A method for sustained hydroquinone production

Abstract

The present disclosure provides a microbiological process for the production of hydroquinone from benzoquinone in a microalgal system. The method is particularly characterized by the extraction of the hydroquinone product at a specific points in the process followed by re- introduction of the benzoquinone substrate into the microalgal system. The said method of the 0 present disclosure allows sustained production of hydroquinone.

Description

[0001] “A METHOD FOR SUSTAINED HYDROQUINONE PRODUCTION”

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of microbiology. Particularly, the present disclosure provides a microbiological process for the production of hydroquinone from benzoquinone in a microalgal system. The method provided herein allows sustained production of hydroquinone.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Benzoquinones are a class of compounds that have the capability to extract electrons from microalgal systems to get reduced to hydroquinones. Hydroquinones are stores of hydrogen. Technologies exist for the generation of energy in the form of hydrogen or electricity from these hydroquinones.

[0006] Known methods for the conversion of benzoquinones to hydroquinones from photosynthetic systems and extraction of energy from the hydroquinones tend to use an electrochemical cell approach. In this approach, the photosynthetic cell is coated or tethered or in other ways attached or suspended in a compartment where the anode is present. The benzoquinone, upon entering the illuminated photosynthetic cell, gets converted into the corresponding hydroquinone. Post this, the hydroquinone traverses to the anode where it gets oxidized back into its benzoquinone form after donating a pair of electrons to the anodic material resulting in the generation of electric current. This process continues in a cyclical manner. This phenomenon is termed Mediated Electron Transfer (MET). However, this MET process is known to suffer from mass transfer limitations since the benzoquinone has to traverse into and outside the cell repeatedly to sustain this process, limiting the same by mass transfer. Hence, it is desirable to convert the benzoquinone to hydroquinone using a photosynthetic cell and extract the converted hydroquinone from the bulk medium.

[0007] The methods that are available to extract energy from microalgae are led by the art of extraction of biodiesel from these species. However, most variants of this process are disruptive, where the cell is disrupted to extract the biodiesel. A process that is able to extract energy from the cell over long periods of time using the cell as a catalyst is therefore desirable, especially from the perspective of a biorefmery approach, where more than one value added compound of commercial interest can be extracted from the photosynthetic cell. SUMMARY OF THE DISCLOSURE

[0008] Addressing the aforementioned need in the art, the present disclosure provides a method of sustained production of hydroquinone in a microalgal system, comprising: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0009] In some embodiments, the benzoquinone is selected from a group comprising benzoquinone, tertiary butyl benzoquinone, phenyl para benzoquinone, duroquinone or any combination thereof.

[0010] In some embodiments, the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 2,000 pM.

[0011] In some embodiments, the microalgal culture is a photosynthetic microalgal culture.

[0012] In some embodiments, the photosynthetic microalgal culture comprises photosynthetic organism(s) of genus selected from a group comprising Synechococcus, Synechocysiis. Chlorella. or any combination thereof.

[0013] In some embodiments, culture media for the microalgal culture is selected from a group comprising treated or untreated seawater, treated or untreated freshwater or established broth recipes, wherein the broth recipe is selected from a group comprising ASN III medium, BG 11 medium, MES Volvox medium, Bold’s basal medium or any combination thereof.

[0014] In some embodiments, the microalgal culture is maintained at a temperature ranging from about 4°C to about 60°C; at a light intensity ranging from about 3,000 lux to about 50,000 lux; a pH ranging from about 2 to 12; and / or an OD ranging from about 0.05 to 5.

[0015] In some embodiments, the hydroquinone is extracted alone or in combination with unutilized benzoquinone. In some embodiments, the extraction of hydroquinone is performed in one or more steps; and wherein the extraction is performed by unit operation(s) selected from a group comprising sedimentation, filtration, adsorption, centrifugation, flocculation or any combination thereof.

[0016] In some embodiments, the extraction of hydroquinone is performed by adsorption; and wherein the adsorption is performed through activated charcoal C 18 and silica or a combination thereof.

[0017] In some embodiments, the extraction of the hydroquinone is followed by re-introduction of benzoquinone into the microalgal culture.

[0018] In some embodiments, the sustained production of hydroquinone is facilitated for about 2 hours to about 8 hours.

[0019] In some embodiments, yield of hydroquinone is at least about 10% higher than a method where the hydroquinone, optionally in combination with the benzoquinone, is not extracted when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0020] In some embodiments, the extracted hydroquinone is used for generation of hydrogen or electricity, wherein the hydroquinone gets oxidized to its corresponding benzoquinone.

[0021] In some embodiments, the corresponding benzoquinone is recycled back into the method as of sustained hydroquinone production as described above.

