AU2021294025A1 - A formulation for stabilization of press mud. - Google Patents

A formulation for stabilization of press mud. Download PDF

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AU2021294025A1
AU2021294025A1 AU2021294025A AU2021294025A AU2021294025A1 AU 2021294025 A1 AU2021294025 A1 AU 2021294025A1 AU 2021294025 A AU2021294025 A AU 2021294025A AU 2021294025 A AU2021294025 A AU 2021294025A AU 2021294025 A1 AU2021294025 A1 AU 2021294025A1
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formulation
press mud
mud
preserving
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Aarohi Atul KULKARNI
Sharad Krishnachandra LALDAS
Aamod Anil NATU
Deepak Pandurang Patil
Shaileshkumar Dhondiram Sawale
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Praj Industries Ltd
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Praj Industries Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Treatment Of Sludge (AREA)

Abstract

The invention relates to a process for the preparation of a formulation for press mud stabilization using combination of microorganisms and enzymes. It particularly relates to increase the stability of press mud 5 for more than 150 days which is further useful for biogas generation.

Description

TITLE: A FORMULATION FOR STABILIZATION OF PRESS MUD.
FIELD OF THE INVENTION
The invention relates to a process for the preparation of a formulation for press mud stabilization using combination of microorganisms and enzymes. It particularly relates to increase the stability of press mud for more than 150 days which is further useful for biogas generation.
BACKGROUND:
Sugar cane is the most important crop from which sugar and ethanol are produced commercially. The bagasse, molasses and sugarcane press mud are the by-products of the sugar industry. Press mud is a by-product obtained after sedimentation/filtration of suspended materials from the cane juice before its crystallization as sugar. It is soft, spongy, amorphous and dark brown solid material. It generally contains 60-85 % moisture, besides sugar, inorganic salts and mineral elements. The chemical composition of press mud varies depending on the cane variety, soil condition, nutrients applied in the field, process of clarification adopted and other environmental factors.
A sugar factory with about 1000 TCD [MT of crushing per day] capacity generates about 35 - 40 MT/day press mud having up to 35 % solids in it. One metric ton of press mud occupies about 56 cubic feet or 2 cubic yards of volume. The pressmud is very useful for agricultural and horticulture crops because of its richness in various micronutrients and hence it is used as a fertilizer after composting it for 45 - 50 days. However, during the season a large amount of press mud is not picked by farmers due to which it keeps on accumulating. In addition, sugar industries are not supposed to discard the press mud into water bodies due to stringent rules and regulation of the pollution control boards. When press mud generated by sugar industries keeps accumulating and if not stored or preserved properly, the carbohydrates and other organic components in it degraded and within few months it has no economic value. However, it has a lot of potential for revenue generation for sugar industries through production of biogas as a fuel. Press mud contains large amounts of biodegradable organic matters, having a good potential to produce biogas. Biogas, a clean and renewable fuel, has vast potential worldwide. It can be a supplement to petroleum products, if used in compressed form in the cylinders. Biogas originates from bacteria in the process of biodegradation of organic materials under anaerobic conditions. It consists of a varying proportion of CH4 (methane) and CO2 (carbon dioxide) and traces of H2S, N, CO, O, etc. The content of CH4 and CO2 is a function of the matter digested and the process conditions like temperature, C/N ratio, etc. Methane is the most valuable component of biogas as a fuel. But the problem with press mud is its stability which directly affects the biogas generation. The press mud generated from the sugar industry and kept accumulatedon the open grounds, starts degrading immediately due action of the natural microbes present in press mud or soil. During this degradation process the moisture and sugar contains reduces leading to non usability of press mud for any further useful activity. The invention presented herein discloses a method for the preparation of a biological formulation for press mud stabilization using combination of two microorganisms and three enzymes, which helps in lowering the exposure of press mud to unfavourable environmental conditions. It increases the stability of press mud when applied to it, for more than 150 days and it is further useful in the process of biogas generation.
DETAIL DESCRIPTION:
In one embodiment of the present invention, the degradation of fresh press mud starts, when left as it is, from day 1 and over next 45 days it gets converted to the compost. This degradation leads to a substantial decrease in carbohydrates, free sugars, fats, total volatile solids and acids present in the press mud, during the period of storage due to natural microbial flora present in it. Here pH of fresh press mud also increased from 4.5 to 8. When such press mud is subjected to biomethanation, the yield of biogas substantially decreases due to the degradation of carbohydrates and organic matters present in the untreated press mud while stored.
