Importance of Ultrafiltration in Countering Climate Change

• Ultrafiltration and Environmental Sampling
  
• Filtration Device Selection
  
• Examples of Ultrafiltration in Environmental Research
  
• Filtration in Averting the Impact of Climate Change
  
• Filtration Products for Environmental Sampling and Research
  

Ultrafiltration and Environmental Sampling

Efforts over the last 50 years have triggered the implementation of many laws, amendments, and regulations for environmental improvements.

Recent publications show that scientists and researchers heavily influence efforts for global climate change improvement. Their discoveries guide environmental awareness initiatives and regulations, affecting mandated local, regional, and country-specific testing and environmental health. Freshwater, seawater, soil, sludge, and other samples are studied and graded to track the Earth’s condition. Levels of pollutants, toxins, nanoparticles, genetic material, and proteins are assessed. These results are important for climate control and improvement, and findings may help to avert negative environmental impact.

Ultrafiltration (UF) methods have been incorporated into environmental research and monitoring workflows, with samples prepared from our Earth’s oceans, rivers, waterways, and soils. Small volume samples (0.5 – 70 mL) typically utilize centrifugal ultrafiltration (cUF) devices such as Amicon^® Ultra filter units or centrifugal microfiltration (cMF), while large volume samples (up to 400 mL) are compatible with pressurized ultrafiltration (pUF) or pressurized microporous filtration (pMF) devices, such as Amicon^® Stirred Cell assemblies. Filtration techniques are widely used in assessing levels of pollutants and evaluating methods for removal, surveying biodiversity in our oceans, identifying biomolecular indicators in soil toxicity, measuring environmental antibiotic resistance, and other studies.

Filtration Device Selection

When using ultrafiltration for sample concentration, particular attention should be paid in choosing the correct filter membrane. Performance may vary by manufacturer, as well as membrane material.

• Ultrafiltration membranes are typically made from regenerated cellulose (RC) or polyethersulfone (PES). The material of choice will greatly depend on sample compatibility. Ultracel^® regenerated cellulose is generally recommended due to lower non-specific binding. PES is used for processing certain plant samples.
• The selection of membrane nominal molecular weight cut-off (NMWCO) will depend on the molecular weight (MW) of the macrosolute to be retained, and is typically defined in kilodaltons (kDa). This selection will have a significant impact on performance. As a rule, the NMWCO filter rating should be 2-3 times smaller than the molecular weight of the solute to be retained when using Ultracel^® regenerated cellulose (RC), and 3-5 times smaller for Biomax^® PES membranes.

Generally, solutes larger than the specified MWCO will remain upstream of the filter (i.e., in the retentate), while water and solutes smaller than the specified MWCO pass through the membrane into the filtrate. This can permit removal or retention of other macromolecules that may affect critical biological and chemical determinations, downstream analysis, and assay performance based on size.

When using centrifugal ultrafiltration (cUF) devices, separation, concentration, or washing functions can be performed. Centrifugal microfiltration (cMF) devices are for pre-filtering and washing only.

• Amicon^® Ultra centrifugal ultrafiltration devices use Ultracel^® regenerated cellulose (RC) membranes, are offered with nominal molecular weight limits (NMWL) of 3 kDa, 10 kDa, 30 kDa, 50 kDa, and 100 kDa, and accommodate sample volumes of 0.5 mL, 2 mL, 4 mL or 15 mL.
• Centricon^® Plus 70 centrifugal ultrafiltration devices use Ultracel^® regenerated cellulose (RC) membranes, are offered with nominal molecular weight limits (NMWL) of 3 kDa, 10 kDa, 30 kDa, 50 kDa, and 100 kDa, and accommodate sample volumes up to 70 mL.
• Microcon^® centrifugal devices use Ultracel^® regenerated cellulose (RC) or Biomax^® PES membranes. Regenerated cellulose membranes are offered with nominal molecular weight limits of 10 kDa, 30 kDa, and DNA Fast Flow for genomic DNA. PES membrane nominal molecular weight limits include 5 kDa, 10 kDa, 30 kDa, 50 kDa, 100 kDa, and 300 kDa. Microcon^® centrifugal devices accommodate sample volumes ≤ 0.5 mL.
• Ultrafree^® microfiltration centrifugal devices for pre-filtering applications use microporous Durapore^® (PVDF) or hydrophilic polytetrafluoroethylene (PTFE) membranes, are offered with pore sizes ranging from 0.1 to 5.0 μm, and accommodate samples volumes of 0.5 mL and 2 mL (for PVDF) or 0.4 mL and 2 mL (for hydrophilic PTFE).

