Description:THE PATENTS ACT 1970
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION
Extraction Process of Polyhydroxyalkanoates (PHA) from PHA-rich mixed culture waste activated sludge (WAS) in a Continuous Mode Reactor system using low cost acid recovery method
APPLICANT
Rigel Bioenviron Solutions Private Limited
Flat 2B, 64 Garia Station Road, Kolkata, Pincode- 700084.
The following specification particularly describes the invention and the manner in
which it is to be performed
Field of the invention:
The present invention relates toengineered Continuous Mode Reactor System for extraction of Polyhydroxyalkanoates (PHA) from PHA-rich mixed culture waste activated sludge (WAS) using low cost acid recovery method.
Background of the Invention:
One of the biggest threats to our planet earth is the environmental pollution caused by continuous piling of plastic waste in land and water. Therefore, It is witnessed tremendous development in recent years in the use of biodegradable polymers to combat concerns over constant plastic waste accumulation. Today, researchers have developed bioplastic alternatives for almost every single conventional plastic material and corresponding application is being inspected. The most prominent polymers are polylactic acid (PLA), PHA (Polyhydroxyalkanoates), PAs (polyamides) etc. which will show promising production capacities in the next 5 years. The global bioplastics production volume will rise from 2.18 million tonnes in 2023 to estimated 7.43 million tonnes at the end of 2028 as per EUBP Market Data Report 2023.
PHAs are the biodegradable bioplastics that can successfully replace conventional petrochemical plastics due to their similar material properties. The natural polymers PHA is an assembly of biodegradable polyesters of varied chain length. The polymeric unit pf PHAs can be denoted by formulae:
Where in 'x' generally ranges from 1 to 5 and n is the polymer chain length. These are produced inside a wide range of microorganisms owing to intracellular carbon and energy storage. Accumulation of PHA granules is guided by the survival mechanism of the bacteria itself. For this reason prompt extraction process is needed to lyse the organism and produce PHA granules.
In current development of industrial practices, the use of mixed microbial cultures or MMCs is important for various bioprocesses. In industrial research, production of PHAs is a recognized example of MMC application. PHA-rich mixed culture waste activated sludge utilisation in PHA production using renewable resources from wastes (as a substitute of fossil fuels) as carbon source has potential to reduce production cost.
Current industrial practice is production of PHA from pure-culture and based on refine feedstock with sterile cultivation settings - these conditions essentially increase the PHA production cost to larger extent. Previously an integrated system of PHA biomass production from dairy waste water was achieved by Patent number 288052 at laboratory pilot scale where regular chloroform extraction process was employed. Other recent examples of attempts on PHA productions from mixed culture system are with municipal wastewater treatment facility, food-processing waste streams and municipal solid waste etc. However, for industrial viability the present invention proceed with acid extraction instead of the chloroform use.
Most importantly, to achieve the target of replacing the conventional plastics, it is necessary to reduce PHA production costs which is otherwise 7 to 10 times higher compared to fossil based plastics. Till date almost 50% of the total PHA production charge mounts from the recovery and purification process. That is why the process of extraction of PHA from cell biomass are determining step for this bioplastic production in a cost-effective and environment-friendly manner. Limiting the use of halogenated solvents, specifically chloroform are essentials for industrial process development. Use of acid and base as lysing step of biomass is thus a promising technique in PHA recovery process. Laboratory scale research has been done by López-Abelairas et.al. [Comparison of several methods for the separation of poly(3-hydroxybutyrate) from Cupriavidus necator H16 cultures, Biochemical Engineering Journal, Volume 93, 2015, Pages 250-259] on acid-base extraction of PHA polymer which are chemically viable for industrial use. The chemical lysis method reported by them is choosen to build the present invention, a novel engineered continuous mode pilot process for extraction of PHA polymer from PHA-rich mixed culture waste activated sludge (WAS).
The present invention is a novel 'engineered continuous mode reactor system' process employing acid digestion of the bacterial biomass for the effective extraction of PHA bioplastic. The novelty lies in the (a) reactor stream design (b) first time continuous mode engineering in PHA extraction instead of batch mode reactor system (c) reporting optimised condition for industrial production plant (d) achievement of more than 50% yield from biomass in industrial mode continuous reactor system (e) industry ready and cost savingdue to the use of continuous mode design (f) integartable to any continuous mode ETP system.
