the intensity of PPCP treatment and sensitivity of plant species to PPCPs, and may be used as indicators for early plant response to trace organics introduced into agroecosystems. Furthermore, plants may detoxify PPCPs through different mechanisms, including enhanced antioxidant defense systems to prevent oxidative damage and increased activities of xenobioticmetabolizing enzymes. These mechanisms help maintain plant physiological, biochemical and molecular functional integrity, offering the possibility to use some of these endpoints as biomarkers for predicting phytotoxicity induced by PPCPs and likely other man-made chemicals. Further research is needed to evaluate the physiological and biological responses of plants in realistic field practices, such as irrigation with treated wastewater or fertilization with biosolids and animal wastes in agriculture.The Paharpur Business Centre and Software Technology Incubator Park is a 7 story, 50,400 ft2 office building located near Nehru Place in New Delhi India. The occupancy of the building at full normal operations is about 500 people. The building management philosophy embodies innovation in energy efficiency while providing full service and a comfortable, safe, healthy environment to the occupants. Provision of excellent Indoor Air Quality is an expressed goal of the facility, and the management has gone to great lengths to achieve it. This is particularly challenging in New Delhi, where ambient urban pollution levels rank among the worst on the planet.

The approach to provide good IAQ in the building includes a range of technical elements: air washing and filtration of ventilation intake air from rooftop air handler, the use of an enclosed rooftop greenhouse with a high density of potted plants as a bio-filtration system, dedicated secondary HVAC/air handling units on each floor with re-circulating high efficiency filtration and UVC treatment of the heat exchanger coils, additional potted plants for bio-filtration on each floor, and a final exhaust via the restrooms located at each floor. The conditioned building exhaust air is passed through an energy recovery wheel and chemisorbent cartridge, transferring some heat to the incoming air to increase the HVAC energy efficiency. Flooring is a combination of stone, tile and “zero VOC” carpeting. Wood trim and finish appears to be primarily of solid sawn materials, with very little evidence of composite wood products. Furniture is likewise in large proportion constructed from solid wood materials. The overall impression is that of a very clean and well-kept facility. Surfaces are polished to a high sheen, probably with wax products. There was an odor of urinal cake in the restrooms. Smoking is not allowed in the building. The plants used in the rooftop greenhouse and on the floors were made up of a number of species selected for the following functions: daytime metabolic carbon dioxide absorption, nighttime metabolic CO2 absorption, and volatile organic compound and inorganic gas absorption/removal for air cleaning. The building contains a reported 910 indoor plants. Daytime metabolic species reported by the PBC include Areca Palm, Oxycardium, Rubber Plant, and Ficus alii totaling 188 plants . The single nighttime metabolic species is the Sansevieria with a total of 28 plants . The “air cleaning” plant species reported by the PBC include the Money Plant, Aglaonema, Dracaena Warneckii, Bamboo Palm, and Raphis Palm with a total of 694 plants .