[0022] In some embodiments, the generation of electricity is in an electrochemical or a fuel cell.

[0023] In some embodiments, the generation of hydrogen comprises introducing the extracted hydroquinone into a catalyst system(s), wherein the hydroquinone gets oxidized to its corresponding benzoquinone with concomitant production of the hydrogen; and wherein the catalyst system is composed of material(s) selected from a group comprising transition metals, amino acids, protein complexes or any combination thereof.

[0024] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0025] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments, non-limiting in nature, as illustrated with reference to the accompanying figure. The figure together with detailed description below, is incorporated in and forms part of the specification, and serves to further illustrate the embodiments and explain various principles and advantages, where:

[0026] Figure 1 depicts a general schematic of the method of the present disclosure.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] General definitions

[0029] The term “sustained production” in the context of the present disclosure refers to the continuous and long-term production of hydroquinone from a microalgal system.

[0030] The terms “microalgal culture” and “microalgal system” have been used interchangeably to denote cultures of microscopic algae. Said reference encompasses in scope both pure single strain cultures as well as mixed cultures.

[0031] Reference to decrease in “productivity” of the microalgal culture implies decrease in yield of hydroquinone by the microalgal culture.

[0032] Reference to “allowing conversion of the benzoquinone to hydroquinone” in the present disclosure implies allowing the microalgal culture to produce hydroquinone using benzoquinone as substrate, wherein the said conversion is facilitated by providing to the culture optimum conditions for growth.

[0033] Reference to “extraction” of hydroquinone in the present disclosure encompasses extraction of hydroquinone alone or in combination with unutilized benzoquinone, wherein the said extraction may be from the bulk medium, supernatant or from intracellular space, wherein the extraction of the hydroquinone product arising from the method is based on one or more parameters such as but not limited to productivity of the microalgal culture, solubility of benzoquinone added to the microalgal culture or by duration of hydroquinone production by the microalgal culture, such that a continuous and long-term production of hydroquinone is achieved.

[0034] As used herein the phrase “accumulated hydroquinone” refers to the hydroquinone that builds up within the culture tank over a period of time, which interferes with the ongoing reaction by slowing down the reaction rate, altering equilibrium conditions, or negatively impacting the efficiency of the microalgal culture. The phrase “unutilized benzoquinone” refers to the benzoquinone that remains in the microalgal system as unutilized substrate after conversion of benzoquinone added to the system to hydroquinone.

[0035] As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

[0036] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

[0037] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0038] Numerical ranges stated in the form ‘from xto y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0039] The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher / lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0. 1% or less from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0040] As used herein, the terms “include” (any form of “include”, such as “include”), “such as”, “have” (and “having”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0041] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I;

[0042] B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H;

[0043] C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0044] In addition to the above, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0045] Disclosure

[0046] Addressing the afore-defined need in the art for efficient and sustained production of hydroquinone without disruption of cells, the present disclosure provides a method of sustained production of hydroquinone in a microalgal system.

[0047] Specifically, the present disclosure provides a method of sustained production of hydroquinone in a microalgal system, comprising: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0048] In some embodiments, employable examples of the benzoquinone include but are not limited to, tertiary butyl benzoquinone, phenyl para benzoquinone, duroquinone and combinations thereof. Use of other benzoquinones in the above-described method is envisaged in the scope of the present disclosure.

[0049] In a non-limiting, exemplary embodiment, the benzoquinone is selected from a group comprising tertiary butyl benzoquinone and phenyl para benzoquinone or a combination thereof.

[0050] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; wherein the benzoquinone is selected from a group comprising tertiary butyl benzoquinone, phenyl para benzoquinone, duroquinone or any combination thereof; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0051] In a preferred embodiment, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; wherein the benzoquinone is selected from a group comprising tertiary butyl benzoquinone and phenyl para benzoquinone, or a combination thereof; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0052] In some embodiments, the concentration of benzoquinone added to the microalgal culture may vary depending on the specific benzoquinone being used and the batch size of production. In a non-limiting embodiment, the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 2,000 pM.

[0053] In some embodiments, the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 250 pM, about 250 pM to about 500 pM, about 500 pM to about 750 pM, about 750 pMto about 1000 pM, about 1000 pMto about 1250 pM, about 1250 pM to about 1500 pM, about 1500 pM to about 1750 pM, about 1750 pM to about 2000 pM, about 5 pM, about 250 pM, about 500 pM, about 750 pM, about 1000 pM, about 1250 pM, about 1500 pM, about 1750 pM or about 2000 pM, including values and ranges therebetween.

[0054] In some embodiments, the benzoquinone is provided to the microalgal culture in one shot or at regular intervals.

