Universal or “customized” enzyme cocktail for sugar production

Currently available commercial enzyme preparations are limited in number and composition and have generally been optimized for acid-pretreated stover from maize and other grasses.

However, lignocellulosic feedstocks for sugar production include a variety of biomass: woody biomass such as softwood and hardwood, waste paper; non-wood biomass such as grass stovers (e.g., maize, sorghum, switchgrass or Miscanthus); and other biomass materials such as corn cobs, dried distillers’ grains solubles. All these biomass have different chemical composition, which will response differently to different pretreatments (e. acidic, basic, or oxidation), resulting in pretreated biomass with different chemical composition, even sugar composition. Therefore a general enzyme cocktail may work well for one type biomass or one type of pretreated biomass but not efficiently for other biomass. For example, for grass biomass with branchy arabinose and glucuronic acid, it may need GH10 endo-xylanase, a-arabinosidase, and a-glucuronidase. For woody biomass, in addition to the key endo-and exo-glucanases and endo-xylananse, it may need ferulate esterase and beta-mannanase. For acid pretreatment at a temperature >160 C, some of biomass hemicelluloses will be removed but some lignin may condense or re-deposit on fiber surface; while alkaline pretreatment will delignify but may have hemicelluloses re-deposited on the fiber surface. All of these will impact the efficiency of enzymatic hydrolysis and may require specifically modified enzyme cocktails. Therefore, a modified “customized” enzyme cocktail is more appropriate to adapt the spectrum of biomass and pretreatment combination for target hydrolysis.

A good research paper presents a study on this issue.

In planta expression of highly thermostable enzymes

In planta enzyme expression uses plants instead of microbial bioreactors as a “factory” to produce industrial hydrolytic enzymes for biofuel production. The enzymes that are active at typical and ambient plant growth temperatures will hurt plant normal growth/phenotypes and produce shriveled seeds as reported in the research paper.


However, in planta expression of thermophilic enzymes have been suggested as a method to reduce the detrimental effects on plants as these enzymes have higher temperature optima than encountered during plant growth. As a result, the plant stover can be pretreated at a relatively high temperature to induce their activation during processing.


Since a typical enzyme cocktail will require a combination of different class of enzymes to work together to destroy the cell wall for hydrolysis, is it possible to find all the key enzymes with thermophilibility and express them into a single plant without impacting plant growth?

Hemicelluloses for fuel ethanol-A good source of information but what else can we get?

A good summary in this review paper with the following aspects:

• various hemicelluloses structures present in lignocellulosic biomass
•  the range of pre-treatment and hydrolysis options including the enzymatic ones
•  the role of different microbial strains on process integration aiming to reach a meaningful consolidated bioprocessing
• the recent trends, technical barriers and perspectives of future development are highlighted.

So far, all the review paper on biomass pretreatments cover almost the same: list the acid, alkaline, wet oxidation, organosolv, ionic liquids pretreatments and their advantages and disadvantages, based on which comes a conclusion: no ‘‘ideal” pre-treatment.

 Actually there do exist good pretreatments, which are effective under low temperature and less by-products. In addition, a milder pretreatment with an efficient process configuration is able to overcome some drawbacks.

The pretreatment I have is conducted at a temperature lower than 120 C and near neutral alkaline conditions, which significantly reduce up-front capital cost and operational cost as well as the chemicals required for neutralization. In addition, a simple process configuration is used for more practical application, which will make it more economical competitive.

My advice: do not just look at these review papers when developing your own pretreatments. They are good sources and references. Understand the fundamentals first and jump out of the box. Remember: be simple for the process.

The latest MIT Sloan report on "Sustainability: The‘Embracers’ Seize Advantage"

Here is the latest MIT Sloan report on "Sustainability: The‘Embracers’ Seize Advantage"


Is it "W" shape for sustaibable business development and investiment?

The first engineered trait designed for the ethanol industry

The news: A corn called Enogen,  the first crops genetically engineered to contain a hydrolytic trait that has been approved or commercial growing by the Department of Agriculture. The crop with this self-hydrolysis trait will increase ethanol output while reducing the use of water, energy and chemicals in the production process.

The advantages of in planta cell wall degrading enzyme expression

1. Can  produce biomass and hydrolytic enzymes in plant sisimultaneously
2. Since the enzymes are already embedded into cell wall, the resistance of mass transfer for exogenous enzymes for diffusion is removed and non-selective binding of enzymes on lignin is avoided
3. Allow consolidated low temperature pretreatment and enzymatic hydrolysis
4. Similar or more sugar production in plant by overexpression of hydrolyitc enzymes

As a result, low cost sugar production is possible.

Biomass hydrolyzate fermentation performance:

An economically-attractive cellulosic technology usually requires the strain to achieve ethanol yield, titer and rate higher than 90%, 40 g/L (5.1%v/v), 1.0 g/L/h, respectively. Many metabolically-engineered ethanologens  have been developed to hit the metrics. A research paper published recently by Lau et al compared the fermentation performance of the three engineered microorganisms such as S. cerevisiae 424A(LNH-ST), Z. mobilis AX101 and E. coli KO11, demonstrating that the difficulty to achieve the targets when fermenting  milled AFEX pretreated corn stover using CSL as nitrogen source. 

In a large  commercial scale with a  un-milled biomass biomass at a solids content >= 15%, it will be much harder to achieve the target fermentation conversion through one-pass hydrolysis and fermentation. To avoid harsh pretreatments at extreme conditions, a process with partial recycling  of solid stream from fermentation might be helpful to achieve the target. Without harsh pretreatments and expensive capital cost, the overall cost will be low if the target metrics are achieve.

Biotechnological strategies for hydrolyzate detoxificarion

A revew paper published recently by Parawira et al (Crit Rev Biotechnol. 2010 May 3) describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates.

Of course, the better strategy is to produce fermentable sugars with little toxic compounds, which will eable us to use a whole slurry process.