Pressurized filtration (pUF and pMF) methods are utilized when centrifuges are not available, for gentler or controlled processing, or to accommodate samples volumes up to 400 mL (this volume can be further expanded with the addition of an external reservoir). The larger sample volume capacity makes pressurized ultrafiltration with stirred cells ideal for larger environmental samples such as seawater, wastewater, or freshwater. Flexible, easy-to-use filter housing assemblies can accommodate a wide range of ultrafiltration and microporous filtration membranes, allowing processing of samples at varying pressures and temperatures. Stirred cell devices for pressurized and microporous filtration are reusable and autoclavable, with magnetic stirring for minimizing concentration polarization and shear stress-induced denaturation during filtration.

• Ultrafiltration (UF) membranes enable sample concentration and diafiltration, and are rated by nominal molecular weight limits (NMWL) in kilodaltons (kDa). Amicon^® Stirred Cell-compatible membrane filters include Ultracel^® RC (regenerated cellulose) and Biomax^® PES (polyethersulfone), offered in a broad range of NMWL sizes from 1 kDa to 500 kDa.
• Microfiltration (MF) membranes enable separation and washing, and are rated in microns (µm). Biomolecules or particles larger than the specified rating will remain upstream in the retentate after filtration. Amicon^® Stirred Cells can be loaded with a broad selection of disc filters. Durapore^® PVDF disc filters are available in 0.1 µm, 0.22 µm, and 0.45 µm for 200 mL and 400 mL Amicon^® Stirred Cell units.

Factors to consider when selecting an ultrafiltration centrifugal method or membrane for pressurized filtration methods:

1. Biological size: This can be estimated from published sources or by various measurement techniques.
2. Size of key separation targets in solution: The size of sample components (proteins, antibodies, toxins, and other particles that need to be separated) will affect membrane size selection. Refer also to “Membrane NMWL or MF rating”.
3. Sample volume: Processing volumes ranging from ≤ 0.5 mL to 70 mL are compatible with centrifugal filtration devices. Pressurized ultrafiltration (pUF) Amicon^® Stirred Cells have capacities of 50 mL, 200 mL, and 400 mL or more (if external reservoir is used).
4. Concentration factor or final volume: Final volumes as low as 20 µL can be achieved with Amicon^® Ultra 0.5 mL devices. Final volumes as low as 5 µL can be achieved with Microcon^® regenerated cellulose devices. Ultrafiltration concentration factor is calculated with respect to starting and final volume. Microporous filtration separates samples without concentration.
5. Fluid type: Samples may be aqueous, organic, highly viscous, particle-laden, or highly concentrated. In some cases, initial dilution (i.e. buffer or water) may be required. Consult the chemical compatibility or chemical resistance of the device or membrane, referring to the product instructions.
6. Buffer exchange, dialysis, diafiltration, and washing: Different applications (changing one buffer for another, or removal of salt or chelators) may require different filtration techniques or processes. Multiple spin-wash steps can be performed with centrifugal ultrafiltration (cUF) or centrifugal microfiltration (cMF). Multiple washes can be performed using pressurized systems (pUF and pMF).
7. Processing conditions: Conditions for centrifugal ultrafiltration and centrifugal microfiltration such as centrifugal g-force (x g), spin time, processing time, and operating temperature may impact performance with centrifugal devices. Conditions for pUF such as pressure (≤ 75 psi), processing times, and stirring rates may impact performance with pressurized devices. Conditions can be optimized as needed. Follow safety guidelines for centrifuges and when using pressurized systems. Centrifugation at lower temperatures will take longer time to process. Nucleic acid purifications should use reduced g-forces. Follow manufacturer guidelines or published methods.
8. Membrane NMWL or MF rating: For ultrafiltration, to retain a protein, nanoparticle, or other target for concentration, purification, separation, or enrichment, the molecular weight cut-off of the filter membrane needs to be smaller (~2-3 times smaller for regenerated cellulose or ~ 3-5 times smaller for (PES) than the target size, but large enough to allow smaller components to filter through. For small nucleic acids, PCR clean-up reactions, or genomic DNA use 10 kDa, 30 kDa, or Microcon^® DNA Fast Flow devices, respectively. (see Table 1). For MF, select a filter with smaller pore size than the particle or biomolecule.

