RO130691A2 - Process for obtaining bioethanol from starchy plant wastes with microbial consortia - Google Patents

Abstract

The invention relates to a process for preparing bioethanol from starchy plant wastes. According to the invention, the process consists in the hydrolysis of starch from starchy wastes up to glucose units with an enzymatic complex containing α-amylase, glucoamylase and pullulanase, biosynthetized by recombinant mutants of the species Pichia pastoris.

Description

Process for obtaining bioethanol from plant waste I have donated with microbial consortia

The present invention relates to a biotechnological process for obtaining bioethanol from starch plant waste in which the hydrolysis of starch is carried out in a single phase with an enzymatic complex produced by a consortium of microorganisms.

There is a growing demand worldwide for supplementing classic fuel (gasoline) with other energy contributors, who, if they will not completely replace gasoline, will at least reduce it by additional.

In some countries bioethanol has become an additive in some gasoline products (1). Also, the use of bioethanol reduces greenhouse carbon dioxide emissions.

Starchy vegetable waste, including potato shells that can accumulate in huge quantities, can be an important raw material base in bioethanol production. The ethanol obtained from the treatment of potato waste has a branched structure and several enzymes are needed for its release into glucose units. These are: α-amylase. glucoamylase, pullulanase. Another source of accessible starch waste is the maize processing waste and we are talking about coarse grains, grits, corn kernels. The starch obtained from the processing of maize waste has a linear structure, with only amylase and glucoamylase required for hydrolysis at glucose units. Alcoholic yeast is added and the process continues under anaerobic conditions until the total carbon source consumption (below 1% reducing sugar) (2).

The increasing demand for ethanol is also considering other industrial purposes such as the solvent industry, cleaning and preserving agents, thus supplementing the chemical synthesis of petrochemical substrates.

Thus, the depletion of oil resources leads to the need to find other renewable energy resources through the use of waste products from agriculture, the food industry, or biodiesel.

(4) The processing of potatoes and their by-products by liquefaction and sugaring using three commercial enzymatic products is communicated, processes followed by fermentation with alcoholic yeast.

The present invention enhances the thorough knowledge of the characteristics of the microorganism Pichia pastoris (Guilliermond) Phaff (1956) that ferment glucose and assimilate ethanol and glycerol (5), being especially strong methylotroph.

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This microorganism has become of considerable interest for biotechnology due to the ability of its genetic system to produce a wide range of proteins of medical and industrial importance. Also, Pichia pastoris can be a stable host for heterologous genes.

The process according to the invention uses three strains of Pichia pastoris, recombinant mutants obtained by the Department of Technical Sciences and Natural Sciences - Sapientia Miercurea Ciuc University. These are carriers of heterologous genes as follows:

for α-amylase YPD + zeacin with pPICZ α-amyl; for glucoamylase YPD + zeacin with pPICZA a-glu; for pullulanase Pichia pastoris III with the indicative pPICZ a + GA;

These Pichia pastoris strains will perform α-amylase, glucoamylase and pullulanase biosynthesis responsible for the dextrinization and saccharification stages in the process of producing ethanol from starch wastes.

The process according to the invention utilizes a consortium of three recombinant microorganisms of the species Pichia pastoris that produce an enzyme complex (a-amylase, glucoamylase. Pullulanase) by cultivation on an inoculum medium containing: glycerin, corn extract, KH ₂ PO ₄ , yeast extract, MgSO ₄ and a biosynthesis medium containing: potatoes, corn + flour, KH ₂ PO ₄ , NaCl, citric acid, MgSO ₄ , trace elements, MeOH. The enzymatic complex obtained is added to the fermentation medium, carrying out the hydrolysis of the starch, followed by the anaerobic fermentation of ethanol producing with a consortium of three microorganisms of the genus Saccharomyces.

3 strains of Saccharomyces cerevisiae are used as alcoholic microorganisms: ICCF 310. ICCF 312, ICCF 386, from the collection of microorganisms of industrial interest of ICCF.

