A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

Summary

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Abstract

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD¹ and nitrogen species^2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH₄⁺ and NO₃^- concentrations in the feed did not have an effect on NH₄⁺ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH₄⁺ feed concentration was found to be negatively related with NO₃^- removal (n=44, p=0.000) and NO₃^- feed concentration was found to be positively correlated with NO₃^- removal (n=44, p=0.000). Consistent removal of NH₄⁺, combined with the accumulation of oxidized nitrogen species at high NH₄⁺ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

Introduction

Municipal wastewater is commonly treated with activated sludge processes in order to reduce the suspended solids (SS), biological oxygen demand (BOD), organic and inorganic nitrogen, and phosphorous content^5,6. The activated sludge process, a means of secondary wastewater treatment, entails the oxidation of organic carbon in an aeration tank filled with a mixed liquor of incoming wastewater and recycled heterotrophic microorganism (commonly referred to as activated sludge)^5-7. The mixed liquor then enters a relatively large clarifier (settling tank) where the sludge settles for easier collection, to either be disposed or recycled back to the aeration tank, while the clarified, treated wastewater can continue to tertiary treatment or disinfection before being released into receiving waters^5-7. Efficient separation of the treated wastewater and solids (sludge) in the secondary clarifier is essential for the proper function of a wastewater treatment system, as any activated sludge continuing beyond the clarifiers will increase the BOD and SS in the effluent ^5-8.

A number of alternative biological processes exist for secondary treatment of wastewater, which reduce or eliminate the need for large clarifying tanks, including attached-growth (biofilm) reactors, membrane bioreactors (MBRs), and granular sludge reactors. In biofilm reactors, the formation of biofilms, in which microorganisms naturally aggregate and attach as a layer on a solid surface, allows for biomass retention and accumulation without the need for a clarifying tank. Biofilm reactors can be classified into three types: packed bed reactors, fluidized bed reactors, and rotating biological contactors. Packed bed reactors, such as a trickling filters and biological towers, utilize a stationary solid growth surface^5,6. Fluidized bed reactors (FBRs) depend on the attachment of microorganisms to particles, such as sand, granular activated carbon (GAC), or glass beads, which are kept in suspension by a high upward flow rate^9,10. Rotating biological reactors depend on biofilms formed on media attached to a rotating shaft allowing the biofilm to be alternately exposed to air and the liquid being treated^5,6. MBRs use membrane filtration units, either within the bioreactor (submerged configuration) or externally via recirculation (side-stream configuration)^5,11. The membranes serve to achieve good separation of biomass and solid particles from the treated liquid^11,12. Granular sludge reactors are upflow reactors in which the formation of extremely dense and well-settling granules of microorganisms occurs when they are exposed to high superficial air upflow velocities¹³.

As another alternative to the activated sludge process, a novel upflow reactor system, now called a high density bioreactor (HDBR), was designed and built by Sales and Shieh (2006) to study COD removal by activated sludge from synthetic waste streams in low F/M conditions that are known to cause the formation of poor settling sludge (i.e., bulking sludge)^1,7,14. The HDBR system utilized modified fluidized bed reactors that typically consist of an upflow reactor and an external recycle tank. Fluidized bed reactors are typically operated with recycle stream flow rates high enough to keep the biofilm growth substratum suspended but low enough so that the biofilm-covered substrate is retained. Unlike fluidized bed reactors, the HDBR described in Sales and Shieh (2006) used relatively low recycle stream flow rates which, along with external aeration, prevented disruption of the biomass zone formed within the reactor¹. Subsequent studies have demonstrated this reactor design's ability to successfully treat a range of nitrogen fluxes using nitrifying/denitrifying bacteria^3,4. In all studies the formation of a stable, dense biomass zone within the HDBR eliminated the need for an external flocculation/sedimentation process^1-4.

As we report here, the use of the HDBR to grow dense cultures has also been tested in a photobioreactor (PBR) configuration for the cultivation of algae. We discuss the benefits and drawbacks of this novel reactor system for algal cultivation and its potential for overcoming a large hurdle in the commercialization of algal biofuels associated with biomass harvesting (i.e., good solid-liquid separation^15,16). The following protocol outlines the steps needed to assemble, startup, sample from, and maintain an HDBR with algae as the microbial community of interest. Variations in the startup and operation protocol of heterotrophic and nitrifying/denitrifying cultures will also be mentioned. Lastly, general advantages, disadvantages, and unknowns of this novel reactor design will be highlighted.