[0055] In a preferred embodiment, the benzoquinone is provided to the microalgal culture in a fixed amount at regular intervals.

[0056] In some embodiments, the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 2,000 pM; and the benzoquinone is provided to the microalgal culture in one shot or at regular intervals.

[0057] In a preferred embodiment, the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 2,000 pM; and the benzoquinone is provided to the microalgal culture in a fixed amount at regular intervals.

[0058] In some embodiments, the microalgal culture is a photosynthetic microalgal culture.

[0059] In some embodiments, the microalgal culture comprises one or more photosynthetic organisms. Examples of such photosynthetic organisms include those of genus such as but not limited to Synechococcus. Synechocystis, Chlorella. In some embodiments, the microalgal culture is a monoculture or mixed culture comprising one or more photosynthetic organisms selected from a group comprising but not limited to Synechococcus, Synechocystis, Chlorella or a combination thereof

[0060] In some embodiments, the microalgal culture comprises microalga Synechococcus elongatus.

[0061] Without intending to be limited by theory, the conversion of the benzoquinone to its corresponding hydroquinone occurs by photoreduction of the benzoquinone to hydroquinone in the microalgal system. In some embodiments, PSII (Photosystem II) in the microalgal system acts as an in vivo molecular catalyst in facilitating the conversion of the benzoquinone to hydroquinone.

[0062] In some embodiments, the conversion of the benzoquinone to hydroquinone is facilitated in the presence of a culture medium comprising treated or untreated seawater or comprising treated or untreated freshwater or established broth recipes including but not limited to ASN III medium, BG 11 medium, MES volvox medium, Bold’s Basal Medium, or any combination thereof.

[0063] In some embodiments, the microalgal culture comprises one or more photosynthetic organisms, wherein the photosynthetic organisms are of genus selected from a group comprising Synechococcus, Synechocystis, Chlorella, or any combination thereof; wherein culture media for the microalgal culture is selected from a group comprising treated or untreated seawater or comprising treated or untreated freshwater established broth recipes including but not limited to ASN III medium, BG 11 medium, MES volvox medium, Bold’s Basal Medium, or any combination thereof.

[0064] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; wherein the microalgal culture is a photosynthetic microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM. In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; wherein the microalgal culture is a photosynthetic microalgal culture; wherein the microalgal culture comprises one or more photosynthetic organisms of genus selected from a group comprising Synechococcus, Synechocystis, Chlorella. or any combination thereof; wherein culture media for the microalgal culture is selected from a group comprising treated or untreated seawater or comprising treated or untreated freshwater or established broth recipes including but not limited to ASN III medium, BG 11 medium, MES volvox medium, Bold’s Basal Medium, or any combination thereof; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0065] In some embodiments, the microalgal culture is maintained at a temperature ranging from about 4°C to about 60°C.

[0066] In some embodiments, the microalgal culture is maintained at a temperature of about 4°C to about 10°C, about 10°C to about 20°C, about 20°C to about 30°C, about 30°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 4°C, about 10°C, about 15 °C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C or about 60°C, including values and ranges therebetween.

[0067] In some embodiments, the microalgal culture is maintained at a light intensity ranging from about 3,000 lux to about 50,000 lux.

[0068] In some embodiments, the microalgal culture is maintained at a light intensity of about 3,000 lux to about 10,000 lux, about 10,000 lux to about 20,000 lux, about 20,000 lux to about 30,000 lux, about 30,000 lux to about 40,000 lux, about 40,000 lux to about 50,000 lux, about 3,000 lux, about 5,000 lux, about 10,000 lux, about 15,000 lux, about 20,000 lux, about 25,000 lux, about 30,000 lux, about 35,000 lux, about 40,000 lux, about 45,000 lux or about 50,000 lux, including values and ranges therebetween. In some embodiments, the microalgal culture is maintained at a pH ranging from about 2 to about 12.

[0069] In some embodiments, the microalgal culture is maintained at a pH of about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, about 11 to about 12, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12, including values and ranges therebetween.

[0070] In some embodiments, the microalgal culture is maintained at an OD ranging from about 0.05 to 5.

[0071] In some embodiments, the microalgal culture is maintained at an OD of about 0.05 to about 0.10, about 0.10 to about 0.50, about 0.50 to about 1, about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 0.02, about 0.05, about 0. 10, about 0.50, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5, including values and ranges therebetween.

