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A broad range of themes in Microbial Biotechnology

Craig Daniels and Juan-Luis RamosEstación Experimental del Zaidín, Consejo Superiorde Investigaciones Científicas, C/Prof. Albareda, 1,E-18008 Granada, Spain.

In the November issue of Microbial Biotechnology a seriesof relevant articles were published regarding energy gen-eration, thermostable enzymes, influence of transgenicplants on microbes that live in the rhizosphere of plants,degradation of hormones, identification of pathogens, andthe use of phage to control bacterial populations. Thisabundant selection of articles reflects that Microbial Bio-technology is attracting interest from a wide range of fieldswith the aim of placing science within the prospect offuture industrial developments.

One of the themes that has attracted a good number ofarticles in 2008 has been energy generation. The Novem-ber issue brought three relevant articles. One of themdealt with microbial fuel cells (MFCs), which are based onthe use of microorganisms as biocatalysts to convertchemical energy contained in electron donors to electricalenergy. Microbial fuel cells are considered to have poten-tial application in the domains of power supply, biologicaloxygen demand sensors and especially in sustainablewastewater treatment. However, a number of limitationsappear in the system and one of the approaches to over-come a number of these limitations was described byPham and colleagues (2008) in the previous issue ofMicrobial Biotechnology. These authors show that theapplication of high shear rates enriched an anodophilicmicrobial consortium in MFC that resulted in higher per-formance. Enrichment at a shear rate of about 120 s-1

resulted in the production of a current and power outputtwo to three times higher than those in the case of lowshear rates. A series of taxonomical analyses revealedsubstantial differences in the microbial biofilm coveringthe anode at the high shear rate with respect to the thinnerbiofilm present in the low shear rate bioreactor. Theauthors suggested that differences in microbial communi-ties may explain the superior performance of the highshear MFCs. This article is related to an excellent over-view from the same lab on the use of sediment microbialfuel cells (SMFCs), which allow the production of energyin the form of readily usable electrical power that serves,for instance, to operate a small electrical apparatus (De

Schamphelaire et al., 2008). The authors envisage thatSMFCs could be used to power sensors and data trans-mitters at remote sites.

Lignocellulosic biomass such as straw or waste wood isan abundant low-cost source for production of biofuelssuch as ethanol that does not compete for human nutri-tional needs. Despite these advantages, turning plantbiomass into ethanol remains primarily an economic chal-lenge. For fiscal reasons it is preferable that all sugarspresent in the polymers hemicellulose and cellulose areconverted to ethanol. To make the monomeric sugarscontained in hemicellulose accessible for fermentation bymicroorganisms, biomass is pretreated under rough con-ditions. This pretreatment leaves the cellulose intact, thisis subsequently hydrolysed enzymatically. Pretreatmentgenerates a wealth of compounds, many of which inhibitfermentation by Saccharomyces cerevisiae and othermicroorganisms. Chemical and toxicological analysis ofseveral different lignocellulosic hydrolysates by Heer andSauer (2008) allowed them to identify furfural as the maintoxic agent for yeasts, which influences yeast perfor-mance for ethanol production. The authors designed along-term evolution series of assays where significantlyincreased tolerance of a semi-industrial yeast strain totoxic hydrolysates was achieved by selection for growthin furfural-containing medium. The increased toleranceresulted in three improved parameters: ability to grow inthe presence of higher concentrations of hydrolysates,significantly reduced lag phases and reduced processtimes in process-relevant fermentation set-up.

Evolution of enzymes for better industrial performance isone of the main targets in biotechnology. Glucoamylase(GA) from Aspergillus niger (EC3.2.1.3) is used as anindustrial enzyme to hydrolyse maize starch into glucose.The first step in this process is the liquefaction of starch intostarch dextrin in the a-amylase reaction which is performedat 105°C for 5 min and then at 95°C for 1 h. Glucoamylaseis used in the second step (saccharification) to convert thedextrin into glucose monomers. This step involves coolingthe dextrin to 60°C, the operational temperature of GA. TheClark laboratory presents a series of elegant experimentsdirected to increase thermostability of GA without adecrease in catalytic efficiency (McDaniel et al., 2008). Tothis end, the authors used the combined techniques ofdirected evolution (phenotype screening following random

Microbial Biotechnology (2009) 2(1), 3–5 doi:10.1111/j.1751-7915.2008.00076.x

© 2008 The AuthorsJournal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd

mutagenesis and random recombination) and site-directedmutagenesis to isolate several multiply-mutated GAvariants with enhanced thermostability compared withwild-type GA. The so-called CR2-1 variant is the mostthermostable A. niger GA enzyme described to date.