Table 1. Selection of Ultracel^® regenerated cellulose ultrafiltration membrane NMWL based on target retention size.

Ultrafiltration Ultracel®	Nominal Pore Size (nm)	Recommended Retaining Size Range
Regenerated Cellulose (RC) Membrane NMWL (kDa)		Molecular Weight (kDa) Minimum	Molecular Weight (kDa) Maximum
3	0.3	6	20
10	1	20	90
30	3	60	18
50	5	100	300
100	10	200	900

Table 2. Examples of Ultrafiltration in Environmental Research

Target	Investigation/Impact	Method	cUF or pUF device & MWCO (kDa)
Dissolution of nanoparticles (NP’s) in environmental waters	Studied the dissolution of metal and metal oxide nanoparticles in artificial aqueous media, similar in chemistry to environmental waters, for up to 19 days.1	Concentration of nanoparticles in environmental waters.	Amicon® Ultra 3 kDa
Soil microbials in biosolid-amended soil	Potential exists for microbial communities and the ecosystem services they provide to be disrupted by environmentally-relevant concentrations of silver sulfide nanomaterials (Ag2S). Engineered nanomaterials (ENM’s) were used to determine the environmental risk of nanoparticles (NPs) on mycorrhizal colonization of tomato plants and soil microbial communities in biosolid-amended soil.2	Soluble Ag present in the background of ENM stock suspensions was quantified by filtering samples of each suspension.	Amicon® Ultra 3 kDa
Bioaccumulation of nanoparticle coatings by green alga	Dependence of dissolution and bioaccumulation by alga (C. reinhardtii) on nanoparticle surface coating.3	Centrifugal filter units were used to separate dissolved zinc (and its soluble complexes) from nanoparticulate zinc.	Amicon® Ultra 3 kDa
Aquatic environment	Light condition and its role in processes of nanoparticles dissolution and potential toxicity for aquatic organisms.4	Dissolved metal was separated from nanoparticles.	Amicon® Ultra 3 kDa
Groundwater and food	Contribution of nitrate fertilizer to the presence of perchlorate in ground water and food across the United States.5 One-step sample processing method for the determination of perchlorate in human urine, whole blood and breast milk using liquid chromatography tandem mass spectrometry.6	Internal perchlorate standards were spiked into sample matrix, followed by centrifugation and filtration. The filtrate was collected and subjected to LC-MS-MS analysis.	Amicon® Ultra 0.5 mL 3, 10, 30, and 50 kDa
Ocean’s surface, oil fate, and oil distribution	Protein:polysaccharide ratio in exopolymeric substances controlling the surface tension of seawater in the presence or absence of surrogate Macondo oil with and without Corexit were investigated. Results from this study provide mechanistic insights into the fate and distribution of oil in the surface ocean.7	0.45 µm filtrate was used to collect the colloidal fraction and ultrafiltered.	Amicon® Ultra-15 3 kDa
Exposure of oil to the Atlantic codfish	Highlights include identification of 717 proteins of the Atlantic cod (Gadus morhua) plasma proteome, identification of ten new biomarker candidates for oil exposure in Atlantic cod plasma, and altered immune response in the Atlantic cod after exposure to dispersed crude oil.8	Plasma samples were diluted with 360 µL TBS before desalting using UF.	Amicon® Ultra 3 kDa
Modified clay absorbents for removal of heavy metals	Modified clays using imidazolium-based ionic liquids as green and eco-friendly adsorbents due to substantial increase in their capacity for the removal of heavy metals, with positive economic and environmental impacts.9	Separation of reaction solutions from nanoclays.	Amicon® Ultra 3 kDa
Environmental antibiotic resistance	The transfer of antibiotic resistance genes (ARGs) in the environment as a threat to both human and animal health. Data was used to estimate the contribution of bacteriophages to the dissemination of antibiotic resistance.10	The filtrate from this final step was concentrated and used for phage DNA extraction.	Amicon® Ultra 100 kDa
Water quality and fish embryo toxicology	Fish embryo toxicology of Japanese medaka embryos, and later effects on population growth rate relationship to water quality of silver nitrate, silver nano-colloids, and silver chloro-complexes.11	Separation of silver nanocolloids (SNCs) and dissolved silver from solutions.	Amicon® Ultra 3 kDa
Oligotrophic oceanic environment	Members of Marine Group I (MGI) Thaumarchaeota and considered to play a significant ecological role as a primary producer in biogeochemical elemental cycles in the ultra-oligotrophic abyssal plain.12	DNA contained in the water phase was concentrated and buffer exchanged.	Amicon® Ultra-15 10 kDa and Amicon® Ultra 0.5 mL 30 kDa
Greenhouse gas	Comparison of the nitrifying activity of moving bed biofilm reactors (MBBR) with or without chronic exposure to organic loading by amine-based CO2 capture.13	Amplicon sample was concentrated before sequencing.	Amicon® Ultra 0.5 mL 30 kDa
Soil toxicity	Insecticidal Cry, or Bt, proteins produced by the soil-endemic bacterium, Bacillus thuringiensis, and some genetically modified crops. Adsorption toxicity in soils.14	Toxin was chemically extracted and immunoassayed.	Amicon® Ultra-15 kDa (not specified)
Water temperature impact on aquatic interactions	Effects of daily water temperature fluctuation on the survival of carp infected with Cyprinid herpesvirus 3 (CyHV-3).15	Thawed water samples (10 mL) were concentrated by centrifugation.	Amicon® Ultra-15 30 kDa
Ocean diversity	Diversity and community composition of bacterioplankton in surface waters of the Southern Adriatic sub-basin (SAd) in the Mediterranean Sea, across an environmental gradient from coastal to offshore stations.16	DNA samples were pooled and purified after PCR amplification.	Amicon® Ultra 50 kDa
Pollutant removal	Removal efficiency and effect of valences on removal of anionic pollutants using micellar-enhanced ultrafiltration.17	Permeate stream was sampled for pollutant concentrations (nitrate, CPC, chromate,and ferricyanide) and analyzed after ultrafiltration.	Amicon® Stirred Cell 8400 with regenerated cellulose (RC) membrane