Protocol

1. Reactor Assembly

1. Arrange the reactor components according to the schematic in Figure 1.
   1. Place the reactor (R) on a mixing plate, add a stir bar to the reactor. Place the recycle tank (RT) beside the stir plate and reactor so that the effluent (top) port of the tank is directed towards the edge of the lab bench.
   2. Place the waste container (W) underneath the effluent (top) port of the recycle tank (RT). Place the feed tank (FT) next to the recycle tank (RT).
      Note: The feed tank has a total capacity of 5 L.
2. Secure the reactor (R) against tipping with an appropriately sized stand and clamp. Likewise, secure the recycle tank (RT) to prevent movement.
3. Insert neoprene peristaltic pump tubing in the recycle (Pump A) and feed (Pump B) pump heads. Refer to the Materials table for additional tubing specifications. Install the pump heads onto the pump drives with the screws provided with the pump drives.
4. Connect Pump A's tubing to the ports on the reactor and the recycle tank. Insert the end of Pump B's tubing into the feed tank and the recycle tank. Connect the top reactor port to the recycle tank with tubing. Apply clamps to the tubing at the reactor ports.
   Note: Photosynthetic communities may benefit from artificial illumination provided by lamps.

2. Preparation of Stock Solutions, Influent/Feed Solutions, and Algae Inoculant

1. Prepare the mineral stock solution. Add the following to a 1 L volumetric flask with 500 ml of deionized water: 200 g sodium bicarbonate, 40 g monobasic potassium phosphate, 4 g magnesium sulfate, 4 g ferric chloride, 4 g calcium chloride, 1 g copper chloride, 1 g cobalt chloride hexahydrate, 1 g nickel chloride hexahydrate, 1 g zinc sulfate heptahydrate. Add an additional 400 ml of deionized water. Swirl forcefully to encourage dissolution of salts. Following dissolution of salts, add deionized water to bring the total volume of solution to 1 L.
2. Prepare the ammonia stock solution. In a 1 L volumetric flask, dissolve 38.214 grams of ammonium chloride in approximately 900 ml of deionized water. After dissolution, add deionized water to bring the total volume up to 1,000 ml.
   Note: 1 ml of stock solution diluted to 1 L yields a 10 mg L^-1 NH₄⁺-N solution.
3. Prepare the nitrate stock solution. In a 1 L volumetric flask, dissolve 72.413 g of potassium nitrate in approximately 900 ml of deionized water. After dissolution, add deionized water to bring the total volume up to 1,000 ml.
   Note: 1 ml of stock solution diluted to 1 L yields a 10 mg L^-1 NO₃^--N solution.
4. Prepare feed/influent solution. To make a feed solution containing 20 mg L^-1 NH₄⁺-N and 20 mg L^-1 NO₃^--N, dilute 2 ml of ammonia stock solution and 2 ml of nitrate stock solution to 1 L total volume. Prior to dilution, add 0.5 ml mineral solution/L of solution being made. Prepare 5 L of influent in total to start up the reactor.
5. Prepare the algae inoculant.
   1. Collect a large volume (at least 10 L) of water from an algae-containing water body such as a stream or pond. Allow the algae to settle by leaving the water samples undisturbed for 24 hr.
   2. Decant and discard the clear (non-algae containing) water at the top of the samples, leaving a concentrated algae suspension within the sample bottles. Combine the algae suspension from all of the samples into one container and repeat the settling and decanting steps.
   3. Measure the biomass within the concentrated sample.
      1. Dry a paper vacuum filter (0.45 µm MCE vacuum filter) and aluminum weigh boat O/N in an oven which has been set to 103 °C After cool-down in a desiccator for 30 min at RT measure the combined mass of the filter and weigh boat.
      2. Vacuum filter 20 ml of concentrated algae suspension and return the filter and weigh boat to oven to dry O/N.
      3. Measure the combined mass of the filter and weigh boat. Calculate the biomass density within the concentrated sample.
         Note: The total volume of water sample that investigators will need to collect will depend upon the source water body.

