Bioremediation of Chlorate and Chromium in Soil Columns Using Contaminated Site Native Culture

Chlorate and hexavalent chromium (chromate) are both widely used in different industries, and the improper waste management in the past left many sites with elevated concentrations in groundwater that pose potential risk to human and/or ecological health. Bioremediation is a sustainable management solution that can reduce both of these contaminants to less toxic species. In our earlier microcosms experiments, we have demonstrated that native microorganisms collected from a site contaminated with chlorate and chromate can lower the concentration of these chemicals in groundwater to acceptable regulatory levels provided sufficient electron donor, nitrogen, and phosphorous are provided. In this study, continuous flow column experiments were performed using soil from the site impacted by both chlorate and chromate in the Province of Manitoba (Canada) and synthetic groundwater amended with acetate, nitrogen, and phosphorous. The objective was to evaluate at a bench scale possibility of in‐situ groundwater treatment. Concentrations of chromate and chlorate measured in the columns' effluent water dropped by 86% and 96%, respectively. However, increased biomass and precipitation of trivalent chromium reduced the water flow rate in the columns, a concern for implementing this method as a long‐term in‐situ remediation solution.


Introduction
Evaporative lagoons have been used as a common method for managing waste materials at industrial facilities, a practice that results in potential risk to groundwater and surface water sources.At an industrial site in Manitoba, Canada, old lagoons from the 1960s were covered with soil to prevent the spread of chemicals from the site into the environment via surface runoff.However, water infiltration from precipitation events gradually dissolved some chemicals and transferred them with seepage water to the deeper layers of soil beneath the lagoons.
Chlorate and chromium are common co-contaminants in groundwater at pulp and paper mills, sodium chlorate facilities, and electroplating industries.Given the scale of some of the contamination plumes, these sites often go unremediated (e.g., brownfields), or use costly, ex-situ techniques to control groundwater contamination (e.g., pump and treat).Stimulating in-situ bioremediation in groundwater can provide a cost-effective, sustainable process, with minimal inputs required for optimizing conditions for decontamination.

Chlorate
Chlorate and perchlorate are two chlorine oxyanions with high oxidation capacity (Taraszki 2009).Chlorate is a semi-stable ion that forms in the perchlorate reduction process, and it is not common to be found in the environment (EnviroGulf Consulting 2007).Common sources of these chemicals are bleaching agent-producing factories, herbicide factories, the pulp and paper industry, and agriculture which uses chlorates found in sodium chlorate, calcium chlorate, or magnesium chlorate (US EPA 2016).
Chlorate has been identified as posing potential risk to human and environmental health.The applicable Canadian federal criteria for chlorate concentrations is 1 mg/L for drinking water (Health Canada 2022).There are no federal criteria for surface water or groundwater.However, the Province of British Columbia has published water quality guidelines that are protective of both agricultural (3 mg/L) and freshwater aquatic receptors (30 mg/L) (Warrington 2002; B.C. Ministry of Environment and Climate Change Strategy 2020).In 2020, the European Union decreased the chlorate concentration in drinking water from 0.7 to 0.25 mg/L.The higher value is only acceptable if "a disinfection method that generates chlorate, in particular chlorine dioxide, is used for disinfection of water" (European Parliament and the Council 2020).
Since chlorate is an intermediate ion in perchlorate reduction, and the structure and chemical properties are similar, groundwater remediation approaches commonly applied to perchlorate are also applicable to chlorate.Effective physicochemical removal methods include ion exchange, sorption on activated carbon, filtration, capacitive deionization, and electrodialysis.These approaches are primarily feasible for water and wastewater treatment processes given the cost of constructing, maintaining, and operating a system.An in-situ, stimulated bioremediation approach is preferred for groundwater management as it minimizes both cost and can be used to target both the source and the downgradient plume (ITRC 2008).
Perchlorate-reducing microorganisms exist in soils, sediments, groundwater aquifers, sludges, wastewater, animal waste, and other environments.(Per)chlorate reduction is a physiological trait found primarily in the beta-Proteobacteria, although Chloroflexi, Bacteroidetes, the Firmicutes, and the Euryarchaeota are other major (per)chlorate-reducing phyla (Liebensteiner et al. 2016;Wan et al. 2016).These dissimilatory (per)chlorate-reducing bacteria (DPRB) consist mainly of facultative anaerobes or microaerophiles, and organic carbon is their preferred electron donor.DPRB have been found to oxidize acetate, organic acids and alcohols, aromatic hydrocarbons, hexoses, and reduced humic substances (Coates and Achenbach 2004).Specific enzymes produced by these microorganisms help them switch from oxygen to chlorate under anaerobic conditions.Chlorate acts as an oxygen source in an extracellular reaction transferring electrons from the electron donor to chlorine oxyanions (Wang and Coates 2017).The electron donor could be selected from simple, pure chemicals like acetate, ethanol, glucose, or more commercial products with complex compositions like vegetable oil, whey, molasses, and yeast extract (Hatzinger et al. 2002;ITRC 2005;Nilsson et al. 2013).There are three major enzymes typically involved in (per)chlorate reduction: perchlorate reductase (Pcr), chlorate reductase (Clr), and chlorite dismutase (Cld) (Liebensteiner et al. 2016).
In perchlorate-reducing bacteria, Pcr catalyzes the reduction of ClO4 to ClO3, as well as the reduction of ClO3 to ClO2.Thus, all microorganisms that utilize Pcr as their major perchlorate-reducing enzyme are also able to reduce chlorate.In contrast, chlorate reductase only recognizes chlorate, which is reduced to chlorite just as with Pcr.Finally, in most DPRB the enzyme chlorite dismutase catalyzes the final step producing chloride and diatomic oxygen.O2 can either freely diffuse through the outer membrane or undergo reduction by a terminal oxidase.The electrons for both perchlorate and chlorate reduction come from NADH derived from the oxidation of organic compounds, including acetate (Wang and Coates 2017).

