DEVICE AND METHOD FOR MICROORGANISM CELL DISRUPTION BY EXTRUSION

Abstract

Aspects are provided in relation to devices and systems for microorganism cell wall disruption. In this scenario, a device is provided for cell disruption of a microorganism suspension comprising (i) an inlet duct (1) of microorganisms, (ii) an annular channel (13) downstream of inlet duct (1) and in communication therewith, adapted for disruption of microorganism cells, the annular channel (13) being formed by an external part (7) and an internal part (8), the internal part being positioned inside the cavity formed by the external part (7) and (iii) an outlet duct (9) downstream of annular channel (13) and in communication therewith, for output of the ruptured microorganisms. A method is further provided that is associated with the device described above.

Claims

1. Device for cell disruption of microorganisms by extrusion, characterized in that the device comprises: an inlet duct of a microorganism suspension; an annular channel downstream of the inlet duct and in communication therewith, adapted for disruption of microorganism cells, the annular channel being formed by an external part and an internal part, the internal part being positioned inside a cavity formed by the external part; and an outlet duct downstream of annular channel and in communication therewith, for output of ruptured cells.

2. Device according to claim 1, characterized in that the device further includes a pump adapted to pump a suspension of microorganisms through annular channel.

3. Device according to claim 2, characterized in that the pump is a positive displacement pump positioned in inlet duct.

4. Device according to claim 1, characterized in that the position of the internal part is adjustable with respect to the cavity formed by the external part by means of an adjustment mechanism in order to regulate the diameter of annular channel.

5. Device according to claim 4, characterized in that the adjustment mechanism is at least one pneumatic, hydraulic, mechanical, electric and manual device.

6. Device according to claim 1, characterized in that the device further includes one pressure gauge adapted to check the inlet pressure of the device.

7. Device according to claim 4, characterized in that the device further includes an automated control system adapted to control the adjustment mechanism.

8. Device according to claim 1, characterized in that the internal part includes the cavity positioned near the outlet duct.

9. Device according to claim 8, characterized in that the device includes a sealing element adapted to seal the end of the annular channel.

10. Device according to claim 9, characterized in that the sealing element is positioned at the end of the annular channel opposite the inlet duct.

11. Device according to claim 1, characterized in that the device additionally includes a coolant system, comprising: a cooling jacket positioned around the external part; a coolant input adapted to inject coolant into the cooling jacket; and a coolant outlet adapted to remove coolant from the cooling jacket.

12. Method for cell disruption of microorganisms by extrusion, characterized in that the method includes the steps of: promoting a forced flow of a suspension of microorganisms through an annular channel downstream of an inlet duct and in communication therewith, the annular channel being adapted for disruption of microorganism cells and formed by an external part and an internal part, the internal part being positioned inside a cavity formed by the external part; and passing the ruptured cells through an outlet duct located downstream of the annular channel and in communication therewith.

13. Method according to claim 12, characterized in that the method includes the additional step of adjusting the position of the internal part in relation to the cavity of the external part by means of an adjustment mechanism in order to regulate the diameter of the annular channel as a function of the species and diameter of the microorganisms.

14. Method according to claim 12, characterized in that the method includes the additional step of cooling the suspension of microorganisms inside the annular channel by means of a cooling system.

15. Method according to claim 14, characterized in that the cooling step of the microorganism suspension includes circulation of coolant through a cooling jacket.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0023] The detailed description presented below refers to the attached figure and its respective reference numbers.

[0024] FIG. 1 shows a cross section of the device according to a preferred embodiment of the present invention.

[0025] FIG. 2 shows results of application of the device and method of the preferred embodiment of the present invention, more specifically a result of analysis of flow cytometry in the microalgae Scenedesmus obliquus BR003 without rupture (0) and with a number of passes of 1, 5, 10, 20 and 40.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Preliminarily, it is emphasized that the description that follows starts from a preferred embodiment of the invention. However, the invention is not limited to this particular embodiment.

[0027] FIG. 1 shows a cross section of the device according to a preferred embodiment of the present invention. The cell disruption device of the preferred embodiment of the present invention comprises an inlet duct 1 of a suspension of microorganisms. An annular channel 13 is provided downstream of the inlet duct 1 and in communication therewith, whose size can be adjusted according to the species and diameter of the microorganism, for disruption or cells by extrusion, as will be seen below.

[0028] The stream of the microorganisms is forced into the annular channel 13 so that the cell walls are ruptured by extrusion. The forced flow into the annular channel 13 is preferably promoted by means of a positive displacement pump 2, preferably positioned in inlet duct 1.

[0029] A pressure gauge 3 is preferably provided at any point between point 2 and annular channel 13 to measure the inlet pressure of the device.

[0030] Alternatively, a negative displacement pump (not shown) is used in the device downstream of the annular channel 13 to draw the suspension of microorganisms into the device.

[0031] The annular channel 13 is formed by an external part 7 and an internal part 8, the internal part 8 being positioned inside the cavity formed by the external part 7. Internal part 8 preferably has precisely the same cavity-shaped shape formed by external part 7 so that the annular channel 13 has essentially parallel walls. More preferably, external part 7 has a female truncated cone shape. The internal part 8 in this embodiment has the same truncated cone shape but with a male fitting.