3. Seeding and Starting the Reactor

1. Add 750 ml of feed solution to the reactor. Fill the recycle tank with 500 ml of feed solution.
2. Use a long pipette to gently add an inoculate suspension containing 1.5 g of algae near the bottom of the reactor. Allow the inoculum to settle to the bottom of the reactor, ensure this by visual observation, before proceeding to the next step.
3. Once the cells have settled, remove the tube clamps and turn on Pump A to a slow flow rate (10 revolutions min^-1/38 ml min^-1). Air trapped in the tubing will be expelled into the reactor.
   Note: The addition of 750 ml to the reactor will prevent any biomass disturbed by the pump from leaving the reactor. Squeeze the tubing to ensure that all air has been expelled.
4. Gradually add feed solution to the recycle tank as the solution is pumped into the reactor. Continue the addition until both the reactor and the recycle tank are at capacity and effluent starts to exit the recycle tank via the top port.
   Note: The volume of feed solution to be added to the recycle tank will vary with the volume of the inoculant added to the reactor.
5. Pour the remaining feed solution into the feed tank.
6. Set the recycle pump (Pump A) to 19 revolutions min^-1, establishing a recycle flow rate of 72.5 ml min^-1. Observe the algae begin to loft from the bottom of the reactor. Using the gradations on the reactor, determine the algae biomass zone height. Ensure that the height is constant before proceeding to the next step.
7. Turn on the mixing plate at very low speed; a setting of 1 or 2 is appropriate to start. The mixing bar will assist in lofting biomass further, but aggressive mixing will cause algae to leave the reactor, enter the recycle tank, and leave in the effluent. Set mixing speed at a setting needed to establish a clear algae boundary within the reactor (Figure 2A); the algal biomass zone should be approximately 10-15 cm in height.
8. Start the feed pump after observing a clear boundary between the algal plug and the reactor fluid. Set the pump to 25 revolutions min^-1, establishing a flow rate of 1.5 ml min^-1. Observe the reactor fluid exit the effluent port due to gravity and displacement caused by the incoming influent stream.

4. Sample Collection and Analysis

1. Carry out sample collection activities prior to performing maintenance on the reactor system. Collect 20 ml of effluent and influent samples daily. Collect effluent samples from within the recycle tank. Collect influent samples directly from the feed tank.
2. Vacuum filter samples to remove suspended solids prior to storage and analysis.
3. Store the influent and effluent samples at -20 °C until further analysis. Limit the number of freeze thaw cycles samples are subjected to. If needed, samples can be split into aliquots to maintain sample integrity.
4. Carry out sample analysis for nitrate, nitrite, and ammonia using standard techniques¹⁷.
   Note: The authors utilized Ion Chromatography (IC) to produce the results presented herein. Refer to the Materials table for specification.

Results

The HDBR was used to cultivate algae over several ratios of influent ammonia and nitrate concentrations, while maintaining a total nitrogen content in the feed at 40 mg –N L^-1. Influent and effluent samples were taken daily; biomass density samples were taken at the beginning and end of each condition. The reactor took on average 3-5 days to reach steady state equilibrium after conditions were changed. Over a wide range of influent conditions a distinct biomass zone was established, as observed previous ...

Discussion

This section will start with a discussion of protocol variations needed to address possible operational issues as well as using different microbial communities. The strengths of this reactor design will be discussed, including the ability to govern control of oxygen flux and the formation of high density flocs within the reactor. Current challenges and possible avenues of investigation will also be mentioned.

Protocol nuances and variations
The operation of HDBRs for cultivation...

Materials

Name	Company	Catalog Number	Comments
Aeration stone	Alita	AS-3015C
Aerator	Top Fin	Air-1000
Ammonium chloride	Sigma Aldrich	A9434
Anion analysis column	Shodex	IC SI-52 4E
Beaker (600 mL)	Corning Pyrex	1000-600	Used as mixing vessel (MV). Addition of hose barbs at the bottom and 500 mL levels. Outside diameter of hose barbs 3/8".
Calcium chloride	Sigma Aldrich	C5670
Cation analysis column	Shodex	IC YS-50
Cobalt chloride hexahydrate	Sigma Aldrich	C8661
Copper chloride	Sigma Aldrich	222011
Ferric chloride	Sigma Aldrich	157740
Filter (vacuum)	Fisherbrand	09-719-2E	0.45 um membrane filter, MCE, 47 mm diameter
Graduated cylinder (1000 mL)	Corning Pyrex	3025-1L	Used as reactor vessel (R). Addition of hose barbs at bottom, 500 mL, and 1 L levels. Outside diameter of hose barbs 3/8".
HPLC/IC	Shimadzu	Prominence
Magnesium sulfate	Sigma Aldrich	M2643
Masterflex L/S variable speed drive	Masterflex	07553-50	Drive for recycle and feed pumps (2 needed)
Nickel chloride hexahydrate	Sigma Aldrich	N6136
Potassium nitrate	Sigma Aldrich	P8291
(Monobasic) Potassium phosphate	Sigma Aldrich	P5655
Pump head	Masterflex	07018-20	Recycle pump head
Pump head	Masterflex	07013-20	Feed pump head
Pump tubing	Masterflex	6404-18	Recycle pump tubing
Pump tubing	Masterflex	6404-13	Feed pump tubing
Sodium bicarbonate	Sigma Aldrich	S5761
Zinc sulfate heptahydrate	Sigma Aldrich	Z0251