Chromate
Most chromium consumption relates to the metallic form, Cr 0 , in the metallurgy industry.The ions have broad uses in other industrial facilities like pigment production, anti-corrosion coatings, wood preservatives and fungicides, stainless steel, and other high chromium alloys, and leather tanning (Kotas and Stasicka 2000).Chromium exists in different oxidation states with six valence electrons but is predominantly found in the environment in two stable forms-the trivalent and hexavalent state (chromate).The high natural chromium deposits in the earth's crust, feasible to mine, contain trivalent chromium.Elevated chromate concentrations only appear around urban or industrial areas and have anthropogenic sources (Brandhuber et al. 2005).
Trivalent and hexavalent chromium ions are different in their effects on the human body.While trivalent chromium is considered beneficial in trace amounts for living tissues, hexavalent chromium has adverse health effects on biological systems.Asthma and respiratory systems allergies are reported in case of inhalation (Kotas and Stasicka 2000).Bleeding in the gastrointestinal system and liver and kidney damage with diarrhea, vomiting, and indigestion are some health issues that could happen by ingesting hexavalent chromium (Guertin et al. 2005).Health Canada regulated the chromium content of drinking water to be lower than 0.05 mg/L to prevent the health effects of hexavalent chromium (Health Canada 2022).
Hexavalent chromium could be removed through a physicochemical or biological pathway.The physical removal of the ion from the contaminated environment can be achieved by adsorption in ion exchange units, filtration, or electrocoagulation process.The chemical approach first targets the ion species to reduce them to harmless ions (Bryjak et al. 2016).
Reducing the oxidation state of chromium (VI) to chromium (III) eliminates health concerns and stabilizes the ion in an innocuous form.Chemical reduction of hexavalent chromium occurs when an appropriate electron donor for this reaction is available in the environment.The chemical electron donor could be elemental iron (Fe 0 ), ions like Fe 2+ Mn 2+ , S 2− , and organic materials like humic and fulvic acids (Guertin et al. 2005).
Bioremediation of chromium uses the ability of microorganisms to reduce the oxidation state of chromium in the presence of a wide range of electron donors.It can be an unspecific side reaction of various enzymes (Rahman and Thomas 2021), requiring a high density of cells (Suthersan and Payne 2005).Different chromate reduction pathways occur depending on growth conditions (Dhal et al. 2013).If oxygen is present, Cr (VI) can be reduced via soluble chromate reductases using NADH as the electron donor.Under anaerobic conditions, it can instead be utilized as a terminal electron acceptor by hydrogenases or cytochromes in the periplasm, which is an exothermic process (Rahman and Thomas 2021).However, it is not understood whether any bacteria can grow on chromate as an electron acceptor.
Organic matter in soil or groundwater is the primary natural electron donor.Molasses, lactate, glycerol, acetate, yeast extract, gluconate, or fructose are other electron donors that have been added to the media (water) and successfully utilized by microorganisms.(Rahman and Thomas 2021).Acetate was specifically selected for the experiments described below, because, contrary to the other compounds listed above, it is not fermentable, so its presence under anaerobic conditions primarily selects for microorganisms that use inorganic electron acceptors, rather than fermentative organisms.Acetate is also easily adaptable for in-situ remediation approaches, it does not require as much mixing and is cost-effective to implement compared to other electron donors.

