METHOD FOR INCREASING METHANE YIELD IN ANAEROBIC DIGESTERS BY ADDING CELLULOLYTIC BACTERIA COCKTAIL

(CEBAC-G) PREPARED WITH RUMEN FLUID OBTAINED FROM

HORNLESS GOATS.

Technical Field

This invention is related to a method for increasing methane yield in anaerobic digesters by means of treating lignocellulosic biomass, by adding a cellulolytic bacteria cocktail (cebac-g) prepared from rumen fluid of hornless goats. More particularly, this invention is related to a method for increasing methane yield in anaerobic digesters, by adding a cellulolytic bacteria cocktail (cebac-g) prepared from rumen fluid of hornless goats (Naturally polled goats), into anaerobic digesters that has been developed in order to increase methane yield in a system by increasing the hydrolysis efficiency at a maximum level, in anaerobic digesters treating lignocellulosic biomass, such as animal manure, agricultural waste, energy plants, food residue, sewage sludge, household wastewater and industrial wastewater.

Prior Art

Agriculture and animal husbandry play an important role in the our country's economy in terms of employment opportunities created, exports, input into the industrial sector and meeting national needs. Waste created as a result of activities in the agriculture and animal husbandry sector are used as biomass in anaerobic digesters. In the near future, it is projected to use anaerobic digesters more widely within the framework of integrated biotransformation concept and waste management. Moreover large scale plants that are operated with animal waste/agri cultural waste are present. Besides this, anaerobic digesters are commonly used in Europe. However the most important problem during the anaerobic biodegradation of these wastes which are high in lignocellulosic content is the hydrolysis phase which is considered as a rate limiting step. The recalcitrant characteristic of the biomass makes the hydrolysis of these biomasses is harder and there is a need to apply different strategies such as physical/chemical/biological pre-treatment during anaerobic digestion. The carrying out of hydrolysis with low efficiency prevents from benefiting efficiently from the biomass' energy potential of digesters which also decreases the system methane yield.

Various methods have been developed in order to increase methane yield in anaerobic digesters, in order to eliminate said problems.

The United Nations patent document filed on the date of 23.01.2009 numbered US2009137016 of the known state of the art describes a method which comprises a phase in which the plant biomass is digested anaerobically with at least one kind of a rumen microorganism that effects the anaerobic digestion of a plant biomass. According to the method at least a microorganism is a rumen microorganism and said microorganism is formed of bacteria and fungi.

The Chinese patent document that has been filed on 21.04.2015 numbered CN104845889 of the known state of the art, describes methods of the preparation, storage and application of a rumen microbial agent used for producing biogas and for efficient decomposition of cellulosic organic waste. According to the method, the rumen content which comprises rumen bacteria has been inoculated into the anaerobic fermentation system between 10% to 50% by volume and the pH has been adjusted to 7.0-7.5 and it has been enriched and has been added to organic waste. The Chinese patent document that has been filed on 02.04.2009 numbered CN102459099 of the known state of the art, describes a method which accelerates methane production during anaerobic digestion processes. According to this method, glycerol-digestive microbes or pentose is added to anaerobic digesters and they substantially convert biogas into methane. However the applications mentioned in these documents are not sufficient enough to increase methane production and hydrolysis yield at the desired amounts inside anaerobic digesters treating lignocellulosic waste. As a result, the need to develop the method for increasing methane yield by adding cellulolytic bacteria cocktail (cebac-g) prepared with rumen fluid obtained from hornless goats, to anaerobic digesters subject of the invention has risen.

Objects and Brief Description of the Invention

The aim of the invention is to carry out the method for increasing methane yield in anaerobic digesters by adding cellulolytic bacteria cocktail (cebac-g) prepared from rumen fluid of hornless goats, in order to increase the yield and hydrolysis rate of the anaerobic digester treating lignocellulose-rich waste.

Another aim of the invention is to carry out a method to increase methane yield by adding the cellulolytic bacteria cocktail comprising bacteria species such as Firmicutes, Clostridiaceae, Bacteroidales, Ruminococcaceae, Prevotellaceae, Lachnospiraceae, Synergi stales, Fibrobacter, Lentisphaeria, Spirochaetes and Actinobacteria to anaerobic digesters,.

By means of the method subject to the invention, the hydrolysis rate of lignocellulose-rich waste in the anaerobic digester has been increased.

