Previous expression kinetics of CMG2-Fc in N. benthamiana suggested that protein accumulated in leaf increased from day 1 to day 6, supporting the first explanation. It is likely that kifunensine will remain stable even longer if longer incubation period is desired to maximize protein yield. Kifunensine is known to inhibit enzymatic activity of class I α-mannosidases, and thus should stop mannose trimming in the first place to yield single Man9 N-glycan structures. However, we observed multiple oligomannose-type N-glycans with mannose residues ranging from 3 to 9, although the most abundant structure was Man. This observation is consistent with cell culture kifunensine studies, where multiple oligomannose-type N-glycans were detected under kifunensine treatment. This could potentially due to the difference in inhibition efficacy of kifunensine towards class I α-mannosidases isoforms, which results in an incomplete inhibition of mannose trimming from Man9 structure. Also taking enzyme kinetics into consideration, depending on the ER concentrations of Man9 glycoprotein substrate, class I α-mannosidases, and kifunensine, enzymatic mannose trimming from Man9 could take place even if the amount of active ER α-mannosidase I is low. Although mannose trimming was not completely inhibited at Man9 structure, this method still showed the ability to significantly modify glycosylation using a simple bio-processing approach. It is likely that Man9 abundance can be further increased if treated with higher concentration of kifunensine, but it is not necessary if the goal is to eliminate the production of plant-specific complex N-glycans. Although in this case, CMG2-Fc can be purified easily from whole-leaf extracts through a one-step purification with Protein A chromatography, in many cases,vertical rack multiple steps of chromatography are required to purify a target protein from a large pool of host native proteins when a highly selective affinity tag is not present.

This could result in low protein yield and difficulties to achieve high purity, which is typically required for therapeutic recombinant protein products. Targeting proteins to the apoplast allows the collection of target protein in AWF, which contains much lower levels of plant native proteins than whole-leaf extract since only secreted proteins are collected, thus lowers the downstream process complexity. In this case, CMG2-Fc purity and concentration increased by 3.9-folds and 4.4-folds, respectively, when collected in AWF versus in whole-leaf extract. A similar trend was observed in kifunensine-treated samples, which confirms that kifunensine does not affect protein secretion, allowing secretion of CMG2-Fc with oligomannose-type glycoforms. The increase in purity and concentration was consistent with a previous study on harvesting a target protein from plant AWF. Hence, AWF collection is a feasible method for recombinant protein harvesting, which avoids contamination with intracellular host cell proteins, and is particularly valuable when target protein is hard to purify. Together with kifunensine treatment, apoplast-targeted recombinant protein without any plant-specific glycoforms can be transiently produced in N. benthamiana, and likely in other plants as well. Products can be collected at high concentration and purity from AWF, containing predominantly oligomannose-type N-glycans. Further studies should focus on determining how long the inhibition effect of kifunensine lasts after the one-time vacuum infiltration by monitoring the protein glycoform profile at multiple time points after vacuum infiltration, and the threshold concentration of kifunensine that results in a complete N-glycan shift from plant complex-type to oligomannose-type for other glycoproteins, particularly those with more N-linked glycosylation sites. In addition, the protein expression kinetics should be compared between kifunensine-treated and untreated groups to maximize target protein yield. Depending on the desired glycoform, this method can also be applied to other N-glycan processing inhibitors such as castanospermine, deoxynojirimycin, and swainsonine. Contaminants of emerging concern are chemicals and other substances with no regulatory standards but have been recently detected in the environment and have the potential to cause adverse effects at environmentally relevant concentrations.