[0072] In anon-limiting, exemplary embodiment, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; wherein the microalgal culture is a photosynthetic microalgal culture comprising one or more photosynthetic organisms of genus selected from a group comprising Synechococcus, Synechocystis and Chlorelky wherein culture media for the microalgal culture is selected from a group comprising treated or untreated seawater or comprising treated or untreated freshwater or established broth recipes including but not limited to ASN III medium, BG 11 medium, MES volvox medium, Bold’s Basal Medium or any combination thereof; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; wherein, the microalgal culture is maintained at a temperature ranging from about 4°C to about 60°C, at a light intensity ranging from about 3,000 lux to about 50,000 lux, at a pH ranging from about 2 to about 12, and at an OD ranging from about 0.05 to 5, including values and ranges therebetween.

[0073] Without intending to be limited by theory, in some embodiments, a balance exists between the light intensity, the number of photosynthetic cells (for which OD is a measure) and amount of quinone added. This balance has to be maintained during the process to reduce or even eliminate the wastage of benzoquinone and thus optimize the production of hydroquinone. In a non-limiting embodiment, at a lower OD, the PBQ molecules will be subject to a relatively higher light intensity. Both the cell and PBQ compete for light; the lower the cell concentration, the lower is its competition with PBQ and thereby higher is the light absorption by PBQ, thereby leading to a higher autocatalytic activity.

[0074] In some embodiments, the hydroquinone is extracted alone or in combination with unutilized benzoquinone.

[0075] In some embodiments, the extraction of hydroquinone is performed in one or more steps.

[0076] In some embodiments, the extraction of hydroquinone alone and a mixture of the hydroquinone and the unutilized benzoquinone are performed in separate steps.

[0077] In a non-limiting embodiment, the extraction of hydroquinone alone is performed first followed by extraction of the mixture of hydroquinone and unutilized benzoquinone.

[0078] In some embodiments, the hydroquinone is extracted alone or in combination with unutilized benzoquinone; wherein the extraction of hydroquinone alone and a mixture of the hydroquinone and the unutilized benzoquinone are performed in separate steps; and wherein the extraction of hydroquinone alone is performed first followed by extraction of the mixture of hydroquinone and unutilized benzoquinone.

[0079] In a non-limiting embodiment, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM, wherein the hydroquinone is extracted alone or in combination with unutilized benzoquinone; wherein the extraction of hydroquinone alone and a mixture of the hydroquinone and the unutilized benzoquinone are performed in separate steps; and wherein the extraction of hydroquinone alone is performed first followed by extraction of the mixture of hydroquinone and unutilized benzoquinone.

[0080] In some embodiments, the extraction is performed by unit operation(s) such as but not limited to sedimentation, filtration, adsorption, centrifugation, flocculation or any combination thereof.

[0081] In a non-limiting embodiment, the adsorption may be performed through material(s) such as but not limited to activated charcoal and Cl 8 silica or a combination thereof.

[0082] In a preferred embodiment, the adsorption is performed through activated charcoal.

[0083] In a non-limiting embodiment, the hydroquinone alone is precipitated into the culture medium. Accordingly, in some embodiments, the hydroquinone alone is extracted or separated from the microalgal culture by unit operation(s) such as but not limited to sedimentation, filtration, centrifugation, flocculation or any combination thereof.

[0084] In some embodiments, the extraction of the mixture of the hydroquinone and the unutilized benzoquinone may be performed by unit operation(s) such as but not limited to sedimentation, filtration, centrifugation, flocculation or any combination thereof.

[0085] In some embodiments, the extraction of the mixture of the hydroquinone and the unutilized benzoquinone may be performed by adsorption, wherein the adsorption is performed through material(s) such as but not limited to activated charcoal and C 18 silica or a combination thereof.

[0086] Accordingly, in some embodiments, the extraction comprises extraction of the hydroquinone alone and / or in combination with unutilized benzoquinone in a single or multiple steps; wherein both the extractions are performed by adsorption; and wherein the adsorption is performed through materials including but not limited to activated charcoal, C18 silica column and combinations thereof. In some embodiments, the extraction comprises extraction of the hydroquinone alone and / or in combination with unutilized benzoquinone in a single or multiple steps; wherein the extraction of the hydroquinone alone is performed by unit operation(s) such as but not limited to sedimentation, adsorption, flocculation, centrifugation, filtration or any combination thereof and the extraction of the hydroquinone in combination with unutilized benzoquinone is performed by adsorption; and wherein the adsorption is performed through material(s) such as but not limited to activated charcoal and C 18 silica or a combination thereof.

[0087] In some embodiments, the extraction of the hydroquinone from the microalgal culture may be performed based on parameters such as productivity of the microalgal culture, solubility of benzoquinone added to the microalgal culture or by duration of hydroquinone production by the microalgal culture.