Batch (Microcosms) Bioremediation of Chlorate and Chromate with Microbial Culture Collected from a Contaminated Site
We conducted several anaerobic microcosms experiments with synthetic groundwater, microorganisms collected from chlorate-and chromate-impacted site, acetate as an electron donor, nitrogen, and phosphorus.The highest chlorate and chromate concentrations in the groundwater at the contaminated site were observed at 3900 and 3 mg/L, respectively.However, we used lower chlorate concentration (1000 mg/L) in the microcosms experiments to observe the results in a shorter timeframe.
The microorganisms utilized 2200 mg/L acetate to remove 1000 mg/L of chlorate and 3 mg/L of hexavalent chromium entirely from the media provided that the groundwater is supplemented with additional nitrogen and phosphorous (with the carbon:nitrogen:phosphorous molar ratio of 100:10:5).Native microbial culture derived from the contaminated site removed the chlorate and chromate from the synthetic groundwater at 20 °C in about 40 days.The same removal was achieved at 10 °C, but in a longer timespan of 80 days (Motevasselin et al. 2023).Therefore, the bioremediation of the two ions, chromate and chlorate, has been demonstrated in the laboratory batch (microcosms) conditions.

Objectives
The objectives of this paper were to evaluate whether bioremediation of chlorate-and chromate-contaminated water is possible in a continuous flow soil column, which is closer to the actual conditions in the in-situ treatment.
To ensure that the process would rely on the presence of microorganisms rather than chemical reactions, first we investigated whether reducing chlorate and chromate simultaneously is possible under sterile conditions (i.e., in the absence of biological activity).
Finally synthetic groundwater, supplemented with acetate (electron donor) and nitrogen and phosphorus, was filtered through columns filled with the soil from the contaminated site.The changes in concentrations of chromate and chlorate in the columns' effluents were observed.

Materials and Methods
A synthetic groundwater, as shown in Table 1, was prepared based on site-specific groundwater analytical data.The components of the synthetic groundwater were purchased from Sigma-Aldrich chemicals, and they were dissolved in pure deionized water produced by the Millipore Elix-10 system with total organic carbon (TOC) <10 ppb and resistivity of 10 to 15 MΩ•cm.Nitrate and nitrite were not added to the solution since they compete with the other oxyanions to receive electrons.Using synthetic groundwater makes it easier to control the concentration of materials since groundwater from the site contains various trace materials with unknown effect on the process (Motevasselin et al. 2023).
Synthetic media, the solution used in the column study, was prepared by adding the electron donor and nutrients to the synthetic groundwater, as shown in Table 2. Acetate was selected as an electron donor to enhance chlorate-reducing bacteria based on our earlier experiments (He et al. 2019).Based on the stoichiometry of the chlorate reduction reaction, every 4 mol of chlorate need 3 mol of acetate to complete the catabolic and biosynthetic reactions and produce 1 mol of chloride ion (Rikken et al. 1996).
The acetate concentration added to the synthetic groundwater feeding the soil columns was higher than that used in the earlier microcosms study as the microbial population in the columns was likely larger than that in the microcosms.The microcosms inoculum contained only small amount of soil while the columns were filled entirely with that soil (Motevasselin et al. 2023).Ammonium phosphate was used as nitrogen and phosphorus sources, the C:N:P ratio was 100:10:5 to avoid any limitation of the nutrients.Soil for the column study was collected from the contaminated site at the same depth as the groundwater table (about 4 m) and was sieved using 2 mm pore-size sieves.All experiments were conducted at room temperatures of approximately 20 °C.

