Critical review of phytoremediation for the removal of antibiotics and antibiotic resistance genes in wastewater.

Antibiotics in wastewater are a growing environmental concern. Increased prescription and consumption rates have resulted in higher antibiotic wastewater concentration. Conventional wastewater treatment methods are often ineffective at antibiotic removal. Given the environmental risk of antibiotics and associated antibiotic resistant genes (ARGs), finding methods of improving antibiotic removal from wastewater is of great importance. Phytoremediation of antibiotics in wastewater, facilitated through constructed wetlands, has been explored in a growing number of studies. To assess the removal efficiency and treatment mechanisms of plants and microorganisms within constructed wetlands for specific antibiotics of major antibiotic classes, the present review paper considered and evaluated data from the most recent published research on the topics of bench scale hydroponic, lab and pilot scale constructed wetland, and full scale constructed wetland antibiotic remediation. Additionally, microbial and enzymatic antibiotic degradation, antibiotic-ARG correlation, and plant effect on ARGs were considered. It is concluded from the present review that plants readily uptake sulfonamide, macrolide, tetracycline, and fluoroquinolone antibiotics and that constructed wetlands are an effective applied phytoremediation strategy for the removal of antibiotics from wastewater through the mechanisms of microbial biodegradation, root sorption, plant uptake, translocation, and metabolization. More research is needed to better understand the effect of plants on microbial community and ARGs. This paper serves as a synthesis of information that will help guide future research and applied use of constructed wetlands in the field antibiotic phytoremediation and wastewater treatment.

Natural wetlands contribution on phosphorus removal in small northern communities in Canada.

This study focuses on understanding the role of passive wastewater treatment (wastewater lagoon plus wetland) in reducing the phosphorus discharge levels in a northern small community in Manitoba, Canada. The facultative lagoon system of that small community treats domestic wastewater and seasonally discharges effluent into a wetland that connects to Lake Manitoba. This research assesses phosphorus removal efficiency through the natural wetland during the vegetation growing season. The average total phosphorus (TP) concentration reduction for the observed treatment area of 1.3 ha was more than 70%, achieving the desired TP discharge concentration below 1 mg/L. Data analysis showed that the main accumulation of TP occurred at the 21-40 cm soil depth, which indicates the potential of natural wetland treatment applications under cold continental climate conditions as an effluent polishing step to satisfy regulatory requirements for phosphorus reduction.

Optimization of Typha Fibre Extraction and Properties for Bio-Composite Applications Using Desirability Function Analysis.

The effect of extraction time, temperature, and alkali concentration on the physical and mechanical properties of cattail (Typha latifolia L.) fibres were investigated using five levels of time (4, 6, 8, 10, and 12 h), four levels of temperature (70, 80, 90, and 95 °C), and three levels of NaOH concentration (4, 7, 10%, w/v) in a 3 × 4 × 5 factorial experimental design. The extraction parameters were optimized for bio-composite application using a desirability function analysis (DFA), which determined that the optimum extraction time, temperature and NaOH concentration were 10 h, 90 °C, and 7%, respectively. A sensitivity analysis for optimal treatment conditions confirmed that the higher overall desirability does not necessarily mean a better solution. However, the analysis showed that the majority of optimum settings for time, temperature, and concentration of NaOH found in the sensitivity analysis matched with the optimum conditions determined by DFA, which confirmed the validity of the optimum treatment conditions.

A meta-analysis reveals that operational parameters influence levels of antibiotic resistance genes during anaerobic digestion of animal manures.

