Biodegradation of the Endocrine-Disrupting Chemical 17α-Ethynylestradiol (EE2) by Rhodococcus zopfii and Pseudomonas putida Encapsulated in Small Bioreactor Platform (SBP) Capsules.


Ofir  Menashe*, Yasmin Raizner, Martin Esteban Kuc, Vered Cohen-Yaniv, Aviv Kaplan, Hadas Mamane, Dror Avisar and Eyal Kurzbaum, Applied Sciences 2020.

In this study, we present an innovative new bio-treatment approach for 17α-ethynyestradiol (EE2). Our solution for EE2 decontamination was accomplished by using the SBP (Small Bioreactor Platform) macro-encapsulation method for the encapsulation of two bacterial cultures, Rhodococcus zopfii (R. zopfii ) and Pseudomonas putida F1 (P. putida). Our results show that the encapsulated R. zopffi presented better biodegradation capabilities than P. putida F1. After 24 h of incubation on minimal medium supplemented with EE2 as a sole carbon source, EE2 biodegradation efficacy was 73.8% and 86.5% in the presence of encapsulated P. putida and R. zopfii, respectively. In the presence of additional carbon sources, EE2 biodegradation efficacy was 75% and 56.1% by R. zopfii and P. putida, respectively, indicating that the presence of other viable carbon sources might slightly reduce the EE2 biodegradation efficiency. Nevertheless, in domestic secondary effluents, EE2 biodegradation efficacy was similar to the minimal medium, indicating good adaptation of the encapsulated cultures to sanitary effluents and lack of a significant effect of the presence of other viable carbon sources on the EE2 biodegradation by the two encapsulated cultures. Our findings demonstrate that SBP-encapsulated R. zopfii and P. putida might present a practical treatment for steroidal hormones removal in wastewater treatment processes. https://doi:10.3390/app10010336


LP-UV-nano MgO2 pretreated catalysis followed by small bioreactor platform capsules treatment for superior kinetic degradation performance of 17α-Ethynylestradiol

Lakshmi Prasanna Vaddadi, Dror Avisar*, Vinod Kumar Vadivel, Ofir Menashe, Eyal Kurzbaum, Vered Cohen-Yaniv and Hadas Mamane, Materials 2019

A successful attempt to degrade synthetic estrogen 17_-ethynylestradiol (EE2) is

demonstrated via combining photocatalysis employing magnesium peroxide (MgO2)/low-pressure ultraviolet (LP-UV) treatment followed by biological treatment using small bioreactor platform (SBP) capsules. Reusable MgO2 was synthesized through wet chemical synthesis and extensively characterized by X-ray di_raction (XRD) for phase confirmation, X-ray photoelectron spectroscopy (XPS) for elemental composition, Brunauer-Emmett-Teller (BET) to explain a specific surface area, scanning electron microscopy (SEM) imaging surface morphology, and UV-visible (Vis) spectrophotometry. The degradation mechanism of EE2 by MgO2/LP-UV consisted of LP-UV photolysis of H2O2 in situ (produced by the catalyst under ambient conditions) to generate hydroxyl radicals, and the degradation extent depended on both MgO2 and UV dose. Moreover, the catalyst was successfully reusable for the removal of EE2. Photocatalytic treatment by MgO2 alone required 60 min (~1700 mJ/cm2) to remove 99% of the EE2, whereas biodegradation by SBP capsules alone required 24 h to remove 86% of the EE2, and complete removal was not reached. The sequential treatment of photocatalysis and SBP biodegradation to achieve complete removal required only 25 min of UV (~700 mJ/cm2) and 4 h of biodegradation (instead of >24 h). The combination of UV photocatalysis and biodegradation produced a greater level of EE2 degradation at a lower LP-UV dose and at less biodegradation time than either treatment used separately, proving that synergetic photocatalysis and biodegradation are effective treatments for degrading EE2.


Phenol biodegradation by bacterial cultures encapsulated in 3D microfiltration-membrane capsules

Eyal Kurzbaum, Yasmin Raizner, Martin E. Kuc, Anatoly Kulikov, Ben Hakimi, Lilach Iasur Kruh and Ofir Menashe, Environmental technology, 2019.