CECs consist of many different types of chemicals based on their purposes of use, including flame retardants, pharmaceuticals and personal care products , endocrine- disrupting chemicals , nanomaterials, among others. Flame retardants such as polybrominated diphenyl ethers and tetrabromobisphenol A are added to manufactured materials to prevent or slow the development of ignition. Prescribed pharmaceuticals like amoxicillin and overthe-counter drugs like acetaminophen are widely used by individuals for personal health. PPCPs also contain many types of preservatives and anti-bacterial substances, like triclosan. Antibiotics and veterinary medicines are widely applied to improve the production of livestock. For a long time, these substances were unknown, unidentified, unexpected, or unsuspected pollutants due to limitations in analytical methodologies. It was also challenging to assess the impact of CECs on human health and the environment due to the lack of data or risk assessment tools. After emission from varied sources, including household sewers and industrial effluents, CECs are carried in contaminated wastewater to wastewater treatment plants . The removal efficiency of CECs during treatments depends on the design and performance of individual WWTPs, as well as the physicochemical properties of CECs.Many studies have shown that numerous CECs are present at trace levels in the treated effluent in the ng L -1 to µg L -1 range around the world, including Spain,Germany, the United States, China, and South Africa. The concentrations of CECs are generally higher in bio-solids because of the higher organic matter content, and are in the μg kg-1 to mg kg-1 range.For example, triclosan and triclocarban were detected at 2715 and 1265 μg kg-1 respectively, in bio-solids, in a study conducted in the U.S. The use of TWW and bio-solids in agriculture, and/or their direct discharge into the environment, can introduce CECs to agricultural ecosystems and surface aquatic ecosystems, posing potential risks to ecosystems and human health.Many CECs contain active functional groups such as hydroxyl, carboxyl, and amide groups in their chemical structures, and are susceptible to many biotic and abiotic transformations in the environment and organisms.

Transformation products of CECs can be directly introduced into WWTPs in municipal wastewater, leachates, and surface runoff. For instance, pharmaceuticals can be metabolized in the human body after consumption and are excreted in large portions as metabolites, particularly conjugates.TPs can also be formed during the treatment processes in WWTPs via microbial transformations, photochemical transformations and oxidation and halogenation by disinfection processes.These processes may also transform some TPs back to the parent CECs, such as hydrolysis/deconjugation of the conjugates of estrogens, leading to the “negative removal” for certain CECs in WWTPs.Transformations of CECs may also take place in agroecosystems and aquatic environments after TWW and bio-solids are discharged or applied. Soils,plants,terrestrial organisms,algae,aquatic organisms and photochemical degradation have all been reported to mediate CEC transformations. In some cases, TPs may pose higher ecological risks than their parent compounds, as they may have a greater bio-accumulation potential, increased toxicity to organisms, or longer persistence in the environment.Assuming the majority of irrigated TWW and applied bio-solids are received by soil, roots would serve as the major pathway for CEC uptake into plants.Mechanistic understanding of CEC uptake remains rather limited. Based on the current knowledge, root uptake of CECs occurs primarily through passive diffusion, although an energy dependent active process mediated by transporters is likely for certain hormone-like compounds such as naproxen, clofibric acid, hydrocinnamic acid and perfluoroalkyl acids.Translocation of CECs from roots to above-ground tissues, such as stems, leaves and fruits,vertical farming hydroponic has also been observed by previous studies, with concentrations of CECs generally being more substantial in roots.Both biotic and abiotic factors have been shown to affect the uptake, bio-accumulation and translocation of CECs by plants. These factors include plant physiology, soil pore water chemistry, the physicochemical properties of CECs, and the experimental conditions.Plant physiology plays an important role in plant uptake of CECs.Plants exposed to stressors such as drought, salinity and high temperature can respond with various adaptive mechanisms such as heightened antioxidant defense, hormone regulation, and metabolic modifications.The water and nutrient uptake and photosynthetic efficiency can decrease significantly in plants grown under stressed conditions.Therefore, it may be assumed that non-stressed plants have greater potential for CEC uptake and accumulation. Other than plant physiology, plant species within the same genus, even varieties of the same plant species, have shown different patterns of CEC uptake. For example, different carrot genotypes displayed distinct uptake patterns for metformin, ciprofloxacin and narasin.