Anaerobic digestion (AD) has shown the potential to reduce the numbers and types of antibiotic-resistance genes (ARG) present in animal manures. However, the variability of the results has limited the ability to draw solid conclusions. To address this issue, we performed a series of meta-analyses to evaluate how AD of pig, cattle, and dairy manures affects ARG levels and how different parameters, such as temperature, pH, digestion times, and the addition of other substances (e.g., solids, antibiotics) influence ARG changes. Twenty studies with enough details on changes in ARG levels during the AD process were identified and used for the meta-analyses. The results suggested that AD could significantly reduce ARG levels regardless of the conditions of the process. Also, thermophilic AD was more effective than mesophilic AD at reducing ARGs, although this difference was only significant for pig manures. The results also suggested that long digestion times (>50 days) yielded better ARG reduction rates, and that the addition of solids from an external source (co-digestion) negatively affected the efficiency of ARG reduction. In general, the results suggested that ARG changes during AD could be linked to the abundance and activity of hydrolytic communities.

Zebra mussel shells as an alternative mineral resource for lime production as a phosphorus precipitant.

Zebra mussels are an invasive species to North America and are presently found in many rivers and lakes in prolific numbers. Along with many other issues, zebra mussels present a problem when their shells are deposited on shore, carpeting beaches and reducing beach usability. A possible solution presented in this study is to use the zebra mussel shells as an alternative mineral resource to mined calcium carbonate for the production of lime to remove phosphorus in wastewater. Heat-treated coarse (500-1000 µm) and fine (<75 µm) zebra mussel shell dosed to 10 mg L-1 phosphate solution at 0.50 g L-1 and 0.25 g L-1, removed over 99% phosphorus while maintaining pH levels significantly lower than pure calcium hydroxide dosed under the same conditions. It was found that ground zebra mussel shells (<75 µm) heated for 1 h at temperatures of 600°C, 700°C, 800°C, 900°C, and 1000°C were capable of removing varying levels of phosphorus in water. Shells heated at 800°C and dosed at 1.00 g L-1 reduced phosphorus in collected real wastewater effluent by 99.48%. It was also shown that shells heat treated at 1000°C achieved 98.7% phosphorus removal when dosed at 0.25 g L-1, while maintaining a final effluent pH of 9.13, demonstrating the lowest overall energy costs of any of the effective shell treatments. The results indicate that zebra mussel shells show promise as an alternative resource for phosphorus precipitation in wastewater.

Pilot-scale, on-site investigation of crushed recycled glass as tertiary filter media for municipal lagoon wastewater treatment.

ABSTRACTGlass recycling is a process that faces many obstacles, especially in the closed-loop context. Waste glass separation and transportation can become quite expensive, turning glass collection for remanufacture unfeasible. For this reason, it is important that alternative markets for waste glass are sought. This study evaluated crushed recycled glass as tertiary media in subsurface pilot-scale filters for on-site municipal wastewater treatment, using control sand media filters. Filters were operated over 128 days at a 24-h hydraulic retention time, treating secondary lagoon effluent from the rural municipality of Dunnottar, Manitoba, Canada. Crushed recycled glass filters removed 92%, 90% and 45% of the total suspended solids (TSS), ammonium nitrogen (NH4+-N) and chemical oxygen demand (COD), respectively. Total suspended solids were removed equally well in sand and crushed recycled glass filters (α = 0.05), whereas NH4+-N and COD reductions were 10% and 21% greater in sand media, respectively. Both sand and crushed recycled glass filters failed to achieve phosphorus (P) discharge guidelines. This study shows that there is potential for crushed recycled glass in wastewater filtration, especially to achieve TSS, COD and NH4+-N removal. Small communities served by waste stabilization ponds could benefit from glass media filters, as waste glass could be diverted from curbside collection and utilized locally to polish municipal lagoon effluent.

Crushed recycled glass as a substrate for constructed wetland wastewater treatment: a case study of its potential to facilitate pharmaceutical removal.