[0088] In some embodiments, the extraction of the hydroquinone from the microalgal culture may be performed when the productivity of the microalgal culture reduces significantly. The present disclosure specifically reports that the productivity of the microalgal culture reduces significantly when the accumulation of hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM. The extraction of the hydroquinone from the microalgal culture, at this stage, ensures improved product flow, reduces yield loss, and enhances conversion efficiency.

[0089] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction of the hydroquinone from the microalgal culture is performed when the accumulated hydroquinone in the microalgal culture is about 400 pM to about 450 pM, about 450 pM to about 500 pM, about 500 pM to about 550 pM, about 550 pM to about 600 pM, about 600 pM to about 650 pM, about 650 pM to about 700 pM, about 400 pM, 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM or about 700 pM, including values and ranges therebetween.

[0090] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to hydroquinone by the microalgal culture; and c) extraction of the hydroquinone from the microalgal culture, wherein the extraction is performed when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; wherein the extraction comprises extraction of the hydroquinone alone and / or in combination with unutilized benzoquinone in a single or multiple steps.

[0091] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to hydroquinone by the microalgal culture; and c) extraction of the hydroquinone from the microalgal culture, wherein the extraction is performed when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; wherein the extraction is performed by unit operation(s) such as but not limited to sedimentation, fdtration, flocculation, adsorption, centrifugation, adsorption or any combination thereof; and wherein the adsorption is performed through materials like activated charcoal, Cl 8 silica or a combination thereof.

[0092] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to hydroquinone by the microalgal culture; and c) extraction of the hydroquinone from the microalgal culture, wherein the extraction is performed when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; wherein the extraction comprises extraction of the hydroquinone alone and / or in combination with unutilized benzoquinone in a single or multiple steps; wherein the extraction is performed by adsorption through activated charcoal. In order to facilitate the sustained production of hydroquinone, after extraction of the hydroquinone, optionally in combination with unutilized benzoquinone, fresh benzoquinone may be re-introduced into the microalgal culture to repeat the method as described above. Accordingly, in some embodiments, the extraction of the hydroquinone is followed by reintroducing benzoquinone into the microalgal culture. This also ensures that the benzoquinone addition regime is in sync with the reduction capacity of the microalgal species since excess addition of the benzoquinone when compared with the reduction per unit time of the same by the microalgal cell may result in uncontrolled precipitation of the benzoquinone in the bulk media. The said feature of the method of the present disclosure allows for a higher reduction and a higher overall yield of hydroquinone from the method. The method of the present disclosure is therefore a continuous process for the production of hydroquinone using benzoquinone as substrate.

[0093] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; and d) repeating steps (a) to (c) at least once.

[0094] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; and d) repeating steps 2 to 8 times. Accordingly, in some embodiments, the extraction of the hydroquinone followed by re- introduction of benzoquinone into the microalgal culture allows sustained production of hydroquinone over at least about 2 hours to about 8 hours.

[0095] In some embodiments, the extraction of the hydroquinone followed by re-introduction of benzoquinone into the microalgal culture allows sustained production of hydroquinone for about 2 hours to about 2.5 hours, about 2.5 hours to about 3 hours, about 3 hours to about 3.5 hours, about 3.5 hours to about 4 hours, about 4 hours to about 4.5 hours, about 4.5 hours to about 5 hours, about 5.5 hours to about 6 hours, about 6 hours to about 6.5 hours, about 6.5 hours to about 7 hours, about 7 hours to about 7.5 hours, about 7.5 hours to about 8 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours or about 8 hours including values and ranges therebetween.

[0096] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; wherein the method allows sustained production of hydroquinone over at least about 2 hours to about 8 hours.

[0097] In some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM; and d) repeating steps (a) to (c) at least once, wherein the method allows sustained production of hydroquinone over at least about 2 hours to about 8 hours.

[0098] In some embodiments, the method of the present disclosure is characterized by extraction of hydroquinone based on the decrease in productivity of the culture . Thus, in some embodiments, the method of sustained production of hydroquinone in a microalgal system, comprises: a) introducing benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to hydroquinone by the microalgal culture; and c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when productivity of the microalgal culture is reduced by at least about 10%.

[0099] In some embodiments, the method of the present disclosure provides at least about 10% higher yield of hydroquinone as compared to a method where the hydroquinone optionally in combination with the benzoquinone is not extracted when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0100] In some embodiments, the method of the present disclosure provides at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% about 85%, about 90%, about 95%, about 100%, higher yield of hydroquinone as compared to a method where the hydroquinone optionally in combination with the benzoquinone is not extracted when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

[0101] In some embodiments, the method of the present disclosure provides at least about 10% higher yield of hydroquinone as compared to a method where the hydroquinone optionally in combination with the benzoquinone is not extracted when productivity of the microalgal culture is reduced by at least about 10%. In some embodiments, the method of the present disclosure provides at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% about 85%, about 90%, about 95%, about 100%, higher yield of hydroquinone as compared to a method where the hydroquinone optionally in combination with the benzoquinone is not extracted when productivity of the microalgal culture is reduced by at least about 10%.