Abstract: The aim of the study was to evaluate the performance of batch and semi-continuous treatment systems for phenol degradation using a consortium of bacterial cultures that were encapsulated using the ‘Small Bioreactor Platform’ (SBP) encapsulation method. The maximal phenol biodegradation rate was 22 and 48?mg/L/h at an initial phenol concentration of 100 and 1000?mg/L in the batch and semi-continuous bioreactors, respectively. The initial phenol concentration played an important role in the degradation efficiency rates. The batch bioreactor results could be described by the Haldane model, where the degradation rate decreased under low as well as under very high initial phenol concentrations. Concentration equalization between the two sides of the SBP capsule’s membrane occurred after 80 min. The microfiltration membrane is perforated with holes that have an average diameter of 0.2–0.7?µm. It is therefore suggested that the capsule’s membrane is more permeable compared to other polymeric matrixes used for bacterial encapsulation (such as alginate). This study shows that the encapsulation of phenol degraders within microfiltration-membrane capsules which create a confined environment has a potential for enhancing phenol removal in phenol-rich wastewaters.



Treatment of olive mill wastewater using ozonation followed by an encapsulated acclimated biomass

Yeara Bar Oz, Hadas Mamane, Ofir Menashe, Vered Cohen-Yaniv, Rupak Kumar, Lilach Iasur Kruh, Eyal Kurzbaum, Journal of Environmental Chemical Engineering, 2018

Abstract: The environmental impacts caused by Olive Mill Wastewater (OMWW) are a concern for both developing and developed countries. In this study, an ozone pretreatment combined with a fixed biomass bio-treatment using the Small Bioreactor Platform (SBP) capsules technology encasing a pure culture of a phenol-degrading OMWW isolate named Delftia EROSY was implemented to reduce phenolic compounds and organic matter in OMWW.

Up to 90% of tannic acid (TA), a synthetic phenol model, was removed after the ozonation and biological stages. Ozone pretreatment of TA expedites the biological process by decreasing the time needed for the biodegradation of phenols.

Ozonation (ozone dose?=?765?mg?L−1 O3) of OMWW demonstrated 20% COD and 61% total phenol removal, with an additional 36% increase in COD removal after the biological step (48?h). Interestingly, our results also showed that spectral absorbance can be used as a tool for monitoring ozonation followed by bio-treatment of OMWW. Absorbance results clearly demonstrate that bio-treatment is necessary for degrading not only phenolic compounds, but also phenol transformation products and the high organic load of the OMWW, following the ozonation step.



Encapsulated Pseudomonas putida for phenol biodegradation: Use of a structural membrane for construction of a well-organized confined particle

Eyal Kurzbaum, Yasmin Raizner, Oded Cohen, Ran Y. Suckeveriene, Anatoly Kulikov, Ben Hakimi, Lilach Iasur Kruh, Yair Farber, Ofir Menashe. Water Research, 2017, Vol.121, pages 37-45.

Abstract: Phenols are byproducts from a wide range of industry sectors which, if not treated, create exceptionally environmentally hazardous effluents. This study presents the use of a Pseudomonas putida F1 culture encapsulated within a confined environment particle as an efficient technique for phenol biodegradation. The innovative encapsulation technique method, named the "Small Bioreactor Platform" (SBP) technology, enables the use of a constructed microfiltration membrane as a physical barrier for creating a confined environment for the encapsulated culture. The phenol biodegradation rate of the encapsulated culture was compared to its suspended state in order to evaluate the effectiveness of the encapsulation technique for phenol biodegradation. Encapsulated P. putida exhibited a maximal phenol biodegradation rate at an initial phenol concentration of 100 mg/L, with a biodegradation rate (q) of 2.12 h-1. The biodegradation rate decreased significantly at lower and higher initial phenol concentrations of 50 and up to 3000 mg/L, reaching up to 0.1018 h-1. The results also indicate similar and up to double the degradation rate between the two bacterial states (encapsulated vs. freely suspended). Images of the SBP capsule's membrane morphology using high resolution scanning electron microscopy demonstrated a highly porous microfiltration membrane that enables effcient trafficking of dissolved molecules across the membrane. These results, together with the long-term activity of the SBP capsules and the verification that the culture remains pure after 60 days using 16S rRNA gene phylogenetic affiliation tests, provide evidence for a succsesful application of this new encapsulation technique for bioaugmentation of selected microbial cultures in water treatment processes.



A Novel Bioaugmentation Treatment Approach using a Confined Microbial Environment: A Case Study in a MBR Wastewater Treatment Plant

Ofir Menashe and Eyal Kurzbaum, Environmental Technology, (on-line publication, DOI: 10.1080/09593330.2015.1121293), Jan. 2016.