The use of recycled glass as a substrate for constructed wetlands was assessed through two studies. The first study examined the dissipation of atenolol, carbamazepine, and sulfamethoxazole in mesocosm-modeled wetlands using glass or limestone gravel as substrates, with or without cattails (Typha spp.). Following pseudo-first-order kinetics, atenolol dissipated the fastest from the water surface of the mesocosms (t1/2~1 day), followed by sulfamethoxazole (t1/2~14 days), and carbamazepine (t1/2~48 days), with no significant differences across treatments. Increased half-lives were observed at greater depth, likely due to light screening. A Monte Carlo sensitivity analysis diagnosed sunlight absorption rates and second-order hydroxyl-mediated indirect photolysis rates to be the main sources of uncertainty in our dissipation rate estimates, compared to our observed rates. The second study examined in situ pharmaceutical removal in tertiary pilot-scale subsurface filters made of crushed recycled glass or sand in a wastewater treatment facility in Manitoba, Canada. Glass and sand showed no significant differences for pharmaceutical removals; atenolol and metoprolol were removed below limits of detection, while carbamazepine and sulfamethoxazole persisted over a retention time of 24 h. Overall, recycled glass performed similarly to traditional substrates for wetland-based wastewater treatment.

On the impact of wastewater effluent on phytoplankton in the Arctic coastal zone: A case study in the Kitikmeot Sea of the Canadian Arctic.

We present a case study on the impact of effluent from a wastewater lagoon-wetland system on phytoplankton and local primary production near a coastal Arctic community (Cambridge Bay) over spring to fall 2018. Results are also placed within an interannual and regional context for the surrounding Kitikmeot Sea. We find the shallow, relatively fresh Kitikmeot Sea is one of the most nutrient-deplete regions of the Arctic Ocean with NO3- + NO2- concentrations below the surface mixed layer rarely exceeding 2 µmol L-1 and a N:Si:P ratio of 1:6:1. The fjordal-type bathymetry of the main study site and a persistent pycnocline below the bay's exit sill led to slightly elevated N:Si:P of 3:11:1 through trapping of wastewater-sourced N at depth via sinking and remineralization of primary production. Total production in Cambridge Bay over the 3-month open water period was 12.1 g C m-2 with 70% of this production occurring during the 1-month discharge of wastewater into the system. Local primary production responded rapidly to high NO3- + NO2-, NH4+ and PON concentrations provided by wastewater effluent, comprising up to 20% of the production during the discharge period. Remaining production was mostly explained by the deep nutrient pool in the bay, which was only accessed towards the end of the discharge period as the diatom-dominated deep chlorophyll maximum settled below the pycnocline. Although not yet eutrophic, caution is raised at the rapid response of the marine system to wastewater release with a strong recommendation to develop a research and monitoring plan for the bay.

Response of Microbial Community to Induced Failure of Anaerobic Digesters Through Overloading With Propionic Acid Followed by Process Recovery.

In order to effectively use microbial-based strategies to manage anaerobic digesters, it is necessary to distinguish between community shifts that are part of the natural dynamic of the system and shifts caused by environmental or operational disturbances. The objective of this research study was to evaluate the significance of changes in the microbial community of anaerobic digesters during failure in correlation to operational parameters such as an organic acid overload. Five continuously stirred 0.5 L reactors were set-up as semi-continuously-fed, mesophilic dairy manure digesters with a 30-day hydraulic retention time. After a 120-day stabilization period, two digesters were kept as controls, while the organic loading rates in the triplicate set were increased step-wise to ultimately provide a shock-load leading to failure using propionic acid spikes. Acidosis resulting in near cessation of biogas and termination of methane production occurred between 4 and 7 weeks, after which all the digesters continued to be fed only dairy manure. The shock loading of propionic acid led to an accumulation of mainly acetate and propionate, with low levels of iso-butyrate, butyrate, iso-valerate, and valerate. High-throughput Illumina sequencing of the V4 region of the bacterial and archaeal 16S rRNA gene in digester samples showed a significant change in the microbial community composition during propionic acid overload, followed by a return to the original composition with regular feedstock. Bacterial genera whose relative abundance decreased during the inhibition stage included Sedimentibacter, Syntrophomonas, TSCOR003.O20, and Marinilabiaceae, while the relative abundance of Lachnospiraceae, Ruminococcus, Mogibacteriaceae, Pyramidobacter, and Bacteroides increased. The relative abundance of dominant methanogens, Methanosarcina and Methanobacterium, although initially resistant, were decreased (from 91.71 to 12.14% and from 2.98 to 0.73%, respectively) during inhibition, while Methanobrevibacter and Methanosphaera that were prominent in the manure feedstock increased from 17.36 to 79.45% and from 0.14 to 1.12%, respectively. Shifts in bacterial and archaeal compositions, back to their pre-shock steady state after failure, highlight the digester's microbial resilience and recovery potential.