[0102] In some embodiments, the method of the present disclosure enables sustained production of hydroquinone without disruption of the microalgal culture.

[0103] The method of the present disclosure allows sustained production of hydroquinone from the microalgal culture over long periods of time without requiring its disruption. The microalgal system, thus, acts as a catalyst in the method of the present disclosure. Moreover, since the cells remain undisrupted, it provides an opportunity to re-utilize the cells for production and extraction of other compounds, including but not limited to biodiesel. The method of the present disclosure is therefore of particular interest from a commercial perspective since more than one value added compound of commercial interest may be produced using the same culture.

[0104] Further, in some embodiments, the method of the present disclosure may further comprise subjecting the extracted hydroquinone to one or more methods for generation of hydrogen or electricity, wherein such methods may be known in the art. Post the extraction of hydrogen or electricity, the hydroquinone gets oxidized to its corresponding benzoquinone which can thus be utilized again in the method of the present disclosure, thus making the method cyclic. Therefore, in some embodiments, the corresponding benzoquinone formed during the generation of hydrogen or electricity is recycled back into the method of sustained production of hydroquinone as described in the above embodiments.

[0105] In some embodiments, post extraction, the hydroquinone produced by the method of the present disclosure may be used as a fuel in an electrochemical or a fuel cell to generate electricity. Thus, in some embodiments, the extracted hydroquinone is used for generation of hydrogen or electricity, wherein the hydroquinone gets oxidized to its corresponding benzoquinone. In some embodiments, the generation of electricity is in an electrochemical or a fuel cell.

[0106] In a non-limiting embodiment, the generation of hydrogen comprises introducing the extracted hydroquinone into a catalyst system(s); wherein the hydroquinone gets oxidized to its corresponding benzoquinone with concomitant production of the hydrogen; and wherein the catalyst system is composed of material(s) selected from a group comprising transition metals, amino acids, protein complexes or any combination thereof.

[0107] In a non-limiting embodiment, the oxidized benzoquinone is reused in the microalgal system as described above for subsequent reduction.

[0108] It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

[0109] Descriptions of well-known / conventional methods / steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

[0110] EXAMPLES:

[0111] EXAMPLE 1: Effect of accumulated PBQH2 on the yield of the process

[0112] A photosynthetic organism, Synechococcus elongatus UTEX 2973 in BG11 culture media at an OD730 of 0.5 (volume 100 ml) was taken in two separate cuboid photobioreactors (reactors 1 and 2) with an impeller setup. The dimensions of both the reactors were - length, breadth and height - 6.5 cm, 6.5 cm and 7.5 cm respectively (thickness of each side - 0.5 cm). The impeller was a 2-blade impeller with a shaft height of 5 cm and the distance between the far end of the blades was 5 cm. A 12-volt motor was used to drive the impeller to ensure adequate mixing. A 45 W CFL lamp was used as the light source. The light intensity on the side of the reactors facing the light was about 9,000 lux. In reactor 1, about 850 pM of PBQFE was added to the culture initially whereas in reactor 2 no PBQH2 was added. After this, about 300 pM of PBQ was added to each of the reactors. The experiment was monitored for 1 hour after which the extent of reduction of PBQ was ascertained by HPLC (mobile phase - water and acetonitrile in the ratio of 60:40 respectively). The PBQH2 content in reactor 1 was arrived at after correcting for the initial amount of PBQH2 that was added. After about 1 hour, the amount of PBQ reduced in reactors 1 and 2 was about 225. 1 pM and about 289. 1 pM respectively. Thus, the reduction in reactor 2 was about 28.4% greater than that of reactor 1. A lower amount of PBQ was reduced in reactor 1 due to the presence of PBQH2 that was already added before the addition of PBQ.

[0113] The above example demonstrates the effect of accumulated PBQH2 in decreasing the yield of the process.