The plants in the greenhouse numbering 161 of those in the building are grown hydroponically, with the room air blown by fan across the plant root zones. The plants on the building floors are grown in pots and are located on floors 1-6. We conducted a one-day monitoring session in the PBC on January 1, 2010. The date of the study was based on availability of the measurement equipment that the researchers had shipped from Lawrence Berkeley National Lab in the U.S.A. The study date was not optimal because a large proportion of the regular building occupants were not present being New Year’s Day. An estimated 40 people were present in the building all day during January 1. This being said, the building systems were in normal operations, including the air handlers and other HVAC components. The study was focused primarily on measurements in the Greenhouse and 3rd and 5th floor environments as well as rooftop outdoors. Measurements included a set of volatile organic compounds and aldehydes, with a more limited set of observations of indoor and outdoor particulate and carbon dioxide concentrations. Continuous measurements of Temperature and relative humidity were made selected indoor and outdoor locations. Air sampling stations were set up in the Greenhouse, Room 510, Room 311, the 5th and 3rd floor air handler intakes,growing strawberries hydroponically the building rooftop HVAC exhaust, and an ambient location on the roof near the HVAC intake. VOC and aldehyde samples were collected at least once at all of these locations. Both supply and return registers were sampled in rooms 510 and 311. As were a greenhouse inlet register from the air washer and outlet register ducted to the building’s floor level. Air samples for VOCs were collected and analyzed following the U.S. Environmental Protection Agency Method TO-17 . Integrated air samples with a total volume of approximately 2 L were collected at the sites, at a flow rate of <70 cc/min onto preconditioned multibed sorbent tubes containing Tenax-TA backed with a section of Carbosieve. The VOCs were desorbed and analyzed by thermodesorption into a cooled injection system and resolved by gas chromatography. The target chemicals, listed in Table 1, were qualitatively identified on the basis of the mass spectral library search, followed by comparison to reference standards. Target chemicals were quantified using multi-point calibrations developed with pure standards and referenced to an internal standard. Sampling was conducted using Masterflex L/S HV-07553-80 peristaltic pumps assembled with quad Masterflex L/S Standard HV-07017-20 pump heads. Concentrations of formaldehyde, acetaldehyde, and acetone were determined following U.S. Environmental Protection Agency Method TO-11a . Integrated samples were collected by drawing air through silica gel cartridges coated with 2,4-dinitrophenylhydrazine at a flow rate of 1 Lpm. Samples utilized an ozone scrubber cartridge installed upstream from the sample cartridge. Sample cartridges were eluted with 2 mL of high purity acetonitrile and analyzed by high-performance liquid chromatography with UV detection and quantified with a multi-point calibration for each derivitized target aldehyde. Sampling was conducted using Masterflex L/S HV-07553-71 peristaltic pumps assembled with dual Masterflex L/S Standard HV-07016-20 pump heads. Continuous measurements of PM2.5 using TSI Dustrak model 8520 monitors were made in Room 510 and at the rooftop-sampling site from about 13:30 to 16:30 of the sampling day. The indoor particle monitor was located on a desk in room 510 and the outdoor monitor was located on a surface elevated above the roof deck. Carbon dioxide spot measurements of about 10-minute duration were made throughout the building during the afternoon using a portable data logging real-time infrared monitor . Temperature and RH were monitored in the Greenhouse, room 510 and room 311 using Onset model HOBO U12-011 data loggers at one-minute recording rates. Outdoor T and RH were not monitored. The measured VOC concentrations as well as their limits of quantitation by the measurement methods are shown in Table 2. Figures 1-6 show bar graphs of these VOCs. Unless otherwise shown, all measured compounds were above the minimum detection level, but not all measurements were above the LOQ.

Those measurements with concentrations below the LOQ should be considered approximations. These air contaminants are organized by possible source categories including: carbonyl compounds that can be odorous or irritating; compounds that are often emitted by building cleaning products; those associated with bathroom products; those often found emitted from office products, supplies, materials, occupants, and in ambient air; those found from plant and wood materials as well as some cleaning products; and finally plasticizers commonly emitted from vinyl and other flexible or resilient plastic products. The groupings in this table are for convenience; many of the listed compounds have multiple sources so the attribution provided may be erroneous. The carbonyl compounds include formaldehyde that can be emitted from composite wood materials, adhesives, and indoor chemical reactions; acetaldehyde from building materials and indoor chemistry; acetone from cleaners and other solvents. Benzaldehyde sources can include plastics, dyes, fruits, and perfumes. Hexanal, nonanal, and octanal can be emitted from engineered wood products. For many of these compounds, outdoor air can also be a major source. Formaldehyde and acetone were the most abundant carbonyl compounds observed in the PBC. For context, the California 8-h and chronic non-cancer reference exposure level for formaldehyde is 9 µg m-3 and the acute REL is 55 µg m-3 . The 60 minute average formaldehyde concentrations observed in the PBC exceeded the REL by up to a factor of three. Acetone has low toxicity and the observed levels were orders of magnitude lower than concentrations of health concern. Hexanal, nonanal, and octanal are odorous compounds at low concentrations; odor thresholds established for them are 0.33 ppb, 0.53 ppb, and 0.17 ppb, respectively . Average concentrations observed within the PBC building were 3.8±0.8 ppb, 3.5±0.6 ppb, and 1.4±0.2 ppb, for these compounds, respectively, roughly ten times higher than the odor thresholds. Concentrations of these compounds in the supply air from the greenhouse were substantially lower, although stillin excess of the odor thresholds. The concentration of hexanal and nonanal roughly doubled the ambient concentrations as the outside air passed through the greenhouse. Octanal concentrations were roughly similar in the ambient air and in the air exiting the greenhouse. Concentrations of benzene, d-limonene, n-hexane, naphthalene and toluene all increased in the greenhouse air in either the AM or PM measurements. The measured levels of these compounds were far below any health relevant standards, although naphthalene concentrations reached close to 50% of the California REL of 9 µg m-3 . The concentrations of these compounds were generally somewhat higher indoors relative to the greenhouse concentrations.