It has been proved that methane production can be increased significantly, by the addition of a small amount such as 6% of the total microbial community of the digesters, of the specific cellulolytic bacteria cocktail (Firmicutes, Clostridiaceae,

Bacteroidales, Ruminococcaceae, Prevotellaceae, Lachnospiraceae, Synergistales,

Fibrobacter, Lentisphaeria, Spirochaetes, Actinobacteria) that has been developed from the rumen fluid of hornless goats, into anaerobic digesters. The bacteria cocktail that has been added, has improved the hydrolysis phase of wheat straw which is a lignocellulolytic waste, and has increased volatile acid production, and therefore improvement in methane production has also been obtained. Moreover, the findings that have been obtained have shown that the specific cellulolytic bacteria cocktail that has been prepared with the rumen fluid taken from hornless goats, accelerates the hydrolysis phase.

Detailed Description of the Invention

The invention is a method for increasing methane yield by adding cellulolytic bacteria cocktail (cebac-g) prepared with rumen fluid obtained from hornless goats to anaerobic digesters, characterized in that it comprises the steps of; obtaining rumen fluid from healthy hornless goats, obtaining a specific cellulolytic bacteria cocktail by inoculation of the obtained Rumen fluid with Firmicutes, Clostridiaceae, Bacteroidales, Ruminococcaceae, Prevotellaceae, Lachnospiraceae, Synergistales,

Fibrobacter, Lentisphaeria, Spirochaetes and Actinobacteria bacteria species adding the obtained specific cellulolytic bacteria cocktail into digesters.

Ruminant animals, such as sheep, cows, goats, llamas, etc, have the capacity to digest lignocellulosic structures of the plant material found in their diet, by means of their special digestive systems. The diverse microbial communities inside their stomach which is formed of four sections, play an effective role in converting the lignocellulosic structures which are difficult to digest into compounds that can be used by these animals. Although the bacterial community structure of rumen fluid is generally the same, the abundance of these communities are different in the rumen fluid.

Cellulolytic bacteria have been enriched in the method subject to the invention, with serial culturing methods using special medium inoculated with the rumen fluid obtained from a healthy hornless goat and therefore a specific cellulolytic bacteria cocktail (cebac-g) has been prepared. The bacterial community of the rumen fluid obtained from hornless goats comprises 45% Bacteroidetes, 27% Spirochaetes, 19% Firmicutes, 7% Tenericutes, 1% Fibrobacters, and %1 other bacteria. The rumen fluid has been inoculated into the specific medium that has been developed for this method and rapid enriching studies have been carried out. The contents of the obtained cellulolytic bacteria cocktail (cebac-g) are as follows: 17% Firmicutes, 12% Clostridiaceae, 11% Bacteroidales, 11% Ruminococcaceae, 8% Prevotellaceae, 7% Lachnospiraceae, 6% Synergistales, 6% Fibrobacter, 5% Lentisphaeria, 5% Spirochaetes, and 4% Actinobacteria. The ratios that have been provided are the volumetric ratios of the bacteria species inside biomass. According to an application of the invention, in the case that the bacteria species used have the lowest possible percentage ratio, the mixture may comprise different types of bacteria besides said bacteria.

In order to obtain maximum performance from anaerobic digesters microorganisms that play a role during the steps of hydrolysis, acidogenesis, acetogenesis and methanogenesis need to present in the system in certain amounts and they need to function in a symbiotic relationship. The method has been planned within this scope. Besides this, when the ratios to be used were being designed, the applicability thereof in large scale systems, and the competitive relationship thereof with the present microbial community has been taken into consideration. For this reason, the specific bacteria cocktail has been added to digesters at a rate of 2%,4% or 6% of the total microbial community of the digesters. It has been determined that all three rates have a positive effect on methane yield methane production has increased by 35% within the system where 6% of specific cellulolytic bacteria cocktail.

It has been proved that methane production can be increased significantly, by the addition of a small amount such as 6% of the total microbial community of the digesters, of the specific cellulolytic bacteria cocktail that has been developed by means of the rumen fluid obtained from hornless goats, into anaerobic digesters. The bacteria cocktail that has been added, has improved the hydrolysis phase of wheat straw which is a lignocellulosic waste, and has increased volatile acid production, and therefore improvement in methane production has also been obtained. Moreover, the findings that have been obtained have shown that specific cellulolytic bacteria cocktail accelerates the hydrolysis phase.