Martin Filion (2008) has approached the very relevantissue of the effect of genetically modified plants (GMPs)on rhizobacteria. His minireview is a compendium ofapproaches used for analysis and some specific casestudies. One of the most striking conclusions concerningrisk assessment studies is that in spite of the largenumber of parameters studied, confusion still exists as towhich information is really relevant in this context. To date,most studies investigating the impact of GMPs or trans-genic plants have shown at least minor effects on theabundance and/or the diversity of rhizobacteria. Clearrhizobacterial community changes have been shown inthe rhizosphere of a variety of plants using different trans-genes. However, based on the limited information pres-ently available in the literature, it seems that the effects oftransgenic plants, although not negligible, can be consid-ered ‘minor’ when compared with other ‘normal’ sourcesof variation, such as field site, soil type, seasonal varia-tion, plant growth stage, etc. Agricultural practices, suchas irrigation, crop rotation, tillage and use of herbicidesand pesticides, have been shown to influence rhizobac-teria populations with similar impact and in many casesmore significantly than plant genetic transformation.Martin Filion proposed a series of steps and analyses forfurther risk assessment.

Trace pollutants in drinking water with undesirable hor-monal effects on animals and humans are of increasingconcern. In the November 2008 issue of Microbial Bio-technology Sabirova et al. (2008) investigate the biologi-cal treatment of very low concentrations of recalcitrant17a-ethinylestradiol (EE2) using manganese-oxidizingmicroorganisms. They proposed an efficient strategy forthe removal of trace levels of xenoestrogens from envi-ronmental samples through the production of highly oxi-dized Mn oxides by manganese-oxidizing bacteria that, inturn, drive the indirect oxidation and cleavage of EE2. Theauthors discuss the development of a more general strat-egy for the removal not only of estrogens but other trace-level organic contaminants from waste and drinking water.

Fast identification of pathogens is critical for adequatetreatment of patients. The Kauffmann–White scheme iswidely used as a typing method for classification of Sal-monella into serovars on the basis of antigenic variabilityin the outer membrane lipopolysaccharide (O antigen),flagellar proteins (H1 and H2 antigens) and capsularpolysaccharide (Vi antigen). Tankouo-Sandjong and col-leagues (2008) report in Microbial Biotechnology thatthe conventional microbiological method for serotypingSalmonella takes several days and proposed that a

microarray-based Salmonella serotyping method canaddress hundreds of different serovars in a single reactionand deliver results within 24 h, avoiding the need for strainisolation. They report a microarray, based on two house-keeping and two virulence marker genes (atpD, gyrB, fliCand fliB) for the detection and identification of the twospecies of Salmonella (S. enterica and S. bongori), thefive subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S.enterica ssp. enterica serovars. Results demonstrated astrong discriminatory ability of the microarray among Sal-monella serovars and the array was highly sensitive beingable to detect one colony-forming unit (cfu) per 25 g offood sample following overnight enrichment. This biotech-nological advancement in detection of a major food-bornebacterium will undoubtedly aid in the fast and efficienttreatment of infected individuals.

Lactic acid bacteria (LAB) are widely used in themaking of fermented dairy products such as cheeses andyogurts; the global market for these good is huge andnaturally research into these essential bacteria is of sig-nificant importance. In the November issue of MicrobialBiotechnology, Siezen and Bachmann (2008) presentedan up-to-date overview of research into LAB. The articleconcentrates on the current state of genomics in this fieldin relation to mining and analysis of the greater than 20genomes that are presently available. Genome mining isbeing used extensively to annotate the metabolic potentialof these bacteria and this has allowed the construction ofgenome-scale metabolic models which allow energy flowsto be predicted and then experimentally tested. One of themore important angles is the analysis of genomes forenzymes that are involved in proteolysis and amino acidconversions. In fact mining performed on LAB strainsfound that typical dairy LAB such as Lactococcus lactis,Streptococcus thermophilus and Lactobacillus casei con-tained the largest sets of enzymes involved in metabolismof sulfur-containing amino acids, which are known precur-sors of dairy flavours (Liu et al., 2008). In situ transcrip-tome analysis of LAB has mostly been obtained from invitro experiments because the high protein and fat contentof dairy products makes it very difficult to isolate bacterialRNA. The authors detail some of the recent advancestowards solving these problems such as DNA microarraytime series and new methodologies for extraction of RNAdirectly from cheese or separation of bacteria fromcheese prior to RNA extraction (De Jong et al., 2008;Monnet et al., 2008). The authors later discuss recentfindings that LAB suffer extensive gene loss and horizon-tal gene transfer during habitat adaptation (Makarova andKoonin, 2007). Obviously, this evolutionary adaptation isof major importance because it can potentially affect thereproducibility of the production processes. Bacterioph-age is an important mechanism of horizontal gene trans-fer and phage-induced bacterial lysis can impact heavily

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© 2008 The AuthorsJournal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 2, 3–5

on the dairy industry. Resistance to phage infection in LABis sometimes conferred by clustered regularly interspacedshort palindromic repeats. These sequences have beenshown to be responsible for increased phage resistanceduring successive phage challenge (Deveau et al., 2008),a process which could be used to directly evolve multiplephage-resistant strains. Undoubtedly, extensive analysisof the currently available LAB genomes and confirmationof the findings with high-quality experimental techniquessuch as those discussed in this article will greatly aid thedairy industry in the future.