Maximum concentrations of N2O were observed in the soil at 10–15 cm, shallow depths where lateral diffusion away from the dripper could only account for a small loss of N2O compared with losses to the atmosphere. It was also observed that water tended to flow laterally, past the surface wetting front, through a sandy horizon above a clayey horizon which begins at around 50 cm depth, also diminishing net lateral gas diffusion. The model is highly sensitive to variability in N2O concentration data, and at the plot level the calculations showed fluctuations between production and consumption by depth which were not plausible or consistent.Where possible, curves were fit to the concentration data of the form N2O conc = ae, which provided the dc/dz terms. Furthermore, concentrations of N2O measured at 5 cm were generally much lower than at 10 cm and led to frequent estimates of net consumption near surface. It was deemed likely that these samples had been contaminated by atmospheric air. Production of N2O at 10 cm was therefore calculated with reference to ambient N2O concentrations at 0 cm instead of the 5 cm concentration data. Measurements spaced several hours apart determined that an average 4.4% of total production was accounted for by change in concentration over time during the measured days. Ultimately no treatment statistics could be reported with the profile production data, but the model revealed general patterns and treatment effects on the depths of N2O production. The highest emissions, which were seen in summer and fall,hydroponics growing system were associated with the most consistent patterns of N2O distribution in the soil profile. Results from Days 2, 3 and 4 after fertigation, not shown, had similar distributions to those measured on Day 1, although with a slightly higher fraction of N2O concentrated in the deeper soil, 40–60 cm.

The relative stability in depths of production was seemingly contradictory to the changes in N distribution seen in soil solution ; but it was notable that distribution of extractable N , showed less change at the same points . N2O concentration patterns under most days and treatments were bimodal, with a shallow peak at 10–15 cm and a deeper peak around 45–60 cm, in the zone of higher clay content. The deeper peaks were sometimes strong 3–4 days after fertigation, especially in the Standard UAN treatment, illustrating the deeper distribution of N under higher rates of UAN application. Calculations in UAN treatments after winter typically showed points of highest production at 10–15 cm depth, usually underlain directly by the points of greatest consumption, at 15–20 cm . The calculations for 20 and 30 cm might underestimate production, because of more significant lateral diffusion of N2O around 30 cm, where WFPS generally declined . Production was seen at the lower peaks around 45 cm, but calculations suggested that N2O produced in these lower peaks was generally consumed before reaching surface , consistent with the findings of Neftel et al. . This helps to explain why emitted N2O was less per unit applied in Standard UAN than in HF UAN. Calculations in HF NO3 profiles generally showed much lower net N2O production/consumption than the UAN treatments. This is credited to the more even distribution throughout the soil of applied NO3, vertically and laterally, which led to low concentrations. Production profiles further suggest that a high proportion of the N2O produced in this treatment was consumed before it could be emitted from the surface. Overall, surface emissions of N2O decreased more quickly over the days following fertigations than did soil gas concentrations and calculated in-soil production rates, suggesting greater importance of production near surface during the first and second days. Under the driest conditions, seen on Day 3 after fertigation in late summer, increased N2O concentrations at 60 and 80 cm were concurrent with the lowest post-fertigation surface emissions. Calculations of N2O production for that date showed consumption at 45 cm in both HF treatments , supporting the conclusion that N2O produced deeper was being consumed at points immediately above, as well as possibly diffusing downwards.