Abstract: A novel bioaugmentation treatment approach, the Small-Bioreactor Platform (SBP) technology, was developed to increase the biological stabilization process in the treatment of wastewater in order to improve wastewater processing effectiveness. The SBP microfiltration membrane provides protection against the natural selection forces that target exogenous bacterial cultures within wastewater. As a result, the exogenous microorganisms culture adapt and proliferate, thus providing a successful bioaugmentation process in wastewater treatment. The new bioaugmentation treatment approach was studied in a full configuration Membrane Bioreactor (MBR) plant treating domestic wastewater. Our results present the potential of this innovative technology to eliminate, or reduce, the intensity of stress events, as well as shortening the recovery time after stress events, consequently elevating the treatment effectiveness. The effective dose of SBP capsules per cubic meter per day of wastewater was achieved during the addition of 3000 SBP capsules (1.25 SBP capsules per cubic meter per day), which provided approximately 4.5 liters of high concentration exogenous biomass culture within the SBP capsules internal medium. This study demonstrates an innovative treatment capability which provides an effective bioaugmentation treatment in a MBR domestic wastewater treatment plant.


The potential of autochthonous microbial culture encapsulation in a confined environment for phenols biodegradation

Hassan Azaizeh, Eyal Kurzbaum, Ons Said, Husain Jaradat, Ofir Menashe. Environmental Science and Pollutants Research (ESPR), 2015 Vol. 22 (19) pages: 15179-15187.

Olive mill wastewater (OMWW) is claimed to be one of the most polluting effluents produced by agro-food industries, providing high contaminants load that encase cytotoxic agents such as phenolic and polyphenolic compounds. Therefore, a significant and continuous stress episode is induced once the mixed liquor of the wastewater treatment plants (WWTP’s) is being exposed to OMWW. The use of bio-augmentation treatment procedures can be useful to eliminate or reduce such stress episodes. In this study, we have estimated the use of autochthonous biomass implementation within small bioreactor platform (SBP) particles as a bioaugmentation method to challenge against WWTPs stress episodes. Our results showed that SBP particles significantly reduced the presence of various phenolics: tannic, gallic and caffeic acid in a synthetic medium and in crude OMWW matrix. Moreover, the SBP particles succeeded to biodegrade a very high concentration of phenol blend (3000 mg L−1). Our findings indicated that the presence of the SBP microfiltration membrane has reduced the phenol biodegradation rate by 50 % compared to the same suspended culture. Despite the observed reduction in biodegradation rate, encapsulation in a confined environment can offer significant values such as overcoming the grazing forcers and dilution, thus achieving a long-term sufficient biomass. The potential for reducing stress episodes caused by cytotoxic agents through bioaugmentation treatment procedure using the SBP technology is discussed.


Small-bioreactor platform technology as a municipal wastewater additive treatment

Ofir Menashe and Eyal Kurzbaum, Water Science & Technology, 2014, Vol.69 (3), pages 504-510.

The bioaugmentation treatment approach presents both an economical and environmentally friendly solution for wastewater treatment. However, the use of exogenous bacterial cultures presents several limitations: negative interaction between microorganisms and adaptation to new physical and chemical composite environment. These selective forces create a signi?cant challenge for the introduced culture to achieving the required biomass in order to conduct the target biological treatment. Small-bioreactor platform (SBP) technology is aimed at introducing exogenous bacterial culture with some protection to reduce some of the natural selection process. The current study was aimed at validating the use of SBP technology to improve biological treatment, especially during a stress period, by using macro-encapsulated bioaugmentation treatment. The study results indicate that the use of SBP technology elevates the stability of biological treatment, improving operational factors such as the reduction of foaming phenomena and sludge accumulation. Still, a signi?cant study needs to be conducted to understand the potential of this technology; especially the impact on biological treatment by using different types of microorganisms for different types of wastewaters and the relationship between the biomass within the SBP capsules and the natural microorganisms.


 General introduction to the selective bio-treatment process design

The term of bio-augmentation is a generic name for the addition of microorganisms to bioreactors in order to improve the existing biomass activity and performance within the reactor. Bio-augmentation is used for:

  • Preferential degradation of specific compounds (targeting hard-to-biodegrade contaminants and toxic contaminants)
  • Enhancing BOD removal, with emphasis on polymeric BOD
  • Enhancing oil and grease removal, which are considered hard-to-biodegrade COD
  • Enhancing sludge degradation, reducing sludge release
  • Improving settling of solids (sludge) – SSV
  • Enhancing nitrification/de-nitrification
  • Odor reduction
  • Rapid bioreactor start-up
  • Rapid recovery of biomass in the reactor after mortality due to toxicity shocks or other causes
  • Lowering the intensity of bio-process stress episodes

One of the greatest benefits of using a bio-augmentation treatment approach is the ability to target selective contaminants.