Objectives of the Invention:
The objective of the present invention is to provide an optimised engineered process involvingcontinuous mode reactor trainfor extraction of Polyhydroxyalkanoates (PHA) employing acid lysing methodfrom the host biomass, which is generated from agro-based wastewater treatment plant (ETP).
Another objective of the present invention is to provide a novel process for reducing the PHA extraction cost over other extraction processes in an efficient manner employing a designed continuous yield mode instead of batch mode.
Further objective of the present invention is to provide a user-friendly integrationin a continuous mode operation of a wastewater treatment plant designed to use activated sludge process in producing PHA.
Summary of the Invention:
The present invention is directed to an engineered continuous mode extraction process of Polyhydroxyalkanoates (PHA) from the host biomass, generated from agro-based wastewater treatment processes, in a continuous mode reactor train integrated within a continuous mode PHA production wastewater treatment process. In this designed process, sulphuric acid oflow concentration is used for cell lysis at optimized temperature at a specific optimized retention time followed by alkali treatment and subsequent hypochlorite treatment. This process attains the objective of reducing the PHA extraction cost over other extraction processes in an efficient manner on a continuous yield mode. Process feasibility is demonstrated in its operating ease while integrating in a continuous mode operation of a wastewater treatment plant designed to use activated sludge process in producing PHA. The cell debris after PHA extraction is free from any toxicity/pathogenic activitiesdue to acid digestion of cells.
Brief description of the drawings:
The foregoing summary, as well as the following detailed description of preferred embodiments, are better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, exemplary constructions of the invention areshown in the drawings; however, the invention is not limited to the specific methods and system disclosed. The following drawings depict:
Figure 1: A Schematic process flow diagram on continuous mode PHA extraction engineering
Figure 2: A photographic view of the continuous mode PHA extraction pilot at the industrial site
IndexDescriptionIndexDescription
101Dewatered PHA rich sludge125Alkali tank for regeneration
102Acid mixing tank126Alkali recycle pump
103Mixer1127Spent alkali recycle
104Sludge digestion feed pump (P4)128PHA rich solids
105Sludge digestion tank129Hypochloride treatment tank
106Mixer2130Centrifuge feed pump 3
107Heating oil bath131Centrifuge3
108Overflow132Raw PHA
109Sludge separator133Hypochloride tank for regeneration
110Overflow134Transfer pump
111Mixer3135Acetone wash tank
112Transfer tank136Centrifuge feed pump 4
113Spent acid recycle137Centrifuge 4
114Sludge transfer pump 1138Acetone Tank
115Mixer4139Raw washed PHA
116Neutralization tank140Mixer 5
117Centrifuge feed pump 1141Mixer 6
118Centrifuge 1142Mixer 7
119Centrate disposal to ETP143Mixer 8
120Alkali mixing tank144Mixer 9
121Alkali dosing system145Control Box
122Centrifuge feed pump 2146Electrical Speed controller
123Centrifuge 2147Reflux condenser
124Centrate
Detail description of the invention:
The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items.
It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described.
The disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms.
The present invention discloses a process for extraction of Polyhydroxyalkanoates (PHA) from the host biomass, generated from agro-based wastewater treatment processes, in a continuous mode reactor train integrated within a continuous mode ETP operation process. The optimized extraction process having following steps:
STEP1
The PHA rich mixed culture waste activated sludge (WAS) is dewatered by basket centrifuge [Make Avdoot Centrifuge, 3000 rpm, rotor drum SS304 internal, 0.5 HP] to achieve minimum 65% moisture content prior to entering into the extraction process. This dewatered PHA rich sludge [101] first subjected to acid treatment in acid mixing tank [102] at 5% w/v ratio. Tank [102] is fitted with a mixer [103] [Make: REMI, Teflon coated shaft and impeller, 100 rpm, geared motor 0.5 HP] for proper mixing of the Sulphuric Acid (H2SO4) with water to maintain acid solution concentration of 3.5% (v/v) (0.64 M). The pH adjustment is continuously maintained through a pH controller [Make: E&H] and acid dosing system in the Acid Mixing Tank [102].
STEP2
The mixed acidic biomass is then transferred through sludge digestion feed pump [104] to the sludge digestion tank [105]. The tank [105] is fixed with a mixer [106] and is placed inside an oil bath [107] for temperature control. The temperature is kept at a range of 80oC to 90oC to get the ideal conditions for lysing of the bacterial cells.