Although the averaging of soil gas profiles by treatment limited the options for statistical analysis, the factors driving N2O production in the soil could still be assessed. Multiple linear regressions of surface emissions and of production at 15, 30 and 60 cm were carried out using calculated N2O production per treatment per day at those depths, and the corresponding averaged NH4 + in solution, NH4 + in soil extracts, NO3 in solution, NO3 in soil extracts, WFPS and temperature. Treatments were pooled because the dataset was limited within each treatment and the differences seen when HF NO3 was separated were minor. Regressions had little predictive capability at 30 and 60 cm depth. Nevertheless, it was notable that WFPS had negative coefficients at both depths, indicative of more complete denitrification with greater soil moisture. At 15 cm, the Adjusted R2 was only 0.14 but several alternative analyses gave better predictions. When excluding negative production values, an Adj. R2 of 0.58 was seen, which rose to 0.68 when reduced to extractable NH4 + , WFPS and temperature. If production at 10 cm was averaged with that at 15 cm, most negative values were eliminated, and using all data and variables the Adj. R2 was 0.21, or 0.26 with extractable NH4 + , NO3 in solution, WFPS and temperature. These results caused some questioning of the calculations of N2O production and consumption, which were volatile even in averaged forms, so regressions were carried out with soil gas concentrations as well. At 15 cm, all variables regressed to Adj. R2 of 0.26; reduced to NH4 + , WFPS and Temperature, the Adj. R2 was 0.32. Concentration averaged between 10 and 15 cm had an Adj. R2 of 0.41, while reduced to NH4 + , WFPS and Temperature, the Adj. R2 was 0.49. Regression of surface emissions followed the same pattern, being compared to NH4 + and NO3 in soil extracts at 2.5 cm depth, WFPS and temperature, where NO3 was found insignificant. The adjusted R2 of this regression is not reported because it is less complete than the analyses above. The superior predictive capability of extractable NH4 + at 15 cm and near surface was unexpected, since it is usually assumed that only the NH4 + in solution is available for microbial consumption .

However, little relevant investigation has been done in soils and the question can be raised whether microbial foraging on clays can desorb ammonium .The persistence of input effects on the functioning of the soil microbial community is an important agro-ecological concern. Here several assays of nitrification and denitrification capacity tested for persistent treatment effects which could influence N2O emissions. Soils were collected in late August after a month of irrigations without fertilizer. Treatment differences were of interest, not the comparison of assay results to field rates. The most ready metric of a soil’s denitrification response to NO3 amendments is its denitrification enzyme activity ,hydroponic equipment designed to assess soil process rates before they are affected by the synthesis of additional enzymes. Since fertigation applications make a large amount of NO3 available in a short time, the preevent DEA of a soil may play a significant role in denitrification derived N2O emissions. Results showed very similar N2O production by the two HF treatments in a DEA assay, which were significantly higher than Standard UAN . Over 24 h, characterized as Denitrification Potential , this initial difference was persistent, although it lost statistical significance. Given that drip fertigation saturated zones are not entirely dissimilar from the conditions of these assays, it was expected that DEA and DP modified with acetylene might also suggest differences in the product ratio of denitrification in the field treatments. Results were inconclusive, with widely dispersed values. Rates of ammonium oxidation to nitrite, as an index of nitrification potential, supported the importance of frequency and rate of NH4 + application, HF UAN > Standard UAN > HF NO3, but differences were only significant between the HF UAN and HF NO3 treatments . Strict chemoautotrophs typically dominate nitrification in cropped soils , and their numbers are more likely to be affected by availability of NH4 + than are the heterotrophs largely responsible for N2O emission through denitrification. Higher amounts of available nitrite are known to stimulate nitrifier denitrification and associated N2O losses , so a persistent effect of NH4 + application on ammonium oxidation to nitrite could increase N2O emissions under HF fertigation. Ammonium oxidation and DEA assays are predicated upon standard conditions, the former being oxic, open, shaken slurry, and the latter completely anoxic.