[0114] EXAMPLE 2: Effect of intermittent removal of accumulated PBQFhfrom the culture on the yield of the process

[0115] Synechococcus elongatus UTEX 2973 in BG11 culture media at an OD730 of 0.5 (volume 100 ml) was taken in two separate cuboid photobioreactors (reactors 1 and 2) with an impeller setup. The dimensions of both the reactors were - length, breadth and height - 6.5 cm, 6.5cm and 7.5 cm respectively. The impeller was a 2-blade impeller with a shaft height of 5 cm and the distance between the far end of the blades was 5 cm. A 12-volt motor was used to drive the impeller to ensure adequate mixing. A 45 W CFL lamp was used as the light source. The light intensity on the side of the reactors facing the light was about 9,000 lux. In reactor 1, about 300 pM of PBQ was added every hour for 4 hours (i.e. total of 1,200 pM). In reactor 2, about 300 pM of PBQ was added every hour for 3 hours (i.e. for a total of 900 pM) followed by extraction at the end of the 3 hours. The extraction process was performed using activated charcoal (granular; supplied by Akshar chem) in an extraction column. The extraction column was cylindrical in shape with an inner diameter of 3.5 cm and a thickness of 3 mm. The height of the column was 13 cm. There was an inlet and an outlet present on the same side of the cylinder at a distance of 1 cm and 7.5 cm from the base respectively. The inlet and the outlet were connected to a 12V Kamoer pump (model EDLP600 - D12A). 10g of activated charcoal granules was enclosed in a wire gauze. The wire gauze was such that it allowed the culture to flow through the activated charcoal without the activated charcoal escaping out of the gauze. This gauze was then placed inside the extraction column and was equidistant from the inlet and the outlet. The culture, after 3 hours of PBQ reduction, was passed through the extraction column by the connected pump for a period of about 15 minutes which removed almost all of the phenyl-p-hydroquinone that was formed.

[0116] In reactor 2, for the first 3 hours, the extent of PBQH2 produced during the 1st, 2ndand 3rdhour, as ascertained by HPLC, was about 234.1 pM, about 198.4 pM and 0 pM (no reduction). The extent of PBQH2 produced thus was about 432.5 pM (i.e. above the 400 pM threshold). After the extraction, 300 pM of PBQ was added to the reactor 2, and the reaction was monitored for 1 hour. During the 4thhour, the amount of PBQ reduced was about 188.7 pM. The corresponding amount of PBQ reduced in reactor 1 (where no extraction was performed) in the 1st, 2nd, 3rdand 4thhour was about 252.7 pM, about 121.1 pM, about 106.1 pM and 0 pM.

[0117] At the end of 4 hours, the cumulative PBQH2 produced in reactors 1 and 2, as ascertained by HPLC, was 479.9 pM and 621.2 pM respectively. As one can see, the amount of PBQ reduced in reactor 2 is 29.4% greater than that reduced in reactor 1.

[0118] The above demonstrates the importance of intermittent extraction of phenyl-p-hydroquinone in increasing the overall yield of the process.

[0119] EXAMPLE 3: Impact of light intensity on productivity of the culture

[0120] Synechococcus elongatus UTEX 2973 in BG11 culture media at an OD730 of 0.5 (volume 100 ml) was taken in two separate cuboid photobioreactors (reactors 1 and 2) with an impeller setup. The dimensions of both the reactors were as follows - length, breadth and height - 6.5 cm, 6.5 cm and 7.5 cm respectively. The impeller was a 2-blade impeller with a shaft height of 5 cm and the distance between the far end of the blades was 5 cm. A 12-volt motor was used to drive the impeller to ensure adequate mixing. A 45 W CFL lamp was used as the light source. The light intensity on the side of reactor 1 facing the light was about 20,000 lux and the intensity on the side of reactor 2 facing the light was about 30,000 lux. About 300 pM of PBQ was added every 30 minutes and after about 1 hour, the cultures from both the reactors were subjected to the extraction process detailed in example 2. This process was repeated for 4 hours. Owing to the autocatalytic activity of PBQ at these light intensities, the amount of PBQ wasted (converted to products other than phenyl-p-hydroquinone) at the end of the 1st, 2nd, 3rdand 4th hour was 0 pM. about 6.6 pM. about 30.9 pM and about 64.3 pM for reactor 1 and, about 33.1 pM, about 105.6 pM. about 157 pM and about 205.4 pM for reactor 2 respectively. This demonstrates that at a given OD730 of the culture and the PBQ addition regime, increasing the light intensity results in wastage of PBQ due to its autocatalytic activity.

