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New molecular tools for enhancing methaneproduction, explaining thermodynamically limitedlifestyles and other important biotechnological issues

Craig Daniels,1 Carmen Michán2 andJuan L. Ramos1*1Consejo Superior de Investigaciones Científicas,Estación Experimental del Zaidín, Department ofEnvironmental Protection, C/ Prof. Albareda, 1, E-18008Granada, Spain.2Universidad de Córdoba, Campus de Rabanales, Dept.of Biochemistry and Molecular Biology, Edificio SeveroOchoa C-6, 2a Planta, 14071, Córdoba, Spain.

In this special issue a series of articles appear detailingmethods of energy generation under different conditions;processes which are related to the future vision ofmicrobes as a source of electric power (Lovley, 2009).Methanogenesis is identified as an important part of thebiogeochemical carbon cycle in diverse anaerobic envi-ronments. Methane is an important fuel that can be gen-erated from several wastes in these anoxic environmentsand therefore, understanding how it is produced, whichorganisms produce it and the reactions required toachieve high yields is of major biotechnological interest.Alves and colleagues (2009) present a mini-review onthe use of lipids as substrates for methane production.Conversion of lipids into methane is achieved by thesyntrophic consortia of acetogenic bacteria and methano-genic archeae. The microbes involved are influenced bythe nature of the incoming lipids and examples of differentmicrobial communities when feeding reactors with oleateor palmitate are reviewed. Alves and colleagues (2009)point out that the number of acetogenic microorganismsthat degrade butyrate or higher fatty acids is very low andincludes microbes of the Syntrophaceae and Syntroph-omonadaceae families. These microbes live together withhydrogen-consuming methanogenic archeae and sulfate-reducing bacteria. The syntrophic cooperation seems tobe optimal when microbes are organized as microcolo-nies, which is considered to favour interspecies hydrogentransfer. As in many biotechnological processes, lipid

conversion into methane requires both the appropriatemicrobes and their performance optimization in the reac-tors, each of which still require some improvement. Theauthors note that previous failures to deal with high loadlipids were related mainly to sludge flotation and biomasswashout. To overcome these limitations the authorsdescribe a new reactor concept which involves primarybiomass retention through flotation and secondarybiomass retention via settling. These reactors are beingtested in scale and if, as expected, they solve the problemmay soon come into industrial use.

The seminal review of Bernhard Schinck (Schink, 1997)reveals that catabolic reactions mediated by syntrophicbacteria are thermodynamically unfavourable and onlyfeasible when at least a two-member consortium is inoperation. In this issue of Microbial Biotechnology, Katoand colleagues (2009) present an outstanding study onpropionate utilization by Pelomaculum thermopropioni-cum under syntrophic conditions when associated withthe archeae Methanobacterim thermautotrophus. Tounderstand how archeae affect the metabolism of thebacterium Kato et al. exploit previous group results (Katoet al., 2008; Kosaka et al., 2006) and construct a microar-ray for global transcriptional responses to chemicals thatare used only under syntrophic conditions and chemicals,i.e. fumarate, that can be used in mono-cultures. One ofthe beauties of the work is that expression of the bacte-rium’s central pathways is influenced by the substrate, butthe sheer presence of the archeon leads to the inductionof a number of central metabolism genes including thosefor amino acids and cofactors. The authors indicate thatthis is most likely due to cross-feeding of the bacterium bythe hydrogenotrophic Methanothermobacter, and sup-ports why Pelomaculum as mono-culture only grows ifyeast extract is supplemented. Microarray analysis iden-tified Fe-hydrogenase III as the major hydrogenase in allco-cultures, as well as the pyruvate: formate lyase whichcatalyses the conversion between pyruvate and acetyl-CoA (pyruvate + CoA → acetyl-CoA + formate). In all, theyfound that in co-cultures, regardless of the substrateused, almost 70 genes were upregulated and about

*For correspondence. E-mail juanluis.ramos@eez.csic.es; Tel. 34958 181608; Fax 34 958 135740.

