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.

Synergistic effect of toxic chemical compounds in hydrolyzate

The typical inhibitory compounds from lignocellulosic biomass dilute acid pretreatment are sugar degraded products (furfural, HMF, formic acid, and levulinic acid etc), lignin degraded products (vanillin etc), extractives, and some metals.

The maximum concentration of each inhibitor that a microorganism can tolerate varies depends on the factors of types of microorganisms, its adaption to the medium, process, the numbers of inhibitors, and very importantly, their synergistic effect. For example, ethanol production was stimulated by acetic acid only (up to 10 g/l) in medium without furfural, or by furfural only (up to 2 g/l) in medium without acetic acid. However, the combination of both will negatively affect the cell growth, cell mass yield, and ethanol yield, i.e. the toxicity of biomass hemicelluloses hydrolyzates results from the aggregation of several toxic compounds (alcohols, aldehydes, and acid) rather than individual compounds.

Effect of detoxification of dilute-acid pretreatment hydrolyzate

Before detoxification

Detoxification-1

Detoxification-2

After detoxification, almost no sugar loss but significantly remove furfural (HMF) and simple phenolic compounds.

Extractives: Easily negligible chemical compounds from detoxification

When talking about detoxification of biomass hydrolyzate, almost all the research and studies focus on these compounds such acetic acid, sugar degraded products (furfural, HMF etc). However, small amount of extractives exist in the hydrolyzate, which is often ignored. Actually, some of these extractives, naturally exist in biomass for coloration and as insectcides, which are also toxic to miroorganisms. For example, stilbenes are generally very toxic! So, it is not enough to remove just furfural and acetic acid.

Typical extractives in tree:

- Resin acids (no or little in hardwood)
- Fatty acids
- Monoterpenes (turpentine)
- Phenolics (Gallic acid, vanillin, Stilbenes, Flavonoids, Lignans)

- Others (Alkanes, Proteins, Monosaccharides and derivatives)

Extractive components are “small” molecules that can be extracted with a solvent from wood, bark, or foliage.
-Generally, extractives are present in small amounts.
-Extractives vary tremendously within species, between species, and within trees.
-There are thousands of different extractives present in wood.

Detoxification of hydrolyzate by activated carbon

Activated carbon is known to have high affinities for colored impurities in hydrolyzate of sugar mixtures, and its unit cost is relatively lower and it can be reactivated and reused.

It has been found that the affinities of acetic acid and furfural (and HMF) are much higher than that of the sugars. The key parameters include activated carbon particle size (surface areas), the ratio of activated carbon to hydrolyzate, temperature, and adsorption time, etc.

The activated carbon can be packed into a column and the hydrolyzate will be pumped into the column. The residence time will be determined by the flow rate and the length of the column.

A inline IR or UV detector can be used to monitor the compounds in the eluent.

The misundstanding of lime treatment for detoxification of lignocellulosics hydrolyzate

Traditionally, lime treatment was used to remove phenolic compounds in pulping acid sulfite pulping spent liquor because of the formation precipitate calcium lignosulfonate.Unfortunately, this method was also proposed today to be used widely to remove other by-products such as acetic acid, furfural, and HMF in the hydrolyzate, which is not right from fundamental aspect. These by-products can be removed due to the lime trap from lime treatment, which also traps sugars and leads to 20-35% sugar loss.

Efficiency and cost consideration for detoxification of lignocellulosics hydrolyzate

The hydrolyzate of lignocellulocis need to be detoxified before fermentation. However, there must be a consideration of the efficiency and cost when a method is selected.

Biological and membrane are very effective but not cost-effective for bioethanol production;
Lime treatment is effective and cheap but the sugar loss and by-product (gypsum) handling is another cost and environmental issue.

I like the idea of stripping combined with activated charcoal treatment, which works well with good cost advantage. The charcoal can be easily re-activated and reuse.

A Possible Industrial Solution to Ferment Lignocellulosic Hydrolyzate to Ethanol

It is known that fermentable sugars can be obtained from lignocellulosic biomass through enzymatic or chemical hydrolysis processes. Although there are great interests in the application of enzymatic hydrolysis due to its specific products and high yield, in a short term, the time-consuming process and rather expensive enzyme cost make enzymatic hydrolysis still uncertain for use in large-scale industry.