Ultrafiltration in Averting the Impact of Climate Change

Publications show ultrafiltration and microporous filtration play a critical role in environmental monitoring and research workflows. Applications include eco-analysis for water, seawater, soils, tissues, and biologicals. The utility of Amicon^® cUF, cMF, pUF, and pMF devices has been demonstrated for separation, cleanup, and enrichment for lab-scale preparations. Impurities separated for analysis include ionic salts, chemical pollutants, and genomic, molecular, and physical particulate. The optimal filter choice greatly impacts yields, reproducibility of results, and data quality. Both centrifugal filtration (cUF and cMF) and pressurized filtration (pUF and pMF) processes provide quick, simple, and efficient ways to separate larger materials from smaller constituents and concentrate or purify samples. Physical composition of the sample, as well as size and shape of the target retentate or filtrate, are important attributes that need to be considered in filter selection. The Amicon^® portfolio, Microcon^® portfolio, and Centricon^® Plus 70 portfolio offer ultrafiltration devices, assemblies, and membranes for separation, purification, enrichment, desalting, and buffer exchange. Ultrafree^®-MC and Ultrafree^®-CL filter devices separate by pore size for pre-filtration applications. These devices provide valuable workflow solutions used in studies for the climate challenges of our Earth.

Filtration Products for Environmental Sampling and Research

Table 3. Centrifugal Ultrafiltration (cUF) Product Numbers

Device Nominal Molecular Weight Limit, NMWL (Da)	Amicon® Ultra centrifugal ultrafiltration devices				Centricon® Plus 70 centrifugal devices	Microcon® centrifugal devices
	0.5 mL	2 mL	4 mL	15 mL	15 - 70 mL	0.5 mL Ultracel® RC
3,000	UFC5003	UFC200324	UFC8003	UFC9003	UFC7003	—
10,000	UFC5010	UFC2010	UFC8010	UFC9010	UFC7010	MRCPRT010
30,000	UFC5030	UFC2030	UFC8030	UFC9030	UFC7030	MRCF0R030
50,000	UFC5050	UFC2050	UFC8050	UFC9050	—	—
100,000	UFC5100	UFC2100	UFC8100	UFC9100	UFC7100	—
Genomic DNA-rated	—	—	—	—	—	MRCF0R100

