Бурение грунтовых зондов, установка энергетических колодцев

Metabolic engineering has allowed the development of recombi­nant microorganisms that will ferment glucose and xylose. Some of the most widely considered recombinant microorganisms as can­didates for industrial application include ethanologenic Escherichia coli xylose-fermenting Z. mobilis. The National Renewable Energy Laboratory (NREL), USA, has made significant contributions in recent years to engineer Z. mobilis to overcome its inherent defi­ciencies by expanding its substrate range to include C-5 sugars like xylose and arabinose. In one approach from NREL, two oper — ons encoding xylose assimilation and pentose phosphate path­way enzymes were constructed and transformed into Zymomonas mobilis in order to generate a strain that grew on xylose, and effi­ciently fermented it to ethanol [47]. Thus, anaerobic fermentation of a pentose sugar to ethanol was achieved through a combina­tion of the pentose phosphate and Entner-Doudoroff pathways. Furthermore, this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocel — lulosic biomass to ethanol [47]. The same group from NREL later developed an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering as well [48]. A number of research groups around the world have successfully engineered C-5, C-6 co-fermenting bacteria suitable for industrial applications. Some selected examples from these successes and their references are shown in Table 8.3.

In a more recent example, Agrawal et al. reported the engi­neering of efficient xylose metabolism capabilities into an acetic acid-tolerant Zymomonas mobilis strain by introducing adaptation — induced mutations [53]. They reported that chromosomal mutation at the xylose reductase gene was critical to xylose metabolism by reducing xylitol formation, together with the plasmid-borne muta­tion impacting xylose isomerase activity, and these two mutations accounted for 80% of the improvement achieved by adaptation.

In an attempt to produce new xylose fermenting strain in the presence of high acetic acid concentrations, they transferred the two mutations to an acetic acid-tolerant strain. The resulting strain fermented glucose + xylose (each at 5% w/v) with 1% (w/v) acetic acid at pH 5.8 to completion with an ethanol yield of 93.4%, outper­forming other reported strains [53]. Introduction of xylose metabo­lizing pathways from E. coli is an another example [54]. Modified Z. mobilis has the advantages of requiring a minimum of nutrients, growing at low pH and high temperatures, and it is considered

generally recognized as safe (GRAS). A comparison between genet­ically engineered Z. mobilis by introducing xylose metabolizing pathways from E. coli and E. coli is shown in Table 8.4 [55].

A genetic modification of wild-type E. coli to improve its fermen­tation profile is another approach. Saha and Cotta have reviewed the recent developments in recombinant E. coli strains in the fer­mentation of biomass hydrolyzates [56]. Researchers at Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois, USA, have developed two recombinant E. coli strains (FBR4 and FBR5) that have been used for fermenta­tion of corn fiber hydrolyzates [52]. These strains carry the plasmid pLOI297, which contains the genes from Zymomonas mobilis neces­sary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but no selective antibi­otic. An average of 93-95% of the FBR4 and FBR5 cells maintained pLOI297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermen­tations with 10% (w/v) xylose [52]. Fermentation results were simi­lar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86-92% of theoretical. Maximal ethanol concentrations were 3.9-4.2% (w/v). In order to test the applica­bility in real biomass situations, Dien and coworkers tested these strains for their ability to ferment corn fiber hydrolyzate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than E. coli FBR4 from the corn fiber hydrolyzate. E. coli FBR5 fermented all but

0. 4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, which cor­responds to 90% of the maximum obtainable [52].

Saha et al. from the same research laboratory also reported the use of these two recombinant E. coli strains (FBR 4 and 5) [57,58], and in this case for the fermentation of wheat straw. In these experi­ments ethanol production by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) have been compared using recombinant bacterium E. coli FBR 5, where high solid loading of wheat straw was used. The yield of total sugars from dilute acid pretreated wheat straw after enzy­matic saccharification was 86.3 g/L. Then pretreated wheat straw was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. Ethanol yields and productivity under SHF and SSF conditions using recombinant bacterium E. coli FBR 5 on wheat straw are shown in Table 8.5 [57].

A number of research groups have demonstrated that recombi­nant E. coli has the ability to ferment a wide spectrum of sugars, without the requirements for complex growth factors. However, the major disadvantages associated with using E. coli cultures are a narrow and neutral pH growth range (6.0-8.0), less hardy cultures compared to yeast, and public perceptions regarding the danger of E. coli strains. The lack of data on the use of residual E. coli cell mass as an ingredient in animal feed is also an obstacle to its application.

A variety of ethanol-producing thermophilic microorganisms have been isolated and characterized due to their ability to degrade a broad variety of both hexoses and pentoses. These bacteria include Thermoanaerobacter ethanolicus [59], Thermoanaerobacter math — ranii [60] Clostridium thermohydrosulfuricum [61], Thermoanaerobium brockii [62], Clostridium thermosaccharolyticum [63], etc. These types of thermophilic anaerobic bacteria have a distinct advantage over conventional yeasts for bioethanol production in their ability to use a variety of inexpensive biomass feedstocks and their ability to withstand temperature extremes [64]. Nevertheless, the low bio­ethanol tolerance of thermophilic anaerobic bacteria (< 2%, v/v) is a major obstacle for their industrial exploitation for bioethanol production [65]. Thermoanaerobacterium saccharolyticum is one of the

The dilute acid (0.75% H2S04, v/v) pretreatment of wheat straw (150 g/L) was performed at 160°C for 10 min. Enzymatic saccharification was carried out at pH 5.0 at 45°C for 72 h with a cocktail of three commercial enzyme (cellulase, /І-glucosidase, and hemicellulase) preparations. Fed-batch SSF was performed by adding the substrate 4 times (0, 16, 21, and 24 h) in 4 equal portions. SHF — separate hydrolysis and fermentation; SSF — simultaneous saccharification and fermentation.

thermophilic anaerobic bacteria which is able to directly ferment hemicellulosic oligomers as well as primary sugars found in cel­lulosic biomass, including cellobiose, glucose, xylose, mannose, galactose, and arabinose. The ability to ferment the full spectrum of sugars available in hydrolyzates promises to further enhance the overall fermentation of mixed solutions of hexoses and pentoses to ethanol [66], therefore this group of bacterium can be identified as a hopeful branch in fermentation microbiology.

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