The process according to the invention brings as novelty in the process of recovery of starch waste the following advantages:

commercial enzymes are not used, but an enzyme complex produced in a single bioprocess;

the entire technological process is based on microbial consortia, both in the process of obtaining the enzymes necessary to access the bioconversion substrate, as well as in the main stage of ethanol synthesis;

In detail, the process according to the invention comprises the following steps:

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Pichia pastoris I (for α-amylase), Pichia pastoris II (for glucoamylase) and Pichia pastoris III (for pullulanase) are maintained under laboratory conditions (+4 ° C) on an agarized nutrient medium containing% (w / v) : yeast extract 1; bactopeptone 2; glucose 2; agar-agar 2; pH 6.5. These cultures represent the stock culture but also the pre-inoculum for the initiation of a fermentation process or any other selection or conservation action.

The inoculation phase is the liquid culture phase in which the micro-organisms multiply rapidly and can be taken up for the next bioprocess phase. The liquid inoculum medium was designed based on the species characteristics described above, namely: a rapidly assimilable source of carbon is glycerol. Thus, this medium contains% (w / v): glycerol 2; corn extract 1.5; ΚΉ2ΡΟ4 1.5; yeast extract 0.5; MgSO ₄ 7 H ₂ O 0.05, pH 6.3-6.5, 100 ml medium / 750 ml Erlenmayer flask. Incubation 20 h at 28 ° C with rotary shaking.

From this culture the 7% (v / v) bioprocess medium is inoculated in the next step for enzyme synthesis. The bioprocess medium was designed to be assimilable and induce enzyme biosynthesis (α-amylase, glucoamylase and pullulanase) by the action of heterologous genes carried by these recombinant mutants. This medium contains% (w / v): potato 7; corn 1 + corn break 1; corn extract 5.5; citric acid 0.1; NaCl 0.5; MgSO ₄ 7 H2O 0.05; CaCfl 0.02 + mineral salts: FeSO ₄ 7 H ₂ O 0.001; ZnSO ₄ 7 H ₂ O 0.001; CuSO ₄ 7 H ₂ O 0.0005; methanol 0.4 at 0 h and 24 hours.

The liquid inoculaes collected by Pichia I, II and III in optimal development phase (rotating shaking 20 h at 28 ° C) are passed in the bioprocess environment described above and parameters of agitation, aeration are established, so that the oxygen level dissolved in the environment not to fall below 20%. The process takes 72 hours and the final fermentation medium contains the newly biosynthesized enzyme complex α-amylase and glucoamylase and pullulanase.

The final biosynthesis medium contains an α-amylase concentration. glucoamylase, respectively, pullulanase expressed in units / ml (U / ml) of enzyme activity (AE) protein (P) expressed in mg / ml and specific activity (AS) in U / mg protein.

The determination of enzymatic activity is performed using starch substrate.

The final medium is processed by filtration on celite and concentration by ultrafiltration on membranes with separation limit 10 kDa and 5 kDa. The concentrated volume is stored by freezing.

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The alcohol phase follows the following steps: starch wastes are mechanically processed (minced) and boiled for about 10 minutes. The required amount of enzyme complex is added as follows:

- for potato waste, an enzymatic complex consisting of aamylase, glucoamylase, pullulanase is used;

- an enzyme complex comprising α-amylase and glucoamylase is used for waste from maize processing (milling + grain breaking).

The working parameters are as follows:

0-4 hours continuous stirring at 30 ° C, pH 6;

4-8 hours continuous stirring at 40 ° C, pH 5;

8-24 hours continuous stirring at 50 ° C, pH 5.

Mineral salts% (w / v) are added:

ammonium sulphate 0.07;

monopotassium sulphate 0.07;

magnesium sulfate 0.035.

The pH is adjusted to 4.5 - 5; 30 minutes sterilization at 110 ^° C;

Incubation is carried out with a consortium of Saccharomyces cerevisiae and Saccharomyces carlsbergensis obtained on a liquid inoculum medium containing% (w / v): glucose 4 and pepton 1, pH 6.7. Development is done by incubation at 30 ° C, with continuous stirring for 18-20 hours. It is worth mentioning that each strain of Saccharomices is cultivated separately, and inoculation in the next phase is done with a total inoculum of 7% (v / v). The use of the three strains of Saccharomyces has the role of microbial potentiation of reducing sugar consumption.

The fermentative process is totally anaerobic, having the following parameters:

- 0-2 hours continuous stirring for the stimulation of the yeast population at the passage from the inoculum to the biosynthesis process;

- 2-30 hours intermittent shaking, 5 minutes every 4 hours.

Working temperatures are: 28 ° C for starch wastes from corn processing and 30 ° C for potato starch wastes.