Table 4. Centrifugal Microfiltration (cMF) Product Numbers

			Ultrafree® centrifugal unitss
Device pore size (µm)	Sterility	Durapore® PVDF membrane		Hydrophilic Polytetrafluoroethylene (PTFE) membrane
		0.5 mL	2 mL	0.4 mL	2 mL
0.1 µm	Non-sterile	UFC30HV00	UFC40HV00	—	—
0.2 µm	Non-sterile	—	—	UFC30LG25	UFC40LG25
0.22 µm	Non-sterile	UFC30GV00	UFC40GV00	—	—
0.22 µm	Sterile	UFC30GV0S	UFC40GV0S	—	—
0.45 µm	Non-sterile	UFC30HV00	UFC40HV00	UFC30LH25	UFC40LH25
0.65 µm	Non-sterile	UFC30DV00	UFC40DV25	—	—
0.65 µm	Sterile	UFC30DV0S	—	—	—
5 µm	Non-sterile	UFC30SV00	UFC40SV25	—	—

Table 5. Pressurized Ultrafiltration (pUF) Product Numbers
For use with ultrafiltration or microfiltration membrane discs.

Description	Filter Disc Diameter (mm)	Catalog Number
Amicon® Stirred Cell, 50 mL	44.5 mm	UFSC05001
Amicon® Stirred Cell, 200 mL	63.5 mm	UFSC20001
Amicon® Stirred Cell, 400 mL (optional external reservoir adds capacity)	76 mm	UFSC40001
External 800 mL Reservoir for Amicon® Stirred Cell (includes tube fittings, tubing)	n/a	6028

Table 6. Ultrafiltration and Microfiltration Membrane Discs

Filtration	Membrane Disc Type	Rating (kDa or µm)	Amicon® Stirred Cell, 50 mL (44.5 mm filter)	Amicon® Stirred Cell, 200 mL (63.5 mm filter)	Amicon® Stirred Cell, 400 mL (76 mm filter)
UF	Ultracel® PL regenerated cellulose (RC-PL)	1 kDa 3 kDa 5 kDa 10 kDa 30 kDa 100 kDa	PLAC04310 PLBC04310 PLCC04310 PLGC04310 PLTK04310 PLHK04310	PLAC06210 PLBC06210 PLCC06210 PLGC04310 PLTK06210 PLHK06210	PLAC07610 PLBC07610 PLCC07610 PLGC07610 PLTK07610 PLHK07610
	Biomax® PES	5 kDa 10 kDa 30 kDa 50 kDa 100 kDa 300 kDa 500 kDa	PBCC04310 PBGC04310 PBTK04310 PBQK04310 PBHK04310 PBMK04310 PBVK04310	PBCC06210 PBGC06210 PBTK06210 PBQK06210 PBHK06210 PBMK06210 PBVK06210	PBCC07610 PBGC07610 PBTK07610 PBQK07610 PBHK07610 PBMK07610 PBVK07610
MF	Durapore® Polyvinylidene Difluoride (PVDF)	0.1 µm 0.22 µm 0.45 µm	— — —	VVLP06225 GVWP06225 HVLP06225	VVLP07625 GVWP07625 HVLP07625

References

1. Odzak N, Kistler D, Behra R, Sigg L. 2014. Dissolution of metal and metal oxide nanoparticles in aqueous media. Environmental Pollution. 191132-138. https://doi.org/10.1016/j.envpol.2014.04.010

2. Judy JD, Kirby JK, Creamer C, McLaughlin MJ, Fiebiger C, Wright C, Cavagnaro TR, Bertsch PM. 2015. Effects of silver sulfide nanomaterials on mycorrhizal colonization of tomato plants and soil microbial communities in biosolid-amended soil. Environmental Pollution. 206256-263. https://doi.org/10.1016/j.envpol.2015.07.002

3. Merdzan V, Domingos RF, Monteiro CE, Hadioui M, Wilkinson KJ. 2014. The effects of different coatings on zinc oxide nanoparticles and their influence on dissolution and bioaccumulation by the green alga, C. reinhardtii. Science of The Total Environment. 488-489316-324. https://doi.org/10.1016/j.scitotenv.2014.04.094

4. Odzak N, Kistler D, Sigg L. 2017. Influence of daylight on the fate of silver and zinc oxide nanoparticles in natural aquatic environments. Environmental Pollution. 2261-11. https://doi.org/10.1016/j.envpol.2017.04.006

5. (2001). Urbansky E.T., C. T. . Survey of fertilizers and related materials for perchlorate (ClO4-). EPA/600/R-01/tba..