However, achieving bio-augmentation treatment success is a challenge, due to several factors that prevent the accomplishment of a sufficient selective biomass that is necessary for performing the desired bio-chemical catabolic process. Selective culture accumulation failure is mainly caused by negative interactions of bio-augmented cultures with the natural local micro-flora (i.e., predators and toxicant secretion) and by continuous dilution of the culture due to constant flow of influents into the bioreactors, which leads to the washing out of the introduced culture.

Bacterial immobilization methods have been developed for overcoming most of the abovementioned obstacles. Encapsulation of bio-augmented bacterial cultures and their immobilization in beds overcome the continuous dilution and also provide some protection against predators found in the mixed liquor. These immobilization methods therefore enable the accumulation and achievement of a sufficient biomass for the degradation of selective contaminants.

Small particles, such as bacteria, present some challenges for their implementation. These include bacterial viability, transportation and site control. The SBP (Small Bioreactor Platform) technology presents a successful macro-encapsulation (2.5 cm long) method for microorganism growth and administration. The key component of the SBP capsules is a structural micro-filtration membrane that enables the generation of a confined aquatic environment for microorganism growth and prosperity. The capsule membrane acts as a physical barrier between the internal medium that holds the particle and the external medium. This membrane enables holding the encapsulated microorganisms inside an aquatic environment in a suspended state, while preventing external predators and other microorganisms from infiltrating into the internal medium of the capsule. The membrane allows water-dissolved molecules to traffic across at an unexpected relatively fast diffusion rate, almost at the same rate as without a membrane. This enables application of the technology to the field of water and wastewater treatment. To the best of our knowledge, the SBP technology presents the fastest biodegradation kinetic rate among current encapsulation formulas. Furthermore, to date no-one has succeeded in developing and manufacturing a large and stable particle (bigger than 10 mm) that can resist hostile environments (high shear forces environment) such as those that exist in the mixed liquor of domestic and industrial bioreactors. The SBP capsule retains its activity for at least two months, after which it degrades into a sugar, and thus leaves no environmental fingerprint. The technology allows us, for the first time, to achieve control of three important microorganism parameters: 1. The culture type that will be introduced. 2. The amount of culture (biomass) that will be introduced. 3. Culture controlling site implementation. We have already demonstrated that the SBP technology enables the targeting of selective contaminants, while avoiding the need for removal of other organic matter that can affect treatment cost.


SBP capsule illustration

The SBP capsule contains several elements: the external barrier made of cellulose acetate microfiltration membrane, aquatic interface which include inside the exogenous inoculums and nutrients supplemental agar. The SBP capsules are marketed in a dry state (inactive product), and need to be activated by hydration, prior the capsule activity. Each host bioreactor can have several thousands of capsules which encase in cartridges or perforated cages. The ratio of SBP capsules numbers to wastewater volume, depends mainly on the target contaminants (type & concentration), and the hydraulic time retention.


Illustration of SBP capsule microfiltration membrane

 Microfiltration membrane encasing the selective bacterial culture within a given medium (mixed liquor). The SBP capsule's microfiltration membrane physically separates the introduced microbial culture inside the capsule from the outer microorganisms (i.e predators) while enabling the trafficking across the membrane of the dissolved molecules and organic matter (i.e phenols, hydrocarbons, nitrogen compounds ect.).

 Mechanisms of action

The SBP technology is an innovative technology integrating engineering and microbiology into a solution that enables us to adapt and accomplish a sufficient biomass of unique bacterial cultures. The SBP capsules encase specific microorganism cultures which specialize in the biodegradation of various contaminants.

Role of the micro-filtration membrane: The SBP capsules are coated with a semi-permeable membrane (micro-filtration membrane) which allows only dissolved molecules and compounds to traverse across the membrane, while the microorganisms are kept inside under favorable microcosmos conditions. The physical barrier provides the exogenous bacterial culture protection against preying and negative interactions associated with wastewater microorganisms (i.e., protozoa, bacteria). This leads to a significant reduction in the natural selection forces against the introduced culture.

The SBP membrane contains two dimensions of molecule-penetration mechanisms: fast flow in the funnel-like pores (large molecules and colloidal particles as well as small molecules) and slower diffusion through the nano-mesh structure, due to the concentration gradient which enables trafficking of small molecules, thus enabling the achievement of a relatively high biodegradation rate. As the bacteria consume the contaminant and mineralize it into carbon dioxide, the internal medium concentration of the contaminant decreases and fresh contaminant molecules diffuse from the outer medium into the the internal medium of the SBP capsules. Consequently, there is no need to expend energy in bringing the contaminants into the SBP capsules. They will automatically traffic into the capsule in accordance with the basic physical law of diffusion. Small molecules will diffuse through the large pores and through the nano-mesh membrane wall which enable a rapid molecules trafficking rate across the SBP membrane.