The consumption of reducing sugar (compared to the initial value) and the methanol dosage produced on the basis of biotransformation of reducing sugar resulting from processing of potato waste are periodically dosed.

The following examples are given:

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Example 1

The process according to the invention cultivates Pichia I and Pichia II recombinant mutants on an agarized nutrient maintenance medium containing% (w / v): yeast extract 1; pepton 2; glucose 2; agar-agar 2; pH 6.5, sterilized at 110 ^° C, 30 minutes. Static incubation at 30 ° C for 48 hours. Development takes place on the beach.

The nutrient liquid media are incubated with suspensions from the above cultures at a ratio of 10%. The liquid inoculum medium contains% (w / v): glycerin 2; corn extract 1.5; K.H2PO41,5; yeast extract 0.5; MgSO ₄ 7 H ₂ O 0.05. Incubation 18-20 h with continuous stirring at 2830 ⁰ C.

A liquid culture of Pichia I and Pichia II respectively is developed with the following final determinations: glycerol 0.3 mg / 100 ml; biomass as optical density (DO) at 570 nm (1:50) 27; pH 5.87.

These Pichia I and Pichia II liquid cultures are inoculated in a ratio of 7% (v / v) in the specific environment for amylase and glucoamylase (% g / v) biosynthesis:

Potato processed by grinding and boiling 7.5

Corn flour 2.5

Corn extract 6

Citric acid 0.1

NaCl 0.5

CaCl ₂ 0.02

Oligolously:

FeSO ₄ 7H ₂ 0-0.001

ZnSO.i 7 H ₂ O - 0.001

CuSO ₄ 7 H ₂ O - 0.005

Methanol 1.2% (v / v) (0.4% (v / v) is added initially, 24, 48 hours, as it is consumed in its entirety during that period).

The working parameters are: 100 ml medium / 750 ml bottle; permanent stirring 250 rpm; temperature 28-30 ° C; duration 72 hours.

The final biosynthesis medium of ot-amylase and glucoamylase has the following characteristics:

- AE α-amylase 2.09 U / ml -AE glucoamylase 0.71 U / ml

- pH 6.98

- DO 24.3

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The enzyme complex obtained from biosynthesis is isolated and concentrated by filtration and ultrafiltration. The biosynthesis media from 50 vials are combined, resulting in a volume of 5 1 which is filtered on an adjuvant layer of celite, the obtained filtrate (4.4 1) being concentrated by ultrafiltration on a Millipore module (with PLCGC-Pellicon cellulose membrane having the exclusion limit of 10 kDa and a filter surface of 0.5 m ² )

Two phases are obtained: concentrated I (0,5 1) and filtrate (3,9 1). The enzymatic activities in I. concentrate are: a-amylase 3.8 U / ml, glucoamylase 1.6 U / ml. As part of the enzyme has also passed into the filtrate, it is concentrated by ultrafiltration on the PESU (polyethersulfone) membrane, with the exclusion limit of 5 kDa and a filtration surface of 0.5 m ² (Biomax Membranes). An enzyme concentrate II (0.4 1) is obtained with the following enzymatic activities: a-amylase 3.9 U / ml and glucoamylase 1.8 U / ml. The two concentrates I and II are combined (enzymatic complex I) and are used in the next step of hydrolysis of corn starch waste (dough and grain breakage) to obtain bioethanol.

Example 2

Proceed according to example 1, cultivating the three enzyme-producing microorganisms: Pichia I (mutant for the production of α-amylase). Pichia II (mutant for the production of glucoamylase) and Pichia III (mutant for the production of pullulanase)

The final biosynthesis environment has the following characteristics:

AE α-amylase 2.19 U / ml

AE glucoamylase 0.81 U / ml

AE pullulanase 0.35 U / ml

The enzymatic complex obtained from biosynthesis (6 1) is isolated and concentrated by filtration and ultrafiltration according to example 1. Following the concentration the final enzymatic activities are: for concentrate I (0.94 1): a-amylase 3.7 U / ml, glucoamylase 1.7 U / ml. pullulanase 2.7 U / ml. and for concentrate II (0.36 1) are α-amylase 3.9 U / ml, glucoamylase 1.9 U / ml. pullulanase 2.91. The two concentrates I and II are combined (enzyme complex II) and are used in the next step of hydrolysis of potato starch waste (potato peel) to obtain bioethanol.

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Example 3

The process according to the invention uses starch vegetable waste from corn (grain and yolk splits).