Reduction of Chlorate and Chromate under Sterile Conditions
Abiotic chemical reduction using substances such as iron in various forms (e.g., zero-valent iron (ZVI), nanoscale ZVI (NZVI), goethite, ferrihydrite, schwertmannite, magnetite, iron sulfides, and more recently, green rust) has been demonstrated for successful reduction and/or removal of chromate (Shih et al. 2015;Vilardi et al. 2017;Zhao et al. 2019;Huang et al. 2021).Therefore, it was important to include a non-biological control experiment.Tests under sterile conditions were carried out to evaluate the potential for chemical reduction of chromate in the presence of acetate without the presence of the microorganisms.Solution No.1 (Control) bottles used synthetic groundwater (Table 1), to demonstrate chromate stability in solution.Solution No. 2 (C&A) contained chromate and acetate in deionized water to identify any chemical reaction between only these two reactants.Solution No. 3 (SW&A) consisted of the synthetic groundwater as described above in Table 1 amended with acetate.The three solutions used in this study were prepared in triplicate.
All glassware and DI water were sterilized using an autoclave for 30 min at 121 °C for 20 min.Preparation was performed in a biosafety cabinet, disinfected with DNA away from the solution and a UV lamp to prevent any microorganisms from entering the experimental setup.Nine 120-mL bottles (three bottles of each solution) were filled with 100 mL of prepared solutions and capped.At time 0 (initial concentration, C o ) and after days 1, 3, 7, 15, and 30, 2 mL sample aliquots from each bottle were removed with a clean new syringe.Aliquots were diluted six times with deionized water to attain chromate concentrations within the range of analytical detection limits.Hexavalent chromium analysis was done based on the method 8023 using HACH DR-3900 spectrophotometer (HACH); the detection limit for this method is 0.010 mg/L.Total chromium was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) on a Varian model 725-ES made by Agilent.Chlorate and acetate concentration in the samples were measured using Metrohm 930 Ion Chromatograph (IC) with the detection limit of 1 mg/L, using standard methods (APHA 2012).

Continuous Columns Experiments
Two vessels containing the synthetic groundwater with and without acetate and nutrients were used to feed transparent, acrylic columns with an inner diameter of 4.4 cm and length of 30 cm.The columns were packed with the pre-sieved, contaminated site soil using a constant energy packer to attain uniform bulk density in all the columns.The constant energy packer is a 1-m long tube with a funnel on the top end and two 2-mm sieves positioned 5 cm apart at the bottom.The presieved dry soil sample is poured continuously into the funnel which falls through the tube and strikes the two sieves at the bottom sequentially, causing the falling stream of sand particles to spread evenly.The metal tube is raised slowly as the soil column begins to fill to maintain a 5 cm gap between the bottom sieve and the top of the soil column.The falling stream of soil from a constant height leads to even packing of the soil column (R. Sri Ranjan, personal communication (2019), Soil and Water Engineering Lab, University of Manitoba).Two 0.45-μm filters covered each column's top and bottom to prevent microorganisms and other solids from migrating out of the columns.The vessels were connected to a nitrogen tank with a constant flow to prevent oxygen from entering the media, as shown in Figure 1.The flow in the columns was driven by gravity in the up-flow direction.Both influent reservoirs A and B were kept at a constant head by replenishing the influent to maintain the same level above the inlet to the soil column.Flow direction was upwards through the columns to avoid preferential flow paths.Each vessel was set up with three columns in parallel (i.e., triplicate).After 16,40,88,112,and 136 h, aliquot samples were collected from the outlet stream at the top of each column.The samples were analyzed for chromate and chlorate content; the flowrate was determined by weighing the volume of effluent at different time intervals.