Effect of mesophilic anaerobic digestion on the resistome profile of dairy manure.

The effect of mesophilic anaerobic digestion (AD) on the resistome profile of manures from two different dairy farms was evaluated using a metagenomic approach. A total of 187 unique Antibiotic resistance genes (ARGs) for 17 different classes of antibiotics were detected in raw (undigested) manures. The results indicate that regardless of the origin of the dairy manure, mesophilic AD was capable of reducing or enriching the relative abundance of some ARGs. The main driver of these changes was strongly correlated with the evolution of the microbial community during the AD process. Putative ARG hosts were suggested by analyses of the co-occurrence of microbial groups and ARGs. Finally, network analyses revealed that mesophilic AD could also reduce the co-occurrence of different groups of ARGs potentially located in the same genetic elements. Our results provide valuable insights into the microbial mechanisms driving the diversity and abundance of ARGs during mesophilic AD.

Insitu kinetics of human pharmaceutical conjugates and the impact of transformation, deconjugation, and sorption on persistence in wastewater batch bioreactors.

The fate of selected common pharmaceuticals and four of their major conjugates in wastewater batch bioreactors was evaluated to determine how treatment plant parameters such as addition of air, and the presence of waste activated sludge (WAS) could influence the removal of parent compounds and conjugates. Under a realistic hydraulic residence time (HRT) for each treatment sub-process of approximately 2 h, acetaminophen and its sulfate metabolite were both rapidly degraded (>99%). Propranolol was sulfated and concurrently removed. Deconjugation of N-acetylsulfamethoxazole and sulfamethoxazole-glucuronide contributed to increases of the parent sulfamethoxazole. Thyroxine was resistant to degradation, while thyroxine-glucuronide was rapidly deconjugated (>90% in <2 h). In the absence of WAS, sorption to suspended solids was another major removal mechanism for acetaminophen, propranolol, sulfamethoxazole, and thyroxine. However, with WAS, concentrations associated with suspended solids decreased for all analytes within 24 h. These results indicate that both conjugation and back-transformation are compound-specific and dependent on parameters such as HRT, addition of microbial content, and suspended solids levels. Therefore, conjugation-deconjugation processes may strongly influence the speciation of pharmaceuticals and their fate in wastewater treatment plant effluents.

Effect of ceftiofur on mesophilic anaerobic digestion of dairy manure and the reduction of the cephalosporin-resistance gene cmy-2.

The effect of ceftiofur (CEF), a commonly used antibiotics on dairy farms, on the performance and stability of mesophilic batch anaerobic digestion (AD) of dairy manure was evaluated in terms of methane production, organic matter removal (COD, dCOD, TS, and VS), and synthesis of end-products (VFAs, CO2, and H2). The results indicated that only CEF concentrations of 10 mg/L or higher significantly affected the performance of the AD process, although the overall stability was not compromised. Biochemical analyses suggested that hydrolytic microorganisms were the most affected by the presence of CEF leading to lower COD removal, whereas acetogens were only temporarily slowed down. Methanogens, on the other hand, were not directly affected by any of the CEF concentrations tested (0.2-250 mg/L). Additionally, the presence of CEF was shown to alter the incidence of the cephalosporin-resistance marker, cmy-2, although an overall reduction was achieved in 15-day batch anaerobic digestion trials.