Microbial Biotechnology (2009) 2(5), 533–536 doi:10.1111/j.1751-7915.2009.00134.x

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

50 were downregulated. This represents an excellentexample of microbial interactions within the crystal ballconcept presented by Handelsman (2009), whichincludes the term metagenetics and will allow us in thenear future to achieve in-depth knowledge of the waymicrobes interact to survive in a given niche. This is anexcellent piece of scientific work that will open newresearch avenues beyond methanogenesis.

On the topic of thermodynamic limits; is it possible toconvert polycyclic aromatic hydrocarbons (PAHs) intomethane? This issue is analysed by Dolfing and col-leagues (2009) for two- to four-ring aromatic compoundsusing a theoretical basis and via wet analysis with naph-thalene. The thermodynamic calculations for three poten-tial pathways show that the thermodynamic properties arenot independent to the biodegradation of PAHs undermethanogenic conditions, because the process is exer-gonic. The authors speculate that some PAH degraderswould simultaneously act as PAH degraders and aceto-gens. Degradation of PAHs has experienced an outburst inthe last decade (Atlas and Bragg, 2009; Gieg et al., 2009;Parisi et al., 2009) and a thorough review on the mainadvances in the field will appear in one of the future issueof Microbial Biotechnology (Kanaly and Harayama, 2009).

In this issue of Microbial Biotechnology several otherinteresting aspects regarding the biology of microbes andtheir potential exploitation in biotechnology are consid-ered. Jeon and colleagues (2009) show how moderngenomic and proteomic information can be used to designstrategies to combat Campylobacter, a major food-bornepathogen of animal origin and a leading cause of humangastroenteritis. Among the species being the main causeof infections 92% of the reported cases correspond toCampylobacter jejuni. The authors analysed in detail themain metabolic pathways used by this microorganism inthe chicken gut, as well as adaptation variability, proteinglycosylation systems, mechanisms of adherence andantibiotic resistance. This analysis revealed key featuresof the biology of these microorganisms that are, in turn,used to identify potential targets to prevent and controlCampylobacter colonization in animal reservoirs.

One of the emerging features of the genomic analysisof C. jejuni is that the main source of acetyl-CoA genera-tion and therefore electron transfer to the respiratorychains are amino acids, in particular L-aspartate,L-glutamate, L-serine and L-proline which are preferen-tially used by this microorganism. A characteristic ofCampylobacter is that they have a highly branched elec-tron transport system that allows them to derive energythrough respiration using a wide variety of electron accep-tors, which include oxygen under aerobic conditions orhydrogen peroxide and sulfite under oxygen-depletedconditions. Of particular interest is the ability to respiresulfite, which is currently used as a food preservative.

Colonization of the animal gut requires multiple func-tions and is a complex process that involves attachment,motility and tolerance to stressing agents such as bilesalts. Campylobacter adherence to epithelial cells in theintestine is required to resist intestinal peristalsis, andflagella, CadF, PEB1 and PEB4 are critical surface pro-teins involved in adherence. Resistance to bile is mainlymediated by an RND family efflux pump called CmeABCthat is regulated by the CmeR regulator. The regulatorprevents expression of the efflux pump genes, but in thepresence of bile salts is inactivated and transcriptiontakes place. In addition, this efflux pump removes antibi-otics and other drugs that may be used to prevent growth.Moreover Campylobacter also exhibit an arsenal of toolsto resist different stress conditions, such as acid stress,heat changes and exposure to oxidative conditions.

Fluoroquinolones are often used to combat Campylo-bacter infections; however, it has been found that FQresistance appears in this microorganism with a high fre-quency. This could be related to the fact that a numberof enzymes needed for error-prone correction, such asUvrR, MutL, MutH are not present in the genome of C.jejuni, which in turn seems the basis of a high genetic andphenotypic variability in this species. This review alsoexplores different alternatives to decrease infection byCampylobacter, including the use of bacteriophage.