One of the disadvantages of chemical hydrolysis process such as dilute-acid hydrolysis is the formation of by-products (furan derivatives, phenolic compounds, and aliphatic acids), which inhibit cultivation of yeast in lignocellulosic hydrolyzates. Hence on one hand, a great effort has to be made to reduce the inhibitor formation; on the other hand, detoxification step such as membrane separation is needed in the process. In addition, since the fermenting microorganisms are able to detoxify the hydrolyzates in situ, any process that can provide low concentration of the inhibitors and high cell density may be successful in cultivation of these toxic hydrolyzates. To some extent, continuous cultivation can be one of the approaches to provide low concentration of the inhibitors in the reactors and the high cell density can be provided by using cell recycling, immobilization or encapsulation.

According to s new research Paper, "a toxic dilute-acid hydrolyzate was continuously cultivated using a highcell-density flocculating yeast in a single and serial bioreactor which was equipped with a settler to recycle the cells back to the bioreactors. No prior detoxification was necessary to cultivate the hydrolyzates, as the flocks were able to detoxify it in situ. The experiments were successfully carried out at dilution rates up to 0.52 h-1. The cell concentration inside the bioreactors was between 23 and 35 g-DW/L, while the concentration in the effluent of the settlers was 0.32 ± 0.05 g-DW/L. An ethanol yield of 0.42-0.46 g/g-consumed sugar was achieved, and the residual sugar concentration was less than 6% of the initial fermentable sugar (glucose, galactose and mannose) of 35.2 g/L".

Molecular-weight cut-off for membrane separation

Reverse osmosis or nano-filtration membranes with differing pore sizes are often selected to separate phenolic compounds and sugar degraded by-products in the hydrolyzates. Usually, the pore size of a membrane is given by the molecular-weight cut-off instead of pore diameter. The molecular weight of the typical chemical compounds in the liquors under studies is shown below:

Compounds MW
Hexoses 180.2
Pentoses: 150.1
Acetic acid 60.1
Furfural 96.1
HMF 110.1

Detoxification: a key step in the biorefinery process (2)

The detoxification for bioethanol production from lignocellulosic biomass needs to reduce trace containment levels to the following concentrations: lignin: < 1%; acetic acid: < 1 g/L; furfural:< 0.5 g/L; HFM: <0.8 g/L; formic acid:<5g/L.

Detoxification: a key step in the biorefinery process (1)

It has been recognized that hemicellulose from wood furnish can be pre-extracted using hot water, acid, alkali, and organic solvent and then converted into bioethanol by fermentation. Typically, the wood extracts from hot-water or organo-aqueous extraction consist of monomeric and oligomers of sugars (hexoses and pentoses), acetic acid, methanol, aromatic compounds, and other low molecular-weight (MW) extractable substances. However, the presence of various lignins derived components and phenolics interferes with the enzyme adsorption and function to the target substrate in the subsequent hydrolysis process, as well as potentially toxic to the micro-organisms for fermentation. In addition, small molecules such as acetic acid, furfural and hydroxyl methylfurfural (HMF) produced from these processes are also potent inhibitors for fermentation. Obviously, separation of the toxic products from the pre-extracted liquor mixture is a key step in the biorefinery process.

There are several methods which can be used for separation purpose, including striping, distillation, centrifugation, chemical separation, and filtration. Striping off volatile compounds can be performed right after the extraction stage but doesn’t remove the lignin derived compounds. Multiple fractional distillation is often proposed but this is an energy-intensive operation with high capital cost, especially for the separation of chemicals that can form azeotropes such as water and acetic acid. Chemical separation requires additional chemicals to bond the target compounds such that they can be separated more easily. However it is usually hard to achieve the desirable product stream with high purity for a multiple compound liquor mixture and this also requires additional chemical cost and recovery, which is not practical for a massive production. Centrifugation obviously can not work for the wood extracts or pulping spent liquor. Filtration is a simple physical method that can be easily performed with fair capital cost and most importantly, it can preserve the values of the separated streams. Typically, membrane filtration with specific pore size (molecular weight cut-off) can be used to separate different molecules in the extract liquor. But its captial and operation cost, regeneration are the trade-off.