Based on the current knowledge, the ability of crop plants to uptake and accumulate CECs in the edible tissues decreases in the following order: leafy vegetables > root vegetables > cereals and fodder crops > fruit vegetables.The physicochemical properties of CECs, such as hydrophobicity and speciation, can strongly affect their uptake and translocation in plants.Many CECs in TWW and bio-solids are polar compounds with low volatility and contain ionizable functional groups, like hydroxyl, carboxyl and amide groups.Only the dissolved CEC fraction in soil pore water would be considered available for root uptake.For neutral CECs, root uptake usually involves two pathways: 1) equilibrium between the aqueous phase in plant roots and the peripheral solution such as soil pore water; and 2) chemical sorption by the lipophilic root solids.Ionized CECs, on the other hand, may undergo disassociation in soil pore water depending on the solution pH.The electrical attraction or repulsion to the negatively charged root surface, along with the ion trap effects, which occur when CECs are neutral in the apoplast but ionized inside the cell , can greatly influence their uptake and translocation in plants.A linear relationship has been often observed between the hydrophobicity, e.g., log Kow, and the bioaccumulation of neutral CECs in plants.However, using log Kow to estimate the bio-accumulation of ionizable CECs is not accurate, partly because lipid bilayers can more easily accommodate charged organic species than n-octanol.Different experimental settings, such as hydroponic cultivation, greenhouse soil cultivation and field experiments, have also exhibited great influence on the uptake and accumulation of CECs in plants. Hydroponic experiments provide simplified conditions,while greenhouse soil cultivation and field experiments have more environmental relevance. The uptake of CECs by plants is usually evaluated by bio-concentration factor , which is calculated as the ratio of the concentration of CECs in plant tissues to that in soil pore water, or the growth media for hydroponic experiments. BCF values of CECs in roots can be high up to 840 L kg-1 in hydroponic settings, while the values obtained from soil experiments may be much smaller,suggesting the availability of CECs for plants decreased greatly in soil pore water during to phase partitioning. CECs with active functional groups, such as carboxyl, hydroxyl, and amide groups, are susceptible to metabolism in plants via various enzymatic activities after being taken up. This metabolic process is similar to the hepatic detoxification system and is known as the “green liver”.Three metabolic phases are usually involved in the metabolism of xenobiotics in plants: Phase I metabolism is an activation process that includes hydroxylation, dealkylation, oxidation and reduction, that are catalyzed by cytochrome P450s, esterase, peroxidase, or other enzymes to enhance reactivity and polarity of xenobiotics; Phase II metabolism is predominantly conjugation with polar bio-molecules, such as amino acids, sugars and glutathione, to further increase the hydrophilicity and mobility of xenobiotics; Phase III metabolism refers to the sequestration of conjugated metabolites in plant cells, including the storage in vacuoles and the incorporation into cell walls.There have been only a small number of studies focusing on the metabolism of CECs in plants. Plant cell systems, such as A. thaliana cell culture,carrot cell culture,rice cell cultures and horseradish hairy root cell culture,have been used as a simple and fast approach for characterizing metabolites of various CECs. Whole plants, either hydroponically cultivated or grown in soil, have also been used to understand plant metabolism of CECs. For example, phase I metabolites of carbamazepine, 10,11-epoxide-carbamazepine and 10,11-dihyro-10,11-dihydroxycarbamazepine, were observed in the leaves and fruits of tomato and cucumber, leaves and roots of sweet potato and carrot, and leaves of Typha spp.Diclofenac was found to be hydroxylated to 4’-OHdiclofeanc in barley,horseradish root cell culture and bulrush.Phase I metabolism was also reported for epimers of tetracycline in pinto bean leaves.Phase II metabolism has been found to occur extensively for some CECs in plants. For example, conjugation with amino acids was reported for naproxen,ibuprofen,diclofenac,and gemfibrozil in A. thaliana cells and whole plants.