The sludge digestion tank [105] is made of borosilicate glass [Make: Borosil®] of total 18 litre capacity and round bottom for uniform heat distribution to the biomass mixed acidic solution. Active volume of the solution was 12 litres, maintaining the hydraulic retention time [HRT] in the acid digester for 6 hours, where the pump [104] is operated at 2 litres per hour [33.33 mL/min]. Feed pump [104] is peristaltic type [Make: Kamoer Model: FX STP 2 WiFi] with operation range of 1 - 120 millilitre per min [mL/min] with 24 hours operation capacity. Mixer [106] [Make: REMI, Teflon coated shaft and impeller, 40 rpm, geared motor 0.5 HP] is a slow speed agitator, which continuously stirs the solution to allow uniform concentration in the tank [105]. Oil bath is fabricated by SB Scientific Works, Kolkata comprising of Stainless Steel [SS] internals and Mild Steel [IS 304] outer body with L&T make electrical switchgears. Oil is light liquid paraffin oil [boiling point 150oC].
Continuous pumping of biomass mixed acid solution to tank [105] allows overflow from the tank [105] of same quantity of liquid which is passed through spiral glass [Make: Borosil®] cooling jacket [108] and subjected to sedimentation by gravity settling in the sludge separator [109]. The sludge separator is a closed type separating funnel made of borosilicate glass [Make Borosil®]
STEP3
In the sludge separator [109] tank the acid digested biomass gets settled while the supernatant acidic overflow [110] enters the transfer tank [112] [Volume 3 litre, top open, Make Borosil®]. This transfer tank is also fitted with a mixer [111] [Make: REMI, Teflon coated shaft and impeller, 100 rpm, geared motor 0.5 HP] to uniformly mix the spent acid. This regenerated spent acid is transferred to acid mixing tank [102] preferably by a pump [not in figure] for further reuse. This process helps in minimizing the acid usage for sludge digestion. The pH is continuously maintained by the acid dosing system and the pH controller [Make E&H]
STEP4
The lysed biomass that is settled at the separation tank [109] bottom, is pumped [114] by solids transfer pump [Make: ROTO, Screw type, Rotor nitryl coated, 1 HP] to the neutralization tank [116]. The tank [116] made of borosilicate glass [Make Borosil®] and has a mixer [115] [Make: REMI, Teflon coated shaft and impeller, 100 rpm, geared motor 0.5 HP] for uniform mixing and washing of the biomass with water, where continuous water addition is done for reducing the residual acid content. From tank [116], the mixture is subjected to centrifugation in to the centrifuge 1 [118] [Make Avdoot Centrifuge, 3000 rpm, rotor drum SS304 internal, 0.5 HP] fed with feed pump 1 [117]. The centrate [119] is disposed to waste treatment facility. The centrifuged solid mass with minimum 65% moisture content is taken to alkali mixing tank [120]. This process consisting small quantity of liquid solutions is optimal to use the basket centrifuge system. However for larger operations solid bowl centrifuge [Make: Penwaltt/ Alfa Laval/ Humbolt/ Equivalent] is recommended.
STEP5
The alkali mixing tank [120] [Make: Reliable MS IS 2062 fabricated, Epoxy coated] where NaOH solution of concentration 0.5 M is fitted with a mixer [140] [Make: REMI, Teflon coated shaft and impeller, 100 rpm, geared motor 0.5 HP] for effective mixing. The tank is connected to an alkali dosing system [121] generating NaOH solution of concentration 0.5 M and feeding to the alkali mixing tank [120]. The pH is controlled by pH controller [Make: E&H] to maintain at pH 10 to attain proper extraction conditions.
STEP6
The mixture is next sent to the centrifuge 2 [123] with the help of a centrifuge feed pump 2 [122] and centrifuged to separate the phases.
STEP7
The supernatant liquid is taken to the alkali tank for regeneration [125] of the alkali. This tank is having a mixer [141] for uniform mixing of spent alkali [127] and transferred for recycling to the alkali mixing tank [120] by alkali recycle pump [126]. The pH adjustment is always maintained through the pH controller installed in the alkali mixing tank.
STEP8
The sediment [128] from the centrifuge [123] [Make Avdoot Centrifuge, 3000 rpm, rotor drum SS304 internal, 0.5 HP] is taken to the hypocloride treatment tank [Make: Reliable MS IS 2062 fabricated, Epoxy coated] [129] fitted with a mixer [142] [Make: REMI, Teflon coated shaft and impeller, 100 rpm, geared motor 0.5 HP]. In this tank [129], NaOCl of concentration 3 % (w/v) dosing is done by NaOCl Dosing System on the recovered biosolids. The residence time is kept 1 hour with room temperature in the Tank [129].