Reduction of Chlorate and Chromate under Sterile Conditions
The aqueous chromate and chlorate concentrations withdrawn from the sterile bottles showed no significant difference between the three test conditions after 30 days (T test values for chromate 0.94, 0.63, for chlorate 0.42, 0.05) These relatively constant concentrations show that under sterile conditions, no chemical reactions between acetate and chromate or chlorate occur.Results from Study No. 1 (Sterile Condition) are shown below in Figure 2.
While abiotic chemical reduction using substances such as iron in various forms (e.g., ZVI, NZVI, goethite, ferrihydrite, schwertmannite, magnetite, iron sulfides, and more recently, green rust) has been demonstrated for successful reduction and/ or removal of chromate (Shih et al. 2015;Vilardi et al. 2017;Zhao et al. 2019;Huang et al. 2021) and to more limited suc-cess, chlorate (Westerhoff 2003;Gu and Coates 2006;Xiong et al. 2007;Guan et al. 2015), naturally occurring, abiotic degradation of chromate, and/or chlorate have not be observed under the current experimental conditions.Given the available groundwater and soil chemistries, biological processes are necessary for the reduction of both chlorate and chromate and do not compete with abiotic processes at measurable concentrations.It has been hypothesized that the combined effects of abiotic and biotic processes induced by ZVI could affect chromate removal from groundwater (Zhong et al. 2017).This finding (control samples demonstrating that abiotic processes do not affect oxyanion degradation) has been observed by others evaluating in-situ, stimulated bioremediation of chlorate (Sutigoolabud et al. 2005).

Continuous Column Experiments Chlorate
The chlorate concentration in the columns feeding synthetic groundwater with acetate and nutrients (Table 2) decreased gradually from 1000 to 708 mg/L after 3.7 days.After that, the chlorate concentration fell faster and reached 137 mg/L after 5.7 days.Increasing the population of reducing microorganisms lead to faster chlorate concentrations decrease (shown in Figure 3).
Without acetate and nutrients addition, although the chlorate concentration shows a slight drop initially, it remains constant and does not fall below 90% of the initial concentration.The rationale for the initial "drop" in chlorate concentration is attributed to the fact that the packed soil column most likely contained some small amounts of carbon sources that the reducing bacteria could use as electron donors.This carbon source, however, exhausted quickly and was insufficient to allow for microorganism growth at a level that could significantly reduce chlorate concentrations.
The rate and amount of chlorate reduction (Figure 4) is similar to previous studies that have evaluated various car- bon sources to stimulate local microbiological conditions for chlorate remediation (Hunter 2002;Senoo et al. 2004;Sutigoolabud et al. 2005;Schwarz et al. 2012).In most instances, additional stimulation (e.g., including additional nutrients) was not necessary for sustained microbial reduction of chlorate-either soil chemistry or groundwater chemistry was sufficient in supplying adequate nutrition for the microbial communities.The groundwater in our site was shown to be nutrient deficient (Motevasselin et al. 2023).While this does provide more control with respect to competing oxyanions (e.g., nitrates), these results indicate that the addition of an electron donor is not sufficient to stimulate bioremediation under the given the site conditions.These results re-affirm the need to understand local, site-specific soil, and groundwater conditions when evaluating biological processes for in-situ remediation.

Chromate
Chromate concentrations decreased in both column experiments at a faster pace than chlorate (Figure 5).With acetate and nutrients, chromate concentrations were reduced from 3.0 to 0.1 mg/L (96.7% reduction).Without acetate present, chromate concentrations decreased to 1.2 mg/L after 5.7 days (60% reduction).These results indicate that the amount of carbon source present in the natural soil was sufficient to support microorganisms to partially reduce the chromate.These findings are not surprising-multiple researchers have reported on hexavalent chromium biological reduction in laboratory conditions with isolated, site-specific microbial strain, under a wide range of initial concentrations 10 to 4320 mg/L (Jeyasingh and Philip 2005;Zahoor and Rehman 2009;Somasundaram et al. 2011;Zheng et al. 2015).Similar to our findings, reduction rates for chromate in the literature have been faster than for chlorate.At our site, these are co-contaminants and may have some overlapping, competing mechanisms.
One study that evaluated perchlorate and hexavalent chromium reduction (together) used a perchlorate-enriched facultative anaerobic consortium with the perchlorate and chlorate contamination as electron acceptors, and also successfully (95%) immobilized Cr(VI) at initial concentrations of 16 mg/L (Bardiya and Bae 2005).The study herein differs in several key areas: the authors used different electron donors than those being presented here (the sewage sludge as opposed to acetate) and the facultative anaerobic consortium were derived from sewage sludge as opposed to evaluating bio-stimulation of an in-situ microbial community.Concentrations used in the studies proposed in this paper are similar to the observed environmental concentrations at our site, and the reduction in concentrations is to values that meet Canadian guidelines protective of human and or environmental health.The addition of an electron donors and carbon source (acetate) increases the rate of degradation and will provide an adequate source for the simultaneous degradation of chlorate and chromate.