In another embodiment of the present invention, a formulation comprising certain enzymes with certain microorganisms is disclosed that stabilizes the fresh press mud, which is then storable for several months with substantial preservation of organic matters present in it. This formulation is designated as PMStab and is in liquid form. The degradation of press mud depends upon the conversion of carbohydrates or sugars present in it to acids by metabolic action of natural microbes during the storage. When treated with PMStab this process of metabolic degradation is substantially reduced leading to maintenance of the quality of press mud in good condition that is suitable for a higher yield of biogas when subjected to biomethanation process. The enzymes like amylase, cellulase and xylanases are used in said PMStab to convert carbohydrate chains to free sugars. The microbe Lactobacillus plantarum (NBRC 3070) used that produces lactic acid as well as plantaricin from the sugars and organic matters of press mud, while microbe Pediococcus pentoseasus (NBRC 3182) also produces pediocin. Plantaricin and pediocin are anti-microbial peptides and help in preservation of the press mud treated with PMStab. The substances such ad lactic acid, plantaricin and pediocin produced by the above bacteria arrest the growth of natural microbial flora in the press mud preventing its deterioration. Said Lactobacillus and Pediococcus culture metabolizes the carbohydrates or sugars which is released by the enzymes of PMStab during the storage period to form organic acids which is useful in biogas generation. Said organic acids are directly converted into biogas and decreases the hydraulic retention time of biogas production plant.
In another embodiment of the invention, a strain of Lactobacillus plantarum (NBRC 3070) is used for the preparation of PMStab formulations. Said bacteria is grown initially in conical flask containing pre-sterilized MRS (deMan, Rogosa, Sharp) medium at about 37 QC for about 24 hours. The culture is routinely transferred and stored at about -80 °C in about 20% glycerol by volume between transfers. The total viable count on MRS agar is about 1.0x1012 cfu/ml. A strain of Pediococcus pentosaceus (NBRC 3182) is also used for the preparation of PMStab formulations. Said bacteria are grown initially in conical flask having MRS medium at about 37 QC for about 24 hours. The culture is routinely transferred and stored at about -80 °C in about 20% glycerol by volume between transfers. The total viable count in MRS medium is about 1.0x1010 cfu/ml. Whenever there is need of PMStab formation, said bacterial cultures are revived from the glycerol stock using MRS broth. About 1 ml of culture from glycerol stock is inoculated in 100ml MRS broth for the seed preparation. Said broth is incubated at about 37 QC for about 24 hours. Said prepared seed is further used for fermentation using MRS broth to prepare required quantity of microbes. Said cultures having desired number of Lactobacillus plantarum cells in between 1.0x108 to 1.0x1012 cfu/ml and desired number of Pediococcus pentosaceus cells in between 1 .0x 1 06 and 1 .0x 1 010 cfu/ml are mixed with the desired amounts of amylases, cellulases and xylanases and sorbitol for the preparation of PMStab formulation. Required quantity of each component is described in TABLE 3 PMStab contains amylase between 6 and 8 g/L having strength of about 3,000 lU/g. It contains cellulase between 0.1 and 0.2 g/L having strength of about 110,000 CMC U/g. It contains xylanase between 0.1 and 0.2 g/L having strength of about 100,000 XU/g. And it contains sorbitol is about between 30 and 50 g/L. In another embodiment of the invention, the press mud is collected from a sugar factory. About one litre of PMStab is used for one metric ton of press mud. Said PMStab is mixed evenly with press mud and stored in airtight condition using high density polyethylene sheets (HDPE sheets) at ambient temperature and used for biogas generation as required. The PMStab treated sugarcane press mud preserves its organic matters for between 100 and 150 days with total volatile matter loss of not more than 3 % by volume. PMStab metabolizes the carbohydrates of press mud and produces organic acids up to twice the initial amount reducing its pH and stabilizing it for long term use. In yet another embodiment of the invention, said PMStab maintained the pH of treated press mud between 4 and 5 and inhibits the growth of bacteria that generally increases the pH above 5. This process limits the degradation of organic matters present in the press mud. It is capable of preserving said sugarcane press mud by inhibiting the reduction of total sugars present in it. It is capable of preserving said sugarcane press mud by preferential conversion of carbohydrates in it to organic acids. It is further capable of preserving said sugarcane press mud between 100 and 150 days, with total volatile solids loss of not more than 3 % in it. It is further sprayed to the sugarcane press mud before its preservation.
In yet another embodiment of the invention, the acclimatized cow dung containing about 4% solid is added into the digester with stabilized press mud. Said digester is maintained at about 37 QC and pH about 7 with agitation speed of about 70 rpm. The biogas production is increased from about 50 to 60 m3 to about 100 to 130 m3 per ton when treated press mud is used. The methane and C02 are about 55 - 62 % v/v and about 25 - 33 % v/v respectively. Hydraulic retention time [HRT] for said biogas production is about 24 days.
Embodiments provided above give wider utility of the invention without any limitations as to the variations that may be appreciated by the person skilled in the art. A non-limiting summary of various embodiments is given above, which demonstrate the advantages and novel aspects of the process disclosed herein.
ADVANTAGES:
1. The stabilized press mud is used as substrate for biogas generation where biogas yield is increased from about 50 to 60 m3 to about 100 to 130 m3 per MT. Methane and CO2 are about 55 - 62 % v/v and about 25 - 33 % v/v respectively.
2. The ratio of methane and carbon dioxide is also better with the stabilized press mud.