In fact, the topic of bacteriophage biocontrol is coveredin an extremely insightful mini-review by R.J. Atterbury(Atterbury, 2009). The author reflects on the early prom-ising results from bacteriophage application in so called‘Phage Therapy’ which were performed in the 1930s and1940s while lamenting the paucity of rigorous well-controlled studies in these early trials. The lack of well-controlled and standardized methods for bacteriophagetesting and the discovery and use of antibiotics led to arapid decline in the use of bacteriophage as a therapeuticagent; particularly in the West. However, recently therehas been a revival in the use of bacteriophage in thetreatment of disease in animals, and surface decontami-nation (biotic and abiotic). Bacteriophage being ubiqui-tous in the environment and therefore already a part of thehuman food chain suggests that they can be regularlyconsumed without ill effects on human health. The recentapproval of phage treatments for foods by the US Foodand Drug Administration has opened the door for a pre-ponderance of new applications for bacteriophage in thereduction of zoonotic infections or elimination of foodspoilage bacteria. The author points out that carefulscreening of the bacteriophage is of great importance toensure that only virulent self-limiting phage that cannottransfer bacterial DNA are used. It appears that a new erais dawning for what is essentially an 80-year-old bio-technology; although bacteriophage are unlikely to bea universal remedy for disease treatment and food

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preservation they will almost certainly be a valuable addi-tion to our current arsenal.

Yang and colleagues present a thorough and interest-ing review of virulence factor expression in the entericmodel bacteria Citrobacter rodentium (Yang et al., 2009).When moving from the environment to the host, entericpathogens need to modify their gene expression, oftenupregulating the expression of virulence genes and down-regulating housekeeping genes. The authors describehow C. rodentium is able to modulate gene expressionduring the infection process. Citrobacter rodentiumcarries the locus for enterocyte effacement (LEE), whichis a pathogenicity island that is highly conserved amongdiverse enteric pathogens and is required for the devel-opment of lesions in the intestinal epithelium. By adaptingpre-existing regulatory mechanisms and newly acquiredregulatory genes C. rodentium is able to ensure its sur-vival; the intricate regulatory networks allowing specificcontrol of virulence gene expression in response to thedifferent environments. Yang and colleagues detail howthe bacterium utilizes a positive regulatory loop involvingLEE-encoded regulatory proteins (Ler, GrlA and GrlR) tocontrol LEE expression along with several transcriptionfactors not encoded by LEE; some of which respond tospecific environmental signals. One of these is a recentlydescribed AraC-like regulatory protein, RegA, which iskey to the ability of C. rodentium to colonize the intestine.RegA activates the transcription of numerous operonsthat encode virulence factors, while at the same timerepressing transcription of a number of housekeepinggenes. Of great significance is that RegA is the onlyknown AraC-like regulator shown to respond directly to agut-specific environmental signal, bicarbonate, in order toexert its regulatory effects (Yang et al., 2008). Informationgained regarding regulatory proteins, particularly theimportant and diverse AraC-like regulators will undoubt-edly lead to future advances in the use of these proteinsin biotechnology.

In the upcoming edition of Microbial Biotechnologythere is an article by Linares and colleagues (2009) thatdescribes the first report of transcriptional regulation oftyramine biosynthesis in lactic acid bacteria (LAB).Tyramine is a low-molecular-weight biogenic amine that isformed by the enzymatic decarboxylation of tyrosine;such molecules are of importance because they areformed from the natural action of bacteria on foods suchas cheese and can be toxic in high concentrations.Tyramine formation and secretion in LAB requires theproducts of two genes tdcA (tyrosine decarboxylase) andtyrP (tyramine/tyrosine antiporter). In this article theauthors describe the genetic regulation of tyramine bio-synthesis in Enterococcus durans; showing that tdcA andtyrP form an operon that is transcribed from a singlepromoter PtdcA. They also consolidate previous studies

that noted that decarboxylase gene expression requiresacidic pH and high amino acid concentrations. Their quan-titative gene expression data confirmed that both highconcentrations of tyrosine and acidic pH conditions duringlog phase growth led to increased transcription from thePtdcA promoter. These results together with those previ-ously reported indicate the importance of the decarboxy-lation pathway in the regulation of internal pH in LABwhen placed in an acidic environment; knowledge whichcould prove beneficial for both food production and pre-vention of food spoilage.