Actual oxygen availability in drip zones may cover a wide range between those points, but is expected to be limited. Little data is available, but Gil et al. found 4.97% O2 in the sampled soil air of a clay loam in an avocado orchard under drip. It can be assumed that many surfaces within larger aggregates would have lower O2 , being well suited for nitrifier denitrification, which takes place at <5% O2, while denitrification requires <0.05% O2 . It was therefore deemed useful to test the persistent effects of HF fertigation on potential soil production of N2O at 3% O2 . The only treatment differences were in microcosms with NO3 amendments , where N2O was presumably derived mainly from denitrification inside aggregates, supporting DEA results . The lack of HF treatment effects with NH4 + may be due to high rates of adsorption on soil surfaces expected with this N source , leading to gradual liberation. Nevertheless, emissions of N2O with NH4 + amendments were higher than those with NO3, confirming the large potential contribution of nitrifier denitrification from drip zones. The alternative explanation, being a general, rapid turnover from nitrifier-produced NO2 and NO3 to denitrifier produced N2O, has not consistently been supported by isotopic studies in laboratory . Assessments of N2O/ product ratio using acetylene in DEA, DP, and 3% O2 incubation assays did not give robust support to the hypothesis that greater microbial capacity for nitrification and/ or denitrification should correlate to a higher portion of complete reduction of N to N2 . It must be noted that N2O is more likely to be reduced to N2 when NO3 is limited , which it was not in the DEA test and DP tests. Further, the reduction of N2O to N2 dominates under anoxic conditions , which were not prevalent in the 3% O2 test. The factors affecting the “completeness” of nitrifier denitrification to N2 have been little studied and may be distinct from those affecting denitrifier denitrification. Lastly, tests of residual NO3
suggested that acetylene may have slightly inhibited NO3
reduction. The comparison of N2O from HF UAN with a HFNO3
-based treatment including Ca2 raises the question of whether differences may be ascribable to the opposite pH effects of the fertilizers. HF Ca2 + KNO3 did produce a significantly higher pH than HF UAN within 6 months of the treatment’s inception . This could partly explain lower N2O emissions from the HF NO3 treatment , but the effectis likely not a strong one because all were in neutral range . Our observation of 2.0 greater N2O emissions from HF UAN than from HF NO3 agrees well with Abalos et al. , who saw 2.4 greater N2O emissions from urea than from calcium nitrate in a drip-fertigated melon field in Spain. The greater predictive capacity of extractable NH4 + over NO3
provided evidence of a high contribution of nitrifier denitrification to N2O emitted in the field. This was supported by laboratory tests of our field soils at 3% O2, and concurred with findings by Vallejo et al. , as well as by Sanchez-Martin et al. , who calculated that with dripfertigated ammonium sulfate, 45% of N2O came from nitrification. Considering both field and laboratory data, frequency effects in the application of UAN were only seen in nitrate denitrification rates and in N present at 60 cm depth. Nitrifier capacities do not seem to have been affected, due perhaps to the adsorption of fertilizer NH4 + and its gradual release over time. Still, rates of nitrifier denitrification in the field may have seen concentration effects, as a corollary of frequency differences.

Based on the results, we propose the presence of quantitative trait locus or loci for root traits in the distal 15% of the physical length of 1RS arm. In cereals, most of the gene rich regions for agronomic traits are concentrated in the distal ends of the chromosomes . Kim et al. conducted Weld studies for the agronomic performance of 1R from different sources of origin. They found 1RS increases the grain yield significantly, and interestingly, all the lines with 1RS did not show significant differences for shoot biomass. They did not look at the root traits, which could have also been useful. In a similar study, Waines et al. compared 1RS from different sources to study root biomass in hexaploid as well as tetraploid wheats. The translocated hexaploid wheats with 1RSAmigo and 1RSKavkaz showed 9 and 31% increase in root biomass than Pavon 76, respectively. Similar results were reported for the durum wheat ‘Aconchi’ versus Aconchi with the 1RS arm. These studies point toward the definite presence of gene for greater rooting ability on 1RS, and also the differential expression of alleles from different sources of 1RS in root traits. In a recent study on rice root anatomy, Uga et al. identified a QTL for metaxylem anatomy on the distal end of the long arm of chromosome 10. In another comparative study of rye DNA sequences with rice genome, the distal end of the long arm of chromosome 10 of rice was syntenic to 1RS . Both these studies provide evidence to support the general applicability of our mapping method to locate the probable region on 1RS,hydroponic grow system carrying gene/QTL for root traits. Our present finding on root studies prepares a platform to find gene/QTL for root traits on 1RS.

Future work will focus on use of a larger number of recombinant lines to narrow down the QTL region of 1RS responsible for increased root traits and find the molecular markers linked to these QTL. Ultimately, this would lead to our goal of physical mapping and then positional cloning of the root QTL.The increasing use of NPs in commercial products has led to NP-accumulation in the environment and within the food chain.Chronic exposure to NPs can lead to health issues as some inorganic NPs have biological activity at the cellular and sub-cellular level with an unknown cytotoxicity and genotoxicity.In particular, metal oxide NPs are the most abundant form of NP in the environment with the most potential toxic risks.Locating, quantifying, and imaging NPs in vivo can provide information on bio-distribution and fate of NPs in living systems.However, many challenges to quantitatively assess their bio-distributions under realistic environmental exposure concentrations remain.To date, the visualization of most NPs within plants has relied on the use of micro-X-ray fluorescence spectrometry , confocal microscopy, TEM, SEM, scanning transmission electron microscopy , or scanning transmission ion microscopy .The most prominent quantification techniques have been inductively coupled plasma spectroscopy utilizing either optically emission spectroscopy or mass spectrometry .These techniques required mineralization of the plant material generally with hydrogen peroxide and nitric acid, and may not be sufficiently sensitive enough to quantify small changes in the amount of a metal ion.Notably, high background concentrations of essential nutrients make detecting and quantifying the small variation in NP-related metal ion content an analytical challenge as the measurement is often of the same magnitude as noise or at the detection limit.