3. Biogas generation plant has about 24 days HRT with stabilized press mud compared with unstabilized press mud, it is about 35 days.
4. Due to decrease in HRT, the capital cost of anaerobic digester gets decreased.
5. Due to PMStab, the better quality press mud is available throughout the year which directly leads to increase in days of biogas plant operation, with more than 90% capacity utilization. 6. The post biomethanated waste of stabilized press mud is rich in minerals hence very useful as a soil conditioner for agriculture and horticulture crops.
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by the person skilled in the art. A non-limiting summary of various experimental results is given in the examples, which demonstrate the advantages and novel aspects of the process for preparation PMStab formulations using combination of micro-organisms and enzymes.
EXAMPLE 1 PRESS MUD COMPOSITION
About 40 MT of press mud was collected from a sugar factory for the preservation experiments. Said press mud was analyzed on first day for total solids [TS], total volatile solids [TVS], pH, carbohydrates and organic acids. The complete compositional analysis is given in TABLE 1 below.
TABLE 1 :
EXAMPLE 2:
Press mud samples were kept as-it-is in a storage yard for 150 days and evaluated for compositional analysis. Said compositional analysis is described in detail in TABLE 2.
TABLE 2:
The unpreserved or unstabilized press mud was degraded by its own natural microbial flora. The moisture and TVS of unstabilized press mud were decreased by about 21.55 % and about 36.57 % respectively. Carbohydrates were decreased by about 81.91 % and free sugars and acids were decreased by about 100% with respect to day 1 after the storage of 150 days. The pH of unpreserved press mud also got increases up to 7.2 after 150 days and said press mud was not useful for biogas generation. EXAMPLE 3: PREPARATION OF MICROBIAL STRAINS FOR PMSTAB FORMULATION
A strain of Lactobacillus plantarum (NBRC 3070) was used for the preparation of PMStab formulations. Said bacterium was grown initially in conical flask containing pre-sterilized MRS (deMan-Rogosa- Sharp) medium at about 37 QC for about 24 hours. The culture was routinely transferred and stored at about -80 °C in about 20% glycerol by volume between transfers. The total viable count on MRS agar was about 1.0x1012cfu/ml.
A strain of Pediococcus pentosaceus (NBRC 3182) was also used for the preparation of PMStab formulations. Said bacterium was grown initially in conical flask having MRS medium at about 37 QC for about 24 hours. The culture was routinely transferred and stored at about -
80 °C in about 20% glycerol by volume between transfers. The total viable count in MRS medium was about 1 0x1010cfu/ml.
The bacterial cultures were obtained from NBRC, Japan and revived from the glycerol stock using MRS broth. About 1 ml of culture from glycerol stock was inoculated in 100ml MRS broth for the seed preparation. Said broth was incubated at about 37 QC for about 24 hours. Said seed preparation was further used for fermentation using MRS broth to prepare required quantity of microbes.
EXAMPLE 4: PREPARATION OF PMStab FORMULATION
About 1 liter of PMStab was prepared using commercial enzymes, above microbial culture along with sorbitol. The commercial enzymes were mixed in microbial mixture. Required quantity of each component is described in TABLE 3. TVC count of each microbial culture and unit of enzyme was analyzed before its use.
TABLE 3:
EXAMPLE 5: PRESERVATION OF PRESS MUD
About one liter of PMStab was used for one metric ton of press mud. Said PMStab was mixed evenly with press mud and stored in airtight condition using high density polyethylene sheets (HDPE) at ambient temperature. Said press mud was evaluated after 30 days, 75 days and 150 days for total solids, moisture content, total volatile solids, pH, carbohydrates and acids. Said composition analysisis described in detail in TABLE 4.
TABLE 4:
After 150 days there were significant changes in TVS and the moisture content. The moisture reduction was about 3.41 %. Carbohydrate was decreased about 37.73% and free sugars and acids were decreased about 21.94% and about 40% respectively with respect to Day 1. The pH of preserved press mud was about 4.5. Said stabilized press mud was further used for biogas generation.
EXAMPLE 6: BIOGAS GENERATION ON UNSTABILIZED PRESS MUD
A digester of about 50 L working volume was used in batch mode for biogas generation. The acclimatized cow dung containing about 4% solid was added into the digester. About 1.5 kg of unstabilized press mud was added on 1st, 2nd, 5th and 8th day into the digester. Said digester was maintained at about 37 QC and pH of about 7 with agitation speed of about 70 rpm. The effect of unstabilized press mud on biogas yield, methane, CO2 and hydraulic retention time (HRT) was analyzed after every second day up to 35 days. About 81 m3/MT biogas was generated from un-stabilized press mud having HRT of about 35 days. Said biogas generation was started only after five days. The methane and CO2 were about 45 - 55 % v/v and about 35 - 40 % v/v respectively.
EXAMPLE 7: BIOGAS GENERATION ON STABILIZED PRESS MUD
A digester of about 50 L working volume was used in batch mode for biogas generation. The acclimatized cow dung containing about 4% solid was added into the digester. About 1.5 kg of stabilized press mud was added on 1 st, 2nd, 5th and 8th day into the digester. Said digester was maintained at about 37°C and pH about 7 with agitation speed of about 70rpm. The effect of stabilized press mud on biogas yield, methane, C02 and hydraulic retention time (HRT) was analyzed after every second day up to 27 days. The biogas generation was started on 2nd day. It was about 115 m3/MT and HRT was about 24 days. The methane and CO2 were about 50 - 60 % v/v and about 25 - 33 % v/v respectively.