Enterobacter sakazakii is an opportunistic pathogen,which can cause rare but life-threatening infections inneonates and infants. The group referred to as E. sakaza-kii group 1 strains have a high mortality rate, and severeneurological sequelae are often observed in survivingpatients. Several studies have implicated rehydrated pow-dered infant formula and its preparation in hospital settingswith disease transmission in spite that the contaminationlevel with Enterobacteriaceae is in general very low: lessthan 1 cfu per 100 g of formula. However, outgrowth ofeven very low bacterial contamination may occur if thereconstituted product is kept at room temperature or in anincubator for long periods. Zuber and colleagues (2008)present a challenging article in Microbial Biotechnologydealing with the potential use of bacteriophage to controlthese microorganisms. This follows in line with the Foodand Drug Administration approval of a cocktail of phageagainst Listeria in food production. The authors empha-size the need to isolate specific phage, a process for whichthey consider it is critical to establish the niche of the targetbacterium. Clearly then there is the question of whetherthe isolated set of phage is safe for a food application, astemperate phage from Enterobacteriaceae may carry bac-terial virulence determinants. Another practical question iswhether the isolated phage infects a sufficient percentageof the target. In this report they isolated and characterizedfive phages and showed that when they infected a lowpathogen number with low phage titres, the cells grew out– apparently undisturbed by the phage – until they reacheda titre of 104–105 cfu ml-1. Only after crossing this thresh-old, did they observe phage replication and the lysis ofE. sakazakii cells. The authors consider that productsafety can only be reached with high phage concentra-tions added to the finished product, which raises the ques-tions of phage safety for infants, product availability andphage production costs.

References

De Jong, A., Ejby, M., Kuipers, O.P., Kilstrup, M., and Kok, J.(2008) Gene network reconstruction – time series analysisof Lactococcus lactis growing in milk. In 9th Symposium onLactic Acid Bacteria. Kuipers, O.P., Poolman, B., andHugenholtz, J. (eds). Egmond aan Zee, the Netherlands:FEMS, p. 4589.

De Schamphelaire, L., Rabaey, K., Boeckx, P., Boon, N., andVerstraete, W. (2008) Outlook for benefits of sedimentmicrobial fuel cells with two bio-electrodes. Microb Biotech-nol 1: 446–462.

Deveau, H., Barrangou, R., Garneau, J.E., Labonte, J.,Fremaux, C., Boyaval, P., et al. (2008) Phage response toCRISPR-encoded resistance in Streptococcus thermophi-lus. J Bacteriol 190: 1390–1400.

Filion, M. (2008) Do transgenic plants affect rhizobacteriapopulations?. Microb Biotechnol 1: 463–475.

Heer, D., and Sauer, U. (2008) Identification of furfural as akey toxin in lignocellulosic hydrolysates and evolution of atolerant yeast strain. Microb Biotechnol 1: 497–506.

Liu, M., Nauta, A., Francke, C., and Siezen, R.J. (2008)Comparative genomics of enzymes in flavor-forming path-ways from amino acids in lactic acid bacteria. Appl EnvironMicrobiol 74: 4590–4600.

McDaniel, A., Fuchs, E., Liu, Y., and Ford, C. (2008) Directedevolution of Aspergillus niger glucoamylase to increasethermostability. Microb Biotechnol 1: 523–531.

Makarova, K.S., and Koonin, E.V. (2007) Evolutionary genom-ics of lactic acid bacteria. J Bacteriol 189: 1199–1208.

Monnet, C., Ulve, V., Sarthou, A.S., and Irlinger, F. (2008)Extraction of RNA from cheese without prior separation ofmicrobial cells. Appl Environ Microbiol 74: 5724–5730.

Pham, H.T., Boon, N., Aelterman, P., Clauwaert, P., DeSchamphelaire, L., van Oostveldt, P., et al. (2008) Highshear enrichment improves the performance of the anodo-philic microbial consortium in a microbial fuel cell. MicrobBiotechnol 1: 487–496.

Sabirova, J.S., Cloetens, L.F.F., Vanhaecke, L., Forrez, I.,Verstraete, W., and Boon, N. (2008) Manganese-oxidizingbacteria mediate the degradation of 17a-ethinylestradiol.Microb Biotechnol 1: 507–512.

Siezen, R.J., and Bachmann, H. (2008) Genomics of dairyfermentations. Microb Biotechnol 1: 435–442.

Tankouo-Sandjong, B., Sessitsch, A., Stralis-Pavese, N.,Liebana, E., Kornschober, C., Allerberger, C., et al.(2008) Development of an oligonucleotide microarraymethod for Salmonella serotyping. Microb Biotechnol 1:513–522.

Zuber, S., Boissin-Delaporte, C., Michot, L., Iversen, C.,Diep, B., Brüssow, H., and Breeuwer, P. (2008) DecreasingEnterobacter sakazakii (Cronobacter spp.) food contami-nation level with bacteriophages: prospects and problems.Microb Biotechnol 1: 532–543.

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© 2008 The AuthorsJournal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 2, 3–5