STEP 9
The mixture is transferred for centrifugation to the centrifuge 3 [131] with the help of centrifugal feed pump [130]. The centrate is transferred to the hypochloride regeneration tank [133] which is fitted with a mixer [143]. The recycled NaOCl solution is taken to the hypocloride treatment tank [129] through a transfer pump [134].
STEP 10
The centrifuged solid sediment contains the raw PHA [132] mixed with other proteins which is subjected to acetone wash where the protein remnants are dissolved in the acetone while the pure PHA remains unaffected. Total 3 step acetone wash is required in series for proper protein removal from the PHA. First acetone wash tank [135] is fitted with mixer [144] (Subsequent two step acetone wash tanks are similar in make and not shown in figure). The mixture is subjected to centrifugation [137]. The concentrated solids mass [139] is the extracted washed PHA. The supernatant spent acetone is collected in spent acetone tank and discarded [138]. Acetone wash is done under strict enclosed chamber with proper vent and acetone collection system maintaining air pollution control standard.
Detail of the ratio generated by considering dry biomass is given in Table1. The measured data is generated after complete four months data compiled in the table 1 and its weighted average derived. The Projected PHA bioplastics production (kg/day) is based on the pilot plant operation capacity. The data is shown as per sampling data from Pilot scale PHA production plant for four months period separated in date time scale 1st to 10th (D:1-10), 11th to 20th (D:11-20) and 21st to 30th (in some case 31st , D:21-30) in the table below. Thus , date wise data clubbed in a 10 days time scale on weighted average basis and presented in the table 1.
Table 1: Data achieved from continuous mode PHA extraction system
Period of Test
M: MonthAverage Dry Organic PHA rich sludge production per day (kg/d)MEASURED DATADERIVED DATA
Wet Sludge sample taken for PHA analysis (gm)Dry sludge wt at 110oC (gm) w/o moistureDaily Dry Organic Sludge Load (gm)
APHA extracted (gm)
BPHA% wrt dry organic sludge B/APHA production from System (Kg/d)
M 1: D:1-104.8527041439.12899.45458.4550.97%2.47
M 1: D:11-205.2427001553.24970.77498.7851.38%2.69
M 1: D:21-315.2727081564.99978.12502.4651.37%2.71
M 2: D:1-105.2827011565.76978.60504.2751.53%2.72
M 2: D:11-205.4727001620.051012.53522.2751.58%2.82
M 2: D:21-285.4727001621.801013.62523.8451.68%2.83
M 3: D:1-105.3427011583.42989.63508.8751.42%2.75
M 3: D:11-204.9127001454.71909.19467.7851.45%2.53
M 3: D:21-315.4327001610.431006.52517.0551.37%2.79
M 4: D:1-105.4427081617.701011.06521.0051.53%2.80
M 4: D:11-205.3227011576.69985.43507.2051.47%2.74
M 4: D:21-305.3727001590.99994.37511.9051.48%2.76
Wt. Avg. of above data5.3327011580.05987.53508.0451.46%2.74
The projected production is 2.74 kg/day (median) PHA based bioplastics considering extraction of PHA from 5.55 kg/day (median) dry organic biomass at 51.46% w/w recovery ratio utilizing the said process.
, Claims:The following claims describe the invention:
We claim,
1.An extraction process of Polyhydroxyalkanoates (PHA) from PHA-rich mixed culture waste activated sludge (WAS) in a Continuous Mode Reactor system comprising of a plurality of reactors and atleast a combination of reactors which are interconnected along the flow of the sludge biomass with digestion and extraction chemical systems to execute the following steps:
STEP1:
The said PHA rich mixed culture waste activated sludge (WAS) is dewatered by basket centrifuge to achieve minimum 65¬¬% moisture content prior to entering into the extraction process, the dewatered PHA rich sludge 101 first subjected to acid treatment in acid mixing tank 102 at 5% w/v solid concentration for proper mixing with the Sulphuric Acid (H2SO4) solution of concentration 3.5% (v/v) (0.64 M). The pH adjustment is continuously maintained through a pH controller and acid dosing system in the Acid Mixing Tank 102.