Flowrate
The flow rate through the column was measured every 12 h by dividing the weight of the collected effluent water by the elapsed time.There was no flow from the columns recorded in the initial 5 h of the experiment; only one of the three columns had measurable effluent after about 16 h.The flow rate gradually increased as the soil became saturated in the columns.After about 80 days, the flow rate from the column feed with acetate and nutrients was consistently lower than that from the control columns (Figure 6).After 136 h, the flow rate of synthetic groundwater through the column system without acetate and nutrients was approximately 0.4 L/week compared to a flow rate of 0.2 L/week in the presence of the amendments.The timing of this flow decrease coincided well with increased reduction of chlorate (Figure 3) therefore was likely associated with increasing microbial biomass in the soil columns.An increase in biomass was expected (Orozco et al. 2010) demonstrated that the net biological chromate removal was the consequence of two processes: an initial rapid chromate removal process associated with the biomass growth, and a secondary slower removal process that was observed to be independent of the presence of both the carbonaceous substrate and the nitrogen source.These two "stages" can also be observed in Figure 7 by the difference in how quickly the initial chromate concentration decreases between the condition with acetate and the condition without.Both test conditions then appear to have a similar, slower degradation process that appears to be independent of added carbon sources and/or nutrients.Another reason for the flow decrease was formation of a brown precipitate which was observed at the base of the feed vessel A (Figure 8), indicating chromate transformation through reduction, and subsequent precipitation.This precipitate was also identified and analyzed in our earlier microcosms study (Motevasselin et al. 2023).Trivalent chromium species were anticipated to be present in this precipitate as the bio-reduction of hexavalent chromium to trivalent chromium often results in a precipitate (Palmer and Puis 1994).
The form of precipitate is influenced by the existing site geochemistry and redox conditions present-reducing conditions (e.g., lower pH and dissolved oxygen) are expected to occur with the addition of the electron donor (acetate).Further study is required to evaluate whether the transfor-  mation from trivalent chromium precipitate reversible with a "rebound" in oxidizing conditions after the acetate is used up.There is a potential risk that transformed trivalent chromium could become a future source for hexavalent chromium (Palmer and Puis 1994;Galani et al. 2022).

Conclusion
The results from this study demonstrated that chlorate and chromium could not be reduced by acetate under sterile conditions.Stimulating microorganism growth was necessary to reduce these ions to more benign forms.Chlorate and chromate-reducing bacteria are ubiquitous in the environment and were present in the contaminated site soil.The experiment with columns packed with that soil showed that reduction of chlorate and chromate in continuous flow systems is possible, similarly to the microcosms tests reported earlier (Motevasselin et al. 2023).The effluent chlorate concentrations in the column effluent streams decreased from initial concentration of 1000 mg/L by 86% (137 mg/L) after 136 h.In the columns that were fed with synthetic groundwater without acetate and nutrients, only 15% of chlorate was removed (846 mg/L remaining).The contaminated soil natural carbon content alone was sufficient to serve as an electron donor to reduce chromate concentration from 3 to 1.2 mg/L.When acetate was added to the synthetic groundwater feed the chromate in the column effluent reached 0.1 mg/L after 136 h.The reduction process resulted in a gradual decrease in flow rate through the columns.
Increased biomass concentration as well as Cr(III) precipitates most likely changed the soil pore structure of in the columns causing a reduction in flowrate.Although the average water velocity in the column experiments in this study were lower (4.671 × 10 −7 m/s) compared to those observed in the field (1.8 × 10 −5 m/s), the aquifer permeability could be affected, potentially creating preferential flow paths, or even obstructing groundwater flow.These are concerns that would need to be considered in the in-situ bioremediation of chlorate-and chromate-contaminated groundwater.

Figure 1 .
Figure 1.Column study setup.A series feed was synthetic groundwater with acetate and nutrients, and B series feed was synthetic groundwater without acetate and nutrients.

Figure 2 .
Figure 2. Changes in chromate (Cr (VI)) and chlorate concentrations in batch microcosm studies under sterile conditions over 30 days.A shows chromate concentrations over time, B shows chlorate concentrations over time (0 days, 30 days).

Figure 7 .
Figure 7. Precipitated material settled at the bottom of feed vessel for columns A series (with acetate).