Pichia I and Pichia II are cultivated according to example 1. The working phases are the following:

- 25g starch wastes from corn (crack and grind) + 35 ml of water boil about 3-5 minutes.

- An enzymatic complex I (65 ml) consisting of α-amylase and glucoamylase is added. The process of enzymatic hydrolysis of starch takes place in the first 4 hours at 30 ° C, pH 6, for the action of α-amylase and between 4-24 hours at 50 ° C, pH 5, to allow glucoamylase to continue hydrolysis of starch for alcoholic microorganisms. by its biotransformation into reducing sugar, as a substrate for alcoholic microorganisms.

To achieve the alcoholic phase next to the carbon sources, mineral salts are added as follows (% g / v):

ammonium sulphate 0.07;

monopotassium sulphate 0.07;

magnesium sulfate 0.035.

The pH is adjusted to 4.5-4.7, sterilized 30 minutes at 110 ° C.

For the alcohol phase, the medium prepared above is inoculated with a consortium of microorganisms Saccharomyces cerevisiae 1, Saccharomyces cerevisiae 2, Saccharomyces carlsbergensis in a ratio of 7% (v / v).

These are grown separately in the pre-inoculum and inoculation phase, and inoculation in the next phase is done with a total inoculation of 7% (v / v). The fermentation process takes 48 hours and is carried out with the following parameters:

0-2 hours aeration (continuous shaking of the bottle) 28 ° C;

2-24 hours without stirring, anaerobic, 28 ° C.

The biochemical indicators are the following:

- Initial glucose concentration 9% (w / v). The yield of starch bioconversion to glucose is 62%.

-Bioethanol produced (dosage by gas chromatography) 4.76%. The yield of alcoholic fermentation is 52.88%.

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Example 4

The process according to the invention uses starch waste from agri-food waste (crtofi shells).

Pichia I, Pichia II and Pichia III are cultivated according to example 2. The working phases are the following:

kg starch potato waste (potato peel) + 1090 ml water boil about 3-5 minutes.

An enzyme complex (1700 ml) consisting of α-amylase + glucoamylase + pullulanase is added. The process of enzymatic hydrolysis of starch takes place in the first 4 hours at 30 ° C, pH 6, to act α-amylase, between 4-8 hours at 40 ° C, pH 5, for the action of pullulanase. and between 8-24 hours at 50 ° C, pH 5, for glucoamylase to continue hydrolysis of starch by its bioconversion into reducing sugar.

Then add the mineral salts and proceed according to Example 3.

The biochemical indicators are as follows:

-Initially 6.7% glucose concentration (w / v). The yield of starch bioconversion to glucose is 51.53%.

-Bioethanol product (gas chromatography dosage) 3.31% (w / v). The yield of alcoholic fermentation is 49.40%.

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Claims (4)

A process according to the invention in which the enzymes required for the hydrolysis of starch from starch wastes to glucose units are obtained in the form of an enzyme complex (α-amylase, glucoamylase and pullulanase), in a single bioprocess with a consortium of mutant microorganisms. of the species Pichiapastoris. The process according to claim 1, wherein Pichia I Pichia II and Pichia III are grown separately on an inoculum medium containing% (w / v): glycerin 2; corn extract 1.5; K.H2PO4 1.5; yeast extract 0.5; MgSO ₄ 7 H ₂ O 0.05. 3. Process according to claims 1 and 2, wherein Pichia I and Picha II or Pichia I, Pichia II and Pichia III developed in the inoculation phase are passed to the enzyme biosynthesis phase containing% (w / v): potato processed by grinding and boil 7.5; corn meal 2.5; corn extract 6; citric acid 0.1; NaCl 0.5; CaC12 0.02. trace element FeSO ₄ 7 H2O - lmg / lOOml; ZnSO ₄ 7 H ₂ O - lmg / lOOml; CuSO ₄ 7 H2O 0.5mg / 100ml, MeOH 4 (% v / v). The process according to claim 1, 2 and 3, in which bioethanol is obtained from: - corn starch waste (crack and yellowing) with an enzyme complex containing α-amylase and glucoamylase, resulting from biosynthesis with Pichia I and Pichia II recombinant mutants; - potato starch waste (potato peel) with an enzyme complex containing α-amylase, glucoamylase and pullulanase biosynthesized by recombinant mutants Pichia I, Pichia II and Pichia III.