Optically tagged NPs have also been investigated, but the challenge of overcoming the plants’ own bioluminescence can make quantification difficult.Although used in medical imaging, radio labeled NPs for noninvasive tracking and quantification in plants have not been significantly explored.Prior radio labeling of NPs for medical imaging has utilized three main approaches: post radio labeling via attachment of chelator to NPs first, then reaction with the a metal radio nuclide; preradiolabeling, where a radioactive prosthetic group, a small molecule that the radioisotope is attached, followed by attachment to the NP; and direct radio labeling.Of these three main radio labeling approaches, the third approach has been the only method used to study NP distribution in plants where Zhang et al.reported the use of [141Ce]CeO2-NPs produced via neutron bombardment of CeO2-NPs to study distribution in cucumbers. The produced radionuclide 141Ce has a 32.51 day half-life, and the specific activity of the synthesized [141Ce]CeO2-NPs was 2.7 μCi/mg of NP.Despite numerous approaches to analyze NPs, a combination of technologies is required due to the low detection limits and high resolution needed to address the intact nature of NP-transport into plants. Thus, multiple tools must be utilized to quantify and determine the intact nature of NPs at a given location within biological environments .Previous studies have provided conflicting evidence about the intact nature of NP uptake and transport into plants: some studies indicated intact NP uptake and vascular transport,some observed NPs in plants due to dissolution events and reformation within the plant tissue,and still others have indicated that NPs cannot be transported into plants.These varied observations could be linked to inadequate techniques available to track NP movement in vivo. The main challenge in determining intact NP transport into plants is ruling out NP dissolution, as reductive precipitation and formation of NPs within plant tissue has been documented.Even natural formation of NPs within plants and fungi is known.

Further complicating the picture is the fact that many studies assessing NP uptake exposed plants over long periods of time from 2 to 130 days, with very large amounts of NPs per plant, which could make dissolution events more prevalent.Avoiding excessive exposure to the NPs and carefully analyzing the stability of the administered NPs are key to avoid erroneous conclusions due to NP-dissolution and subsequent reformation. In this study we evaluated an analytical method using a radioactive label to non-invasively track and quantify transport and accumulation of NPs in lettuce seedlings in vivo. This method studied NP-size dependent transport immediately upon exposure , an early time frame that has rarely been explored in plants.The visualization of NP transport and accumulation in lettuce seedlings was done by autoradiography and PET/CT imaging and further confirmed by gamma-counting, SEM, and TEM. Our study was designed to use highly uniform NPs of two size sets , which were theoretically too large for passive transport across plant tissues.To ensure a narrow size distribution with a uniform geometric shape and the ability to thoroughly investigate stability , a preradiolabeling method with the PET-radioisotope copper-64, the “clickable” chelator ADIBO-NOTA, and commercially available spherical Fe3O4- NPs containing azides was explored. This radio labeling approach yielded a high specific activity and allowed for size characterization of the NPs after the plant accumulation and imaging period, and avoided complication from fabricating radioactive NPs and production of less stable NP material with a larger size distribution. Rigorous stability studies were carried out at a variety of pHs to investigate possible dissolution of the 64Cu-radiolabeled NPs and substantiate the intact nature of NP-transport into lettuce seedlings.To date the uptake, bio-accumulation, bio-transformation, and risks of NPs in food crops is not well understood.Most studies on NP uptake in plants have focused on the effects of NPs on plants, and have not focused on the transport or entry of intact NPs. Several studies concluded that NPs do not gain entry into plants,vertical grow rack while those that do show NP uptake in plants have found the NP amounts to vary widely between 0.05 μg/g and 38983 μg/g of plant.In vivo tracking of NP transport in plants has traditionally relied on destructive analytical techniques to quantify NP-uptake and accumulation, requiring mineralization for metal quantification mostly by ICP.These analytical techniques face the challenge of being sensitive enough to reliably measure the small changes in metal concentrations caused by NP-uptake and accumulation within the plant. The extensive range of NP accumulation reported in plants suggests that NP-uptake is dependent on several parameters: quantity of NP administered per plant, plant species, NP-size, NP-composition, and duration of exposure.Further complicating the understanding of NP transport and accumulation in plants are the studies that have observed NP-uptake due to dissolution.Collectively the variations of parameters in every study on NP transport in plants have made direct comparison and accurate conclusions challenging. Thus, it was our goal to develop a noninvasive visualization approach to track and quantify the distribution of intact radiolabeled [ 64Cu]-NPs in lettuce seedlings. Using 64Curadioactively tagged NPs, we employed a range of complementary noninvasive analytical tools including autoradiography and PET/CT imaging to spatially and temporally visualize and quantify intact-NP uptake and accumulation in plants. The stability study described demonstrates dissolution of the [ 64Cu]-NPs did not occur. Ligand effects on NP mobility within the lettuce was minimized by modification of ≤5% of the NP surface with [64Cu]-ADIBO-NOTA as to negligibly change the NP surface properties.