STEP2:
The said mixed acidic biomass is then transferred through sludge digestion feed pump 104 to the sludge digestion tank 105 placed inside an oil bath 107 at a range of 80oC to 90oC to get the ideal conditions for lysing of the bacterial cells, the active volume of the solution was 12 litres, maintaining the hydraulic retention time HRT in the acid digester for 6 hours, where the pump 104 is operated at 2 litres per hour [33.33 mL/min], a continuous pumping of biomass mixed acid solution to tank 105 allows overflow from the tank 105 of same quantity of liquid which is passed through spiral glass cooling jacket 108 and subjected to sedimentation by gravity settling in the sludge separator109.
STEP3
In the sludge separatortank 109 the acid digested biomass gets settled while the supernatant acidic overflow 110 enters the transfer tank 112 with a mixer 111 to uniformly mix the spent acid and this regenerated spent acid is transferred to acid mixing tank 102for further reuse.
STEP4
The lysed biomass that is settled at the separation tank 109 bottom, is pumped 114 by solids transfer pump to the neutralization tank 116 where the biomass is washedwith continuous water addition for reducing the residual acid content, the mixture is subjectedtocentrifugation inthe centrifuge 118 fed with feed pump 117 from the tank 116, the centrate 119 is disposed to waste treatment facility and the centrifuged solid mass with minimum 65% moisture content is taken to alkali mixing tank 120.
STEP 5
The alkali mixing tank120 is connected to an alkali dosing system 121 where NaOH solution of concentration 0.5 M is generated and fedto the alkali mixing tank 120 at pH 10 to attain proper extraction conditions.
STEP6
The mixture is then sent to the centrifuge 123 with the help of a centrifuge feed pump 2 122 and centrifuged to separate the phases.
STEP7
The supernatant liquid is taken to the alkali tank125 for regeneration of the alkali, the spent alkali 127is transferred for recycling to the alkali mixing tank 120 by alkali recycle pump 126.
STEP 8
The sediment 128 from the centrifuge 123 is taken to the hypocloride treatment tank 129 fitted with a mixer 142, NaOCl of concentration 3 % (w/v) dosing in this tank 129 is done by NaOCl Dosing System on the recovered biosolids and the residence time is kept 1 hour with room temperature in the Tank 129.
STEP 9
The mixture is then transferred for centrifugation to the centrifuge 131 with the help of centrifugal feed pump 130, the said centrate is transferred to the hypochloride regeneration tank 133 and the recycled NaOCl solution is taken to the hypocloride treatment tank 129 through a transfer pump 134.
STEP10
The centrifuged solid sediment contains the raw PHA 132 mixed with other proteins which is subjected to acetone wash where the protein remnants are dissolved in the acetone while the pure PHA remains unaffected.
2.The extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1, wherein the pH adjustment is continuously maintained through a pH controller.
3.The extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1, wherein the oil of the said oil bath 107 is light liquid paraffin oil.
4.The extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1, wherein the said acetone wash is done under strict enclosed chamber with proper vent and acetone collection system maintaining air pollution control standard.
5.The extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1, wherein the mixture is subjected to centrifugation 137 and the supernatant spent acetone is collected in spent acetone tank 138 and discarded.
6.The system of the said extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1 is charaterised by
a Tank 102 fitted with an acid mixer 103 and a pH controller,
a Sludge digestion feed pump (P4) 104,
a sludge digestion tank 105 fixed with a mixer 106 and is placed inside an oil bath 107 for temperature control,
a sludge separator tank 109,
a transfer tank 112 fitted with a mixer 111,
a Sludge transfer pump 114,
a Neutralization tank 116 fitted with a mixer 115 and a Centrifuge 118 with centrifuge feed pump 117,
an alkali mixing tank 120 and a pH controller
an Alkali dosing system 121,
a Hypochloride treatment tank 129 fitted with mixer 142 for treating PHA rich solids 128, and a Centrifuge 131 with centrifuge feed pump 130,
a Hypochloride tank 133 fitted with mixer 143 for regeneration of raw PHA 132
a Transfer pump 134
an Acetone wash tank 135 fitted with mixer 144 and a Centrifuge 137 with centrifuge feed pump 136,
an Acetone Tank 138,
a Control Box 145,
an Electrical Speed controller 146 and
a Reflux condenser 147.
7.The extraction process of Polyhydroxyalkanoates (PHA) as claimed in claim 1, wherein the production is 5.55 Kg/day PHA based bioplastics considering 51.46% w/w of biomass.
Dated 22nd January 2024
(Paramita Saha)
Applicant's Agent
IN/PA/2154