Claims (14)

1. A formulation for the preservation of a sugarcane press mud comprising:
(a) a desired number of Lactobacillus plantarum cells; (b) a desired number of Pediococcus pentasaceusceWs;
(c) a desired amount of amylase;
(d) a desired amount of cellulase;
(e) a desired amount of xylanase; and
(f) a desired amount of sorbitol in an aqueous medium.
2. The formulation as claimed in claim 1 , wherein said desired number of Lactobacillus plantarum cells is between 1.0 c 108 and 1.0 c 1012cfu/ml.
3. The formulation as claimed in claim 1 , wherein said desired number of Pediococcus pentasaceusceWs is between 1.0 c 106 and 1.0 x 101°cfu/l.
4. The formulation as claimed in claim 1 , wherein said desired amount of amylase is between 6 and 8 g/L having a strength of about 3,000 lU/g.
5. The formulation as claimed in claim 1 , wherein said desired amount of cellulase is between 0.1 and 0.2 g/L having a strength of about 110,000 CMC U/g.
6. The formulation as claimed in claim 1 , wherein said desired amount of xylanase is between 0.1 and 0.2 g/L having a strength of about 100,000 XU/g.
7. The formulation as claimed in claim 1 , wherein said desired amount of sorbitol is about between 30 and 50 g/L.
8. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud by maintaining pH between 4 and 5.
9. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud by inhibiting growth of bacteria that increase pH above 5.
10. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud by increasing the amount of organic acids in it by up to twice the initial amount.
11. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud by inhibiting the reduction of total sugars present in it.
12. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud by preferential conversion of carbohydrates in it to organic acids.
13. The formulation as claimed in claim 1 capable of preserving said sugarcane press mud between 100 and 150 days with total volatile solids loss of not more than 3% in it.
14. The formulation as claimed in claim 1 spray applied to said sugarcane press mud before its preservation.
AU2021294025A 2020-06-18 2021-06-01 A formulation for stabilization of press mud. Pending AU2021294025A1 (en)

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