No ligand detachment or leaching of 64Cu-ion from the NPs within the imaging time frame and at various pHs was observed, by both HPLC and gamma counting analysis, indicating that the radioactive signal in the lettuce seedlings was due to intact [64Cu]-NPs. Additionally, indirect evidence further supported that the observed uptake was from intact [64Cu]-NPs as control lettuce seedlings given only [64Cu]CuCl2 had much higher radioactivity with 4-fold higher concentrations in the cotyledons and 10-fold higher concentrations in the root . These control plants also had visibly higher amounts of radioactivity in each part of the plant by autoradiography images suggesting that if the observed radioactivity was due to [64Cu]CuCl2, then the uptake should be much higher. Furthermore, the use of covalently bound optical-tagged NPs also exhibiting high stability and illustrated the same NP movement from the root to the cotyledon . Thus, helping to substantiate that NP-uptake and transport to the cotyledon was from intact NPs. TEM-sectioning of the plant tissue also helped to corroborate the presence of NPs within the plant tissue . It should be noted that detection of NPs within plant tissue via TEM is challenging,but based on our stability studies of the 64Cu-radiolabeled NPs along with the short exposure time that the observed uptake was attributed to intact [64Cu]-NP transport through the roots and into the cotyledons. This study has shown that NPs were transported intact into plants and can be tracked non-invasively using a radioactive tag for in vivo imaging by autoradiography and PET/CT and quantification using a gamma counter. This method allows for a highly sensitive method capable of quantifying NP amounts in an individual seedling, a level that would be challenging by the traditional ICP quantification.However, different accumulation patterns for the cotyledons were observed for the two different sized [64Cu]- NPs , while the root and whole plant were similar . Most of the accumulation for the larger [ 64Cu]-NPs was within the first hour, where cotyledon NP amounts were ∼0.35 ± 0.15 μg/g with the only significant increase after 1 h between the 12 and 24 h time point in which accumulation plateaued at around ∼0.7 μg/g . The larger [64Cu]-NPs also had higher accumulation than the [64Cu]-NPs at the early time points up to the 4 h time period. The smaller [64Cu]-NPs had ∼8.8 fold increase in cotyledon accumulation from the 4 h time point to the 24 h time point with an increase of ∼1.6 fold between 12 and 24 h time period appearing to have a linear increase in absorption over time. The differences in cotyledon accumulation between the two sized [ 64Cu]-NPs maybe linked to NP size effects on the lettuce hydraulic conductivity. Our work suggests that [64Cu]-NPs around 20 nm in size appear to clog root cortical cell walls, or pit membrane preventing further uptake, explaining why 11 reaches a plateau, while the smaller [64Cu]- NPs continued to increase in amount over time. Initial studies with duckweed also illustrated [64Cu]-NP accumulation in regions of growth and at the node and apex of the cotyledons , suggesting that [64Cu]-NP transport to the cotyledons could occur via the phloem. The TEM images further shows the appearance of intact NPs in the lettuce tissue within the expected size range for the [64Cu]-NPs , but [64Cu]-NPs had a size that appeared smaller than those administered; suggesting that the plant may filter larger NPs and has a size-threshold for uptake , which may also explain the clogging phenomenon.In summary, the combined analysis of the imaging by autoradiography and PET/CT and TEM suggested that both sized [64Cu]-NPs are transported intact from the root to the cotyledons.