The SBP capsule membrane has a very low affinity for biofilm formation on its surface, and the membrane channels thus do not become blocked. Our experience has shown only a negligible amount of attached biofilm on the SBP membrane after its immersion in a sanitary mixed liquid for one month.


High resolution scanning electron microscope (HRSEM) images of the capsule's outer surface. Based on the images, it is suggested that the cellulose acetate (CA) membrane exhibits two types of channels: Constructed large pores varying in size from 1 µm to 10 µm in diameter (average ~2 µm). These large pores are present mainly on the surface of the membrane. Since the membrane wall thickness is approximately 400-500 µm, these pores are funnel-shaped and their inner hole has a diameter of approximately 0.8 µm. A higher magnification (X50,000-100,000) reveals that the membrane is constructed in a mesh-like packed structure, with nano-metric channels (~50-150 nm) which are critical for the filtration process.

HRSEM images of the outer surface of the CA SBP capsule: A) X1,000, B) X10,000, C) X10,000, and D) X100,000.

Bacterial culture growth state inside the SBP capsule: To the best of our knowledge, the SBP capsule is the only technology that allows the introduced bacterial culture to grow and proliferate in a suspended/planktonic growth state. This affords several advantages:

  1. Fast metabolic rate.
  2. No physiological alteration of the bacterial cell.
  3. Rapid digestion rate.
  4. Achievement of a high bacterial culture concentration within the SBP capsule.

Advantages 1 and 4 enable us to achieve a rapid biodegradation rate of hours that is sufficient for considering the SBP technology as a practical solution in DWWTPs/SWWTPs/STPs.

Once the bacterial culture reaches a certain concentration (approximately log 9 to log 10 CFU/mL), the bacterial culture enters a stationery growth state where the proliferation rate is approximately equal to the death rate. This mechanism allows long-term (months) activity of the bacterial culture in the presence of nutrients. After a period of a few months (depending on medium type and shear forces intensity), the cellulose membranes degrade into sugar and the SBP capsule vanishes, leaving no environmental fingerprint. Thus, it is not necessary to remove the old SBP capsules from the treated medium. This simplifies the implementation of the SBP treatment. All that is needed is a periodical (once a month) addition of new SBP capsules into the treated medium in order to replenish the encapsulated biomass.

Confined environment: The SBP structural micro-filtration membrane creates a confined aquatic environment for the introduced culture. The confined aquatic environment enables the provision of preferable conditions to the introduced culture. This results in better culture adaptation and persistence within the bioreactor medium and allows rapid achievement of a high concentration of the viable biomass (within hours). Approximately 2 mL of concentrated suspended bacterial culture medium are encased by the micro-filtration membrane, creating a basic unit of activity. The SBP capsules encase an internal water body that is connected to the host aquatic medium by water channeling (i.e., capsule membrane pores). These connecting channels enable the transport of outer nutrients into the confined aquatic medium that encases the introduced bacterial culture. The same mechanism serves for the evacuation of the SBP’s internal waste into the outer medium. The bacterial cells are not immobilized to a polymer, or to some other biological or chemicals matrix. They are suspended within the internal aquatic medium of the SBP capsule. The use of a protected suspended bacterial cell culture is assumed to preserve normal cell physiology and activity.

Dormant bacterial culture activated upon water exposure: The SBP capsules are provided in a dry state with a dormant bacterial culture. Once the SBP capsules are introduced into the water which penetrates into the capsules, the bacterial cells become active. It is assumed that the confined water body within the SBP capsules is similar to the host aquatic medium. The internal biomass therefore has an initial adaptation period (few hours), prior to entering the proliferation stage (log phase).

Nutrient supplementation: The SBP capsules contain some nutrients in order to increase the internal biomass recovery and proliferation rate.

SBP technology implementation: The SBP technology is usually implemented in aerobic bioreactors. Introduction devices are used to introduce several thousands of SBP capsules into the WWTP host bioreactor. The introduction devices are composed of two components: a crane and a perforated cage that encases the SBP capsules. Different wastewater sources require specialized products, tailor-made to treat specific contaminants.


Read more about the comparison between the SBP technology encapsulation method and other macro and micro encapsulation methods.

Read more about the SBP technology in scientific publications in peer-reviewed journals.