The Thermal and Mechanical Properties of Medium Chain-Length Polyhydroxyalkanoates Produced by Pseudomonas putida LS46 on Various Substrates.

Medium chain-length polyhydroxyalkanoates (mcl-PHA) were produced by Pseudomonas putida LS46 cultured with a variety of carbohydrate and fatty acid substrates. The monomer compositions and molecular weights of the polymers varied greatly and was dependent on whether the substrate was metabolized via the fatty acid degradation or the de novo fatty acid synthesis pathways. The highest molecular weights were obtained from medium chain-length fatty acids, whereas low molecular weights were obtained from longer chain-length and more unsaturated fatty acids or carbohydrates. The differences in monomer compositions and molecular weights due to the choice of substrate did not affect the polymer thermal degradation point. The glass transition temperatures varied from -39.4°C to -52.7°C. The melting points, when observed, ranged from 43.2°C to 51.2°C. However, a profound substrate effect was observed on the crystallinity of these polymers. Reduced crystallinity was observed when the monomer compositions deviated away from C8-C10 monomer lengths. The highest crystallinity was observed from medium chain-length fatty acids, which resulted in polymers with the highest tensile strength. The polymer produced from octanoic acid exhibited the highest tensile strength of 4.3 MPa with an elongation-at-break of 162%, whereas the polymers produced from unsaturated, long-chain fatty acids remained amorphous. A comparative analysis of the substrate effect on the physical-mechanical and thermal properties of mcl-PHAs better clarifies the relationship between the monomer composition and their potential applications, and also aids to direct future PHA synthesis research toward properties of interest.

Bacteria-triggered release of a potent biocide from core-shell polyhydroxyalkanoate (PHA)-based nanofibers for wound dressing applications.

Bacterial infections are a serious issue in wound healing. Extensive use of biocides in wound dressings have raised concerns of biocide resistance and unnecessary harm to normal skin cells. In this paper, we report a new approach to realize bacteria-triggered release of a biocide to the sites of bacterial infections from core-shell polyhydroxyalkanoate (PHA)-based nanofibers prepared by coaxial electrospinning. The hydrophobic PHA-based shell can prevent the biocide from undesirable payload release in physiological environments without pathogens. However, in the presence of pathogens, the PHA-based shell is degraded by the pathogens, and the encapsulated biocide is released. The released biocide subsequently can exert targeted antimicrobial effects on the bacteria. Using Pseudomonas aeruginosa as a model bacterium and dodecyltrimethylammonium chloride as a model biocide, we demonstrated that the core-shell PHA-based nanofibers effectively released encapsulated dodecyltrimethylammonium chloride in the presence of Pseudomonas aeruginosa, resulting in targeted inactivation of Pseudomonas aeruginosa cells.HighlightsUnique core-shell nanofibers were successfully fabricated from PHAs generated by bacteria.An on-demand release of biocide was achieved from a PHA-based core-shell nanofibours membrane.The membrane's mechanical properties closely match those of the human skin.

Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers.

The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L-1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s-1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L-1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to this behavior, the oxygen transfer performance of the bioreactor, for otherwise constant operating conditions, was reduced by 50% over the cultivation time. This study has shown that the dynamic rheology of HCD cultures is an important engineering parameter that may impact the final outcome in PHA cultivations. Understanding and anticipating this behavior and its biochemical origins could be important for improving overall productivity, yield, process scalability, and the efficacy of downstream processing unit operations.

Development of High Cell Density Cultivation Strategies for Improved Medium Chain Length Polyhydroxyalkanoate Productivity Using Pseudomonas putida LS46.