Saccharomyces cerevisiae is an important microorgan-ism from a biotechnological point of view because of itsuse in industrial processes such as bread making andfermented beverage production. Different compoundsincluding volatile esters are responsible, even at traceamounts, for highly desired flavours for example fruity,mellow, etc. There are two main categories of flavour-active esters in fermented beverages: acetate esters(produced from acetate + ethanol or a complex alcoholderived from amino acid metabolism) and medium-chainfatty acid (MCFA) ethyl esters (formed from MCFA andethanol). The review by Saerens and colleagues (2009)deals with the different tactics that can be used to modifythe production of these esters. The final concentration ofthese products is influenced by the overall rates of syn-thesis (influenced in turn by substrate availability andenzyme activity) and degradation. The authors summa-rize data on acetate ester synthesis, which is mainly per-formed by alcohol acetyl transferases I and II (encodedby ATF1 and ATF2), although double null mutants stillproduce other esters indicating the existence of othersynthases. ATF1 is the most important enzyme in terms ofactivity and is subject to a complex regulation; respondingto unsaturated fatty acids, oxygen, nitrogen, phosphate,ethanol and heat stress. All of these factors provide manu-facturers with several options for external influence.

The participation of enzymes such as Eht1 or Eeb1 inethyl ester synthesis is also discussed and the corre-sponding null mutants present lower levels of particularMCFA ethyl esters; however, yet again they do notaccount for all MCFA derivative synthesis. The regulationof Eht1 is complex because the enzyme participates inboth the synthesis and the hydrolysis of the esters. Theauthors suggest that its activity is influenced by nutrients,oxygen and inositol, and also depends on the geneticbackground.

Phosphorus (P) is an essential element classified as amacronutrient because of the relatively large amountsrequired by plants. Although P is widely distributed innature, it is recognized that the availability of phosphate insoils is a major factor limiting the productivity of manyecosystems. The main biological mechanism for mineralphosphate solubilisation is the production of organic

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acids; for example gluconate and 2-keto gluconate. InSashidhar and Podile (2009), they describe the con-struction of recombinant genetically modified organisms(GMOs) from the free-living N2 fixer and common soilhabitant Azotobacter vinelandii which allow improvedmineral phosphate fertilization of sorghum seeds.Membrane-bound Gcd (quinoprotein glucose dehydroge-nase) from Escherichia coli converted glucose to gluconicacid which was released directly into the periplasmicspace and subsequently released to the cell’s environ-ment. The gcd was introduced into A. vinelandii broad-host-range plasmids in which the gene was under thecontrol of A. vinelandii glnA (glutamine synthetase) or pts(phosphate transport system) promoters. Sorghum seedswere inoculated with the GMOs and gene expression,production of gluconic acid, nitrogenase activity, inorganicphosphate release and plant growth were monitored.The recombinant genes responded to the expectedenvironmental/chemical signal and were affected by nitro-gen availability in the constructions containing the glnApromoter or by available inorganic phosphate in thosewith pts promoters. The GMOs presented differences inmorphology and exopolysaccharide content and a slightdecrease in their nitrogen fixation ability, while at the sametime improving biofertilizer potential and plant growth-promoting activity. Interestingly, significant growthenhancement was seen 3–4 weeks after inoculation of theplants with the GMOs indicating potential future use ofthese types of organisms may provide long-term benefitsto crop plants.

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Atlas, R., and Bragg, J. (2009) Bioremediation of marine oilspills: when and when not – the Exxon Valdez experience.Microbial Biotechnol 2: 213–221.

Atterbury, R.J. (2009) Bacteriophage biocontrol in animalsand meat products (p). Microbial Biotechnol. doi. 10.1111/j.1751-7915.2009.00089.x.

Dolfing, J., Xu, A., Gray, N.D., Larter, S.R., and Head, I.M.(2009) The thermodynamic landscape of methanogenicPAH degradation. Microbial Biotechnol 2: 566–574. doi:10.1111/j.1751-7915.2009.00096.

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