The present disclosure relates to the technical field of biotechnology renewable energy, and more specifically, to a method for producing citric acid by degrading roughage with natural symbiotic mixed culture. The mixed culture YakQH5 is composed of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibacter gottschalkii). It was collected in the China General Microbiological Culture Collection Center on Mar. 9, 2020, with the collection number of CGMCC No. 19299. The mixed culture YakQH5 can degrade 15 kinds of roughage respectively and produce a large amount of citric acid. Especially when alfalfa is used as substrate, the yield of citric acid is as high as 46.0 mm. Adding compound antibiotics in the fermentation process can also prevent the mixed culture from being polluted by bacteria in the fermentation process and further improve the efficiency of anaerobic fermentation. The mixed culture YakQH5 has important industrial application value.

This disclosure provides a genetically-modified bacterium from the genus Pseudomonas that produces itaconate or trans-aconitate. The disclosure further provides methods for producing itaconate or trans-aconitate using a genetically-modified bacterium from the genus Pseudomonas.

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; d) whereby less than 7.5 mg enzyme composition/g glucan (on dry matter and enzyme as protein) or less than 3.0 mg enzyme composition/g feedstock (on dry matter and enzyme as protein) is used; and e) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and f) optionally recovery of a fermentation product;
wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; d) whereby less than 7.5 mg enzyme composition/g glucan (on dry matter and enzyme as protein) or less than 3.0 mg enzyme composition/g feedstock (on dry matter and enzyme as protein) is used; and e) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and f) optionally recovery of a fermentation product;
wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; d) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and e) optionally recovery of a fermentation product; wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.

The invention relates to a process for the preparation of a sugar product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; and d) optionally recovery of a sugar product;
wherein during part of the time of the enzymatic hydrolysis, oxygen is added to the ligno-cellulosic material and during part of the time of the enzymatic hydrolysis less oxygen is added to the ligno-cellulosic material compared to the other part of the time of the enzymatic hydrolysis, preferably no oxygen is added to the ligno-cellulosic material.

The invention relates to a process for the preparation of a sugar product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; and d) optionally recovery of a sugar product;
wherein during part of the time of the enzymatic hydrolysis, oxygen is added to the ligno-cellulosic material and during part of the time of the enzymatic hydrolysis less oxygen is added to the ligno-cellulosic material compared to the other part of the time of the enzymatic hydrolysis, preferably no oxygen is added to the ligno-cellulosic material.

Disclosed is an Aspergillus niger seed continuous culture method, comprising the steps of: (1) at a startup stage, Aspergillus niger spores are inoculated into a seed culture medium to obtain a seed liquid; (2) at a seed continuous culture stage, continuous dispersion treatment is performed on the seed liquid obtained in step (1), continuous culture is performed on the seed liquid obtained by dispersion, and meanwhile, a fresh seed feed medium is replenished; and (3) at a stop stage, the replenishment of the fresh seed feed medium and the dispersion treatment are stopped, continuous culture is performed to obtain a seed liquid, and then the seed liquid is transferred into the fermentation medium for fermentation culture. The method according to the present invention makes breakthrough to solve problems that multi-cellular filamentous bacteria grow slowly and mycelium pellets are easy to lose in continuous culture, thus fully achieving seed continuous culture.

Disclosed is an Aspergillus niger seed continuous culture method, comprising the steps of: (1) at a startup stage, Aspergillus niger spores are inoculated into a seed culture medium to obtain a seed liquid; (2) at a seed continuous culture stage, continuous dispersion treatment is performed on the seed liquid obtained in step (1), continuous culture is performed on the seed liquid obtained by dispersion, and meanwhile, a fresh seed feed medium is replenished; and (3) at a stop stage, the replenishment of the fresh seed feed medium and the dispersion treatment are stopped, continuous culture is performed to obtain a seed liquid, and then the seed liquid is transferred into the fermentation medium for fermentation culture. The method according to the present invention makes breakthrough to solve problems that multi-cellular filamentous bacteria grow slowly and mycelium pellets are easy to lose in continuous culture, thus fully achieving seed continuous culture.

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps: a) optionally pre-treatment of the ligno-cellulosic material; b) optionally washing of the optionally pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61; d) whereby less than 7.5 mg enzyme composition/g glucan (on dry matter and enzyme as protein) or less than 3.0 mg enzyme composition/g feedstock (on dry matter and enzyme as protein) is used; and e) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and f) optionally recovery of a fermentation product;
wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.