[0121] EXAMPLE 4: Impact of OD on productivity of the culture

[0122] Synechococcus elongatus UTEX 2973 in BG11 culture media (volume 100 ml) was taken in two separate cuboid photobioreactors named reactor 1 and 2 at an OD730 of 0.5 and 0.1 respectively with an impeller setup. The dimensions of both the reactors were as follows - length, breadth and height - 6.5 cm, 6.5 cm and 7.5 cm respectively. The impeller was a 2- blade impeller with a shaft height of 5 cm and the distance between the far end of the blades was 5 cm. A 12-volt motor was used to drive the impeller to ensure adequate mixing. A 45 W CFL lamp was used as the light source. The light intensity on the side of reactors 1 and 2 facing the light was about 9,000 lux. About 300 pM of PBQ was added every hour to the reactors for 2 hours and the samples were checked every hour to see if there was any wastage of PBQ due to its autocatalytic activity. The amount of PBQ wasted in reactors 1 and 2 for the 1stand the 2ndhours were 0, 0 pM (reactor 1) and 49 and 82 pM (reactor 2). It was therefore seen that at a given light intensity and the PBQ addition regime, decreasing the OD730 of Synechococcus elongatus UTEX 2973 results in wastage of PBQ due to its autocatalytic activity.

[0123] From examples 3 and 4, it is clear that a balance exists between the light intensity, the number of photosynthetic cells (for which OD is a measure) and amount of quinone added. This balance has to be maintained during the process to reduce or even eliminate the wastage of quinone due to its inherent autocatalytic activity.

[0124] The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the general concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure. Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Claims

We Claim:

1. A method of sustained production of hydroquinone in a microalgal system, comprising: a) introducing a benzoquinone into a microalgal culture; b) allowing conversion of the benzoquinone to its corresponding hydroquinone by the microalgal culture; c) extracting the hydroquinone from the microalgal culture, wherein the extraction is performed when accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

2. The method as claimed in claim 1, wherein the benzoquinone is selected from a group comprising benzoquinone, tertiary butyl benzoquinone, phenyl para benzoquinone, or any combination thereof.

3. The method as claimed in claim 1 or 2, wherein the benzoquinone is added to the microalgal culture at a concentration ranging from about 50 pM to about 2,000 pM.

4. The method as claimed in any of claims 1-3, wherein the microalgal culture is a photosynthetic microalgal culture.

5. The method as claimed in claim 4, wherein the photosynthetic microalgal culture comprises photosynthetic organism(s) of genus selected from a group comprising Synechococcus, Synechocystis, Chlorella. or any combination thereof.

6. The method as claimed in any of claims 1-5, wherein culture media for the microalgal culture is selected from a group comprising treated or untreated seawater or comprising treated or untreated freshwater or established broth recipes, wherein the broth recipe is selected from a group comprising ASN III medium, BG 11 medium, MES Volvox medium, Bold’s basal medium or any combination thereof.

7. The method as claimed in any of claims 1-6, wherein the microalgal culture is maintained at a temperature ranging from about 4°C to about 60°C; at a light intensity ranging from about 3,000 lux to about 50,000 lux; a pH ranging from about 2 to 12; and / or an OD ranging from about 0.02 to 5.

8. The method as claimed in any of claims 1-7, wherein the hydroquinone is extracted alone or in combination with unutilized benzoquinone.

9. The method as claimed in any of claims 1-8, wherein the extraction of hydroquinone is performed in one or more steps; and wherein the extraction is performed by unit operation(s) selected from a group comprising sedimentation, fdtration, adsorption, centrifugation, flocculation or any combination thereof.

10. The method as claimed in claim 9, wherein the extraction of hydroquinone is performed by adsorption; and wherein the adsorption is performed through activated charcoal C 18 and silica or a combination thereof.

11. The method as claimed in any of claims 1-10, wherein the extraction of the hydroquinone is followed by re-introduction of benzoquinone into the microalgal culture.

12. The method as claimed in any of claims 1-11, wherein the sustained production of hydroquinone is facilitated for about 2 hours to about 8 hours.

13. The method as claimed in any of claims 1-12, wherein yield of hydroquinone is at least about 10% higher than a method where the hydroquinone, optionally in combination with the benzoquinone, is not extracted when the accumulated hydroquinone in the microalgal culture is in the range of about 400 pM to about 700 pM.

14. The method as claimed in any of claims 1-13, wherein the sustained production of hydroquinone is without disruption of the microalgal culture.

15. The method as claimed in any of claims 1-14, wherein the extracted hydroquinone is used for generation of hydrogen or electricity, wherein the hydroquinone gets oxidized to its corresponding benzoquinone.

16. The method as claimed in claim 15, wherein the corresponding benzoquinone is recycled back into the method as claimed any of claims 1-14.

17. The method as claimed in claim 15, wherein the generation of electricity is in an electrochemical or a fuel cell.

18. The method as claimed in claim 15, wherein the generation of hydrogen comprises introducing the extracted hydroquinone into a catalyst system(s), wherein the hydroquinone gets oxidized to its corresponding benzoquinone with concomitant production of the hydrogen; and wherein the catalyst system is composed of material(s) selected from a group comprising transition metals, amino acids, protein complexes or any combination thereof.

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