6. Song S, Ruan J, Bai X, Xie L, Zhang B, He Y, Zhang T. 2019. One-step sample processing method for the determination of perchlorate in human urine, whole blood and breast milk using liquid chromatography tandem mass spectrometry. Ecotoxicology and Environmental Safety. 174175-180. https://doi.org/10.1016/j.ecoenv.2019.02.081

7. Bacosa HP, Kamalanathan M, Cullen J, Shi D, Xu C, Schwehr KA, Hala D, Wade TL, Knap AH, Santschi PH, et al. Marine Snow Aggregates are Enriched in Polycyclic Aromatic Hydrocarbons (PAHs) in Oil Contaminated Waters: Insights from a Mesocosm Study. JMSE. 8(10):781. https://doi.org/10.3390/jmse8100781

8. Enerstvedt KS, Sydnes MO, Pampanin DM. 2018. Study of the plasma proteome of Atlantic cod ( Gadus morhua ): Changes due to crude oil exposure. Marine Environmental Research. 13846-54. https://doi.org/10.1016/j.marenvres.2018.03.009

9. Naderi A, Delavar MA, Ghorbani Y, Kaboudin B, Hosseini M. 2018. Modification of nano-clays with ionic liquids for the removal of Cd (II) ion from aqueous phase. Applied Clay Science. 158236-245. https://doi.org/10.1016/j.clay.2018.03.037

10. Wang M, Liu P, Zhou Q, Tao W, Sun Y, Zeng Z. 2018. Estimating the contribution of bacteriophage to the dissemination of antibiotic resistance genes in pig feces. Environmental Pollution. 238291-298. https://doi.org/10.1016/j.envpol.2018.03.024

11. Kataoka C, Kato Y, Ariyoshi T, Takasu M, Narazaki T, Nagasaka S, Tatsuta H, Kashiwada S. 2018. Comparative toxicities of silver nitrate, silver nanocolloids, and silver chloro-complexes to Japanese medaka embryos, and later effects on population growth rate. Environmental Pollution. 2331155-1163. https://doi.org/10.1016/j.envpol.2017.10.028

12. Shiraishi F, Mitsunobu S, Suzuki K, Hoshino T, Morono Y, Inagaki F. 2016. Dense microbial community on a ferromanganese nodule from the ultra-oligotrophic South Pacific Gyre: Implications for biogeochemical cycles. Earth and Planetary Science Letters. 44710-20. https://doi.org/10.1016/j/j.pngl.2016.04.021

13. Henry IA, Einbu A, Svendsen HF, Bakke I, ÿstgaard K. 2016. Inhibition factors in biofilm N removal systems treating wastes generated by amine based CO 2 capture. International Journal of Greenhouse Gas Control. 45200-206. https://doi.org/10.1016/j.ijggc.2015.12.023

14. Hung T, Truong L, Binh N, Frutos R, Quiquampoix H, Staunton S. 2016. Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils. Environmental Pollution. 208318-325. https://doi.org/10.1016/j.envpol.2015.09.046

15. Takahara T, Honjo MN, Uchii K, Minamoto T, Doi H, Ito T, Kawabata Z. 2014. Effects of daily temperature fluctuation on the survival of carp infected with Cyprinid herpesvirus 3. Aquaculture. 433208-213. https://doi.org/10.1016/j.aquaculture.2014.06.001

16. Quero GM, Luna GM. 2014. Diversity of rare and abundant bacteria in surface waters of the Southern Adriatic Sea. Marine Genomics. 179-15. https://doi.org/10.1016/j.margen.2014.04.002

17. Baek K, Yang J. 2004. Effect of valences on removal of anionic pollutants using micellar-enhanced ultrafiltration. Desalination. 167119-125. https://doi.org/10.1016/j.desal.2004.06.119