High cell density (HCD) fed-batch cultures are widely perceived as a requisite for high-productivity polyhydroxyalkanoate (PHA) cultivation processes. In this work, a reactive pulse feed strategy (based on real-time CO2 or dissolved oxygen (DO) measurements as feedback variables) was used to control an oxygen-limited fed-batch process for improved productivity of medium chain length (mcl-) PHAs synthesized by Pseudomonas putida LS46. Despite the onset of oxygen limitation half-way through the process (14 h post inoculation), 28.8 ± 3.9 g L-1 total biomass (with PHA content up to 61 ± 8% cell dry mass) was reliably achieved within 27 h using octanoic acid as the carbon source in a bench-scale (7 L) bioreactor operated under atmospheric conditions. This resulted in a final volumetric productivity of 0.66 ± 0.14 g L-1 h-1. Delivering carbon to the bioreactor as a continuous drip feed process (a proactive feeding strategy compared to pulse feeding) made little difference on the final volumetric productivity of 0.60 ± 0.04 g L-1 h-1. However, the drip feed strategy favored production of non-PHA residual biomass during the growth phase, while pulse feeding favored a higher rate of mcl-PHA synthesis and yield during the storage phase. Overall, it was shown that the inherent O2-limitation brought about by HCD cultures can be used as a simple and effective control strategy for mcl-PHA synthesis from fatty acids. Furthermore, the pulse feed strategy appears to be a relatively easy and reliable method for rapid optimization of fed-batch processes, particularly when using toxic substrates like octanoic acid.

Nitrogen and Phosphorus Phytoextraction by Cattail (Typha spp.) during Wetland-based Phytoremediation of an End-of-Life Municipal Lagoon.

Spreading biosolids on farmland can be an effective and beneficial option for managing end-of-life municipal lagoons. Where the spreading of biosolids on farmland is restricted or unavailable, in situ phytoremediation could be a sustainable alternative. This study examined nitrogen (N) and phosphorus (P) phytoextraction by cattail ( spp.) from biosolids in a wetland constructed within a lagoon cell previously used for primary treatment of municipal wastewater. The effect of harvesting season as well as harvest frequency on N and P removal were evaluated. Forty-eight 4-m plots within the constructed wetland were used to determine the effect of cattail harvest frequency on plant N and P phytoextraction. Harvesting twice per season resulted in a 50 to 60% decrease in phytoextraction of N and P relative to a single harvest per season, which produced biomass yields of 0.58 to 0.6 kg m per year and accumulated 36.7 g N m and 5.6 g P m over the 4-yr period. Compared with August, harvesting cattails in November or April reduced N and P phytoextraction by 63 to 85%. These results demonstrate that phytoextraction of nutrients is more effective with a single harvest compared with two harvests per season. Additionally, we found that while harvesting in November and April is appealing logistically (since the wetland is frozen and provides easier access to harvest equipment), nutrient removal rates are significantly reduced.

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis.

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.

Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46.

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36 g L-1 h-1 under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14 h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14 h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

Lipid and carotenoid synthesis by Rhodosporidium diobovatum, grown on glucose versus glycerol, and its biodiesel properties.

Relationships between lipid and carotenoid synthesis by Rhodosporidium diobovatum were investigated for cell cultures in nitrogen-limited medium (GMY) containing equimolar amounts of carbon of glucose or glycerol. The cultures were also supplemented with additional substrate at 120 h postinoculation (pi) and during a fed-batch experiment. Growth of R. diobovatum on glucose resulted in higher yields of triacyglycerides (TAGs) and carotenoid than when grown on glycerol, even though the cultures contained equimolar amounts of carbon. After the addition of fresh substrate at 120 h pi, total carotenoid concentrations were significantly different from the concentrations measured at 120 h pi in both glucose and glycerol cultures, with no concomitant increase in lipid concentrations, suggesting that carotenoid synthesis is linked to exponential-phase growth, while lipid synthesis is linked to stationary phase. We also compared the calculated properties of biodiesel that could be made with TAGs derived from R. diobovatum with properties of biodiesel made from TAGs of other oleaginous yeasts, microalgae, vegetable oils, and animal fats. This study shows that R. diobovatum can be an effective strain for production of neutral lipids containing high percentages of oleic acid, palmitic acid, and linoleic acid, as well as carotenoids.