[0032] The internal part 8 is optionally adjustable with respect to the inner cavity formed by the external part 7 by means of an adjustment mechanism 12 to regulate the diameter of annular channel 13. Moreover, the adjustment mechanism 12 can be a pneumatic, hydraulic, mechanical, electric or manual adjustment mechanism. Activation of the adjustment mechanism 12 can also be formed by a combination of at least two types of drive.

[0033] An automated control system is optionally provided to control adjustment mechanism 12.

[0034] The device of the preferred embodiment of the present invention further comprises an outlet duct 9 downstream of annular channel 13 and in communication therewith for the output of ruptured cells.

[0035] Internal part 8 preferably comprises a cavity 10 positioned near outlet duct 9. This cavity 10 has the function of generating a low-pressure zone at this point and directing the flow of ruptured material to outlet duct 9. More preferably, cavity 10 is aligned with the outlet duct, as shown in FIG. 1.

[0036] The device of the preferred embodiment of the present invention preferably also includes a sealing element 11 positioned at the end of the annular channel 13 opposite inlet duct 1 in the vicinity of adjustment mechanism 12. Sealing element 11 is preferably a gasket made of flexible material.

[0037] To avoid an excessive increase in temperature of the microorganism suspension, a cooling system is preferably provided in the device of the preferred embodiment of the present invention. The system comprises a cooling jacket 4 positioned around the external part 7.

[0038] The cooling system also includes a coolant input 5 to inject coolant into cooling jacket 4 and a coolant output 6 to remove coolant from cooling jacket 4. The coolant inlet 5 is preferably positioned longitudinally and transversely away from the coolant output 6 to promote coolant flow throughout virtually the entire cooling jacket 4 and external part 7.

[0039] The external part 7 is preferably made from a heat-conductive material, such as metal, permitting efficient heat exchange between the coolant and the microorganism suspension.

[0040] The preferred embodiment of the present invention also provides a method for disruption of microorganism cells, comprising the steps of: [0041] (i) promoting a forced flow of a suspension of microorganisms through an annular channel 13 downstream of an inlet duct 1 and in communication therewith, the diameter of the annular channel 13 being adapted for cell disruption and formed by an external part 7 and internal part 8, the internal part 8 being positioned inside the cavity formed by external part 7; and [0042] (ii) passing the ruptured microorganisms through an outlet duct 9 located downstream of annular channel 13 and in communication therewith.

[0043] The method of the preferred embodiment of the present invention also preferably includes the step of adjusting the position of the internal part 8 in relation to the cavity formed by the external part 7 by an adjustment mechanism 12 in order to regulate the diameter of annular channel

[0044] The method of the preferred embodiment of the present invention preferably comprises the additional step of cooling the microorganism suspension inside annular channel 13 by means of a cooling system. More preferably, the step of cooling the microorganism suspension includes the circulation of a coolant through cooling jacket 4.

[0045] The preferred embodiment of the present invention therefore provides a device and method for cell disruption of a microorganism suspension by extrusion, using low pressures from 76.5 to 153.0 kgf/cm.sup.2 (75 to 150 bar) and thus reduced energy consumption. The device of the preferred embodiment of the present invention even permits the regulation of the diameter of the annular channel as a function of the diameter and width of the cells of the microorganisms, making it fully and efficiently adapted to disruption of the species of interest.

[0046] The cooling system also prevents the loss of physicochemical properties of the extracted material.

[0047] To demonstrate the efficiency of the proposed device and method, the disruption of cells of the species Scenedesmus obliquus BR003 was carried out with the preferred embodiment of the present invention. Flow cytometry (BD Facsverse, BD Biosciences) analysis was used to check cell disruption. The analysis revealed that a cycle of five passes at a pressure of 127.5 kgf/cm.sup.2 (125 bar) was sufficient to cause damage to the cell structure, reducing the relative size of the cells (FSC—forward scatter, FIG. 2) by about 50% when compared to the control (without disruption).

[0048] In addition to the relative size of the cells, there was also a reduction in granulosity, observed by the parameter SSC (SSC—side scatter, FIG. 2). This parameter depends on the internal complexity of the particle, for example, shape of the nucleus, number and type of cytoplasmic granules and roughness of the membrane. Thus, the reduction of this parameter indicates that there was a reduction in the number of intact cells, producing cell fragments (debris). The statistical analyses shown in Table 1 indicated that the variation in number of passes through the device, between 10 and 40, did not result in a significant difference in relative cell size.

TABLE-US-00001 TABLE 1 SSC-A FSC-A FSC-A FSC-A FSC-A Number of passes average average SD VC (%) median 0 34,806 111,871 50,149 44.8 109,368 1 27,016 73,560 39,442 53.6 66,187 5 23,658 62,318 31,500 50.6 55,724 10 22,739 59,217 29,629 50.0 52,950 20 21,507 57,099 27,496 48.2 51,431 40 21,840 57,063 27,858 48.8 51,025

[0049] It should be noted that the FSC index shown in Table 1 is related to cell size. SSC is related to internal complexity of the cells and SD and VC correspond to standard deviation and variation coefficient (%), respectively.

[0050] Numerous variations on the scope of protection of this application are possible. This reinforces the fact that the present invention is not limited to the particular configurations/embodiments described above.