27004192

 

Appl Microbiol Biotechnol (2007) 77:23 – 35 35 DOI 10.1007/s00253-007-1163-x

MINI-REVIEW

Biofuels from microbes Dominik Antoni  Vladimir V. Zverlov Wolfgang H. Schwarz   &

  &

Received: 8 June 2007 /Revised: 9 August 2007 /Accepted: 9 August 2007 / Published online: 22 September 2007 # Springer-V Springer-Verlag erlag 2007 Abstract  Today, biomass covers about 10% of the world ’s

Keywords  Biofuel . Sustainability . Fermentation .

 primary energy demand. Against a backdrop of rising crude oil prices, deple depletion tion of resou resources, rces, poli political tical instability instability in  producing countries and environmental challenges, besides effi ef fici cienc encyy an andd in inte tell llig igent ent us use, e, on only ly bi biom omass ass ha hass th thee  potential to replace the supply of an energy hungry civilisation. Plant biomass is an abundant and renewable

Biomass . Substrate . Energy

source of energy-rich carbohydrates which can be efficiently con conver verted ted by mi micro crobes bes int intoo bio biofue fuels, ls, of whi which, ch, onl onlyy  bioethanol  bioeth anol is produc produced ed on an indus industrial trial scale today. Biomethane is produced on a large scale, but is not yet  utilised for transportation. Biobutanol is on the agenda of  several companies and may be used in the near future as a supplement for gasoline, diesel and kerosene, as well as contributing to the partially biological production of butylt-butylether, BTBE as does bioethanol today with ETBE. Biohydrogen, biomethanol and microbially made biodiesel still sti ll req requir uiree fur furthe therr dev develo elopme pment. nt. Thi Thiss pap paper er rev review iewss microbially made biofuels which have potential to replace our present present day fuels, fuels, eit either her alone, alone, by ble blendi nding, ng, or by chemic che mical al con conver versio sion. n. It als alsoo sum summar marise isess the his histor toryy of 

has pub publi licis cised ed th thee eco econo nomi micc nec necess essit ityy to li limi mitt gl globa oball warming warm ing (Ste (Stern rn   2006). 2006). An acc accele elerat rated ed rel releas easee of fos fossil sil entombed entom bed CO2   due to hum human an act activi ivity ty is now gen genera erally lly accepted as a major factor contributing to the green house effectt (IPC effec (IPCC C   2001). 2001). App Approx roxim imate ately ly 28% of the ene energy rgy availa ava ilable ble fo forr con consum sumpti ption on in th thee EU EU25 25 cou countr ntries ies is attributed to transportation, of which, more than 80% is due to road transport (Eurostat   2007). 2007). Worldwide, about  27% of prim primary ary energy is used for tran transport sportation ation,, which is also the fastest growing sector (EIA 2006 (EIA  2006). ). Transportation fuel fu elss ar aree th thus us pr prom omis isin ingg ta targ rget etss fo forr a re redu duct ctio ionn in greenhouse gas emissions. However, only a limited selection of energy-rich chemical compounds, primarily liquid alcohols and esters, can be produced by microbes, while

 biofuels and provides insight into the actual production in various countries, reviewing their policies and adaptivity to the energy challenges of foreseeable future.

complying with the criteria necessary for moder complying modernn fuels fuels,, suitability for safe storage and high energy efficiency in combustion engines and capacity to power a transportation vehicle. In flight applications where the fuel comprises a significant fraction of the load, the energy density of the fuel also becom becomes es a major factor for consideration. consideration. Biomas Bio masss fuel fuelss have been used throughout throughout man’s long histo hi story ry.. Mo Most st of th them em we were re al alcoh cohol olss pr produ oduced ced by th thee fermentation of substances like starch or sugars, others were  plant oils. Alongs Alongside ide combustion combustion,, they were put to a variety variety of  uses us es as sol solve vent nts, s, gr grea eases ses,, cl clean eaner erss or as ba basi sicc ch chem emic ical alss fo forr th thee emerging chemical industry, until a cheaper source was found in fos fossil sil oil. Today oday,, wit withh ris rising ing prices for crud crudee oil and increasing political instability in oil producing countries, the

D. Antoni Institute of Resource and Energy Technology, Technische Universität München, Weihenstephaner Steig 22, 85350 Freising-Weihenstephan, Germany V. V. Zverlov : W. H. Schwarz (*) Department of Microbiology, Technische Universität München, Am Hochanger 4, 85350 Freising-Weihenstephan, Germany e-mail: [email protected]

Introduction

The Stern-Review on the Economics of Climate Change

use bio-based alcohols as solvents or basic chemicalsand is againofunder consideration. The production of chemicals

 

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Appl Microbiol Biotechnol (2007) 77:23 – 35 35

fuelss fro from m loc locall allyy gro grown wn plan plantt mate material rial supp support ortss pol politi itical cal fuel independence through diversification and a decreased dependencee on a few esse denc essentia ntiall ener energy gy sour sources, ces, a CO2 neutral energy  production  produc tion and a surplu surpluss of gross nation national al produc productt and economic econom ic power being kept in the country. country. This is especially crucial to remote and economically disadvantaged agricultural areas. Beginn Beg inning ing wit withh a sho short rt exp explor lorati ation on of the his histor toryy of   biofuels, the state of microbial fermentation to produce

fuel sales in the 1920s (Giebelhaus  1980  1980;; Finlay 2004 Finlay  2004). ). In 1940, ethanol production was widely abolished due to the unbeatably low price of gasoline in the USA. However, in WWII WW II,, va vari riou ouss wa warr rrin ingg na nati tion onss us used ed a wi wide de ra rang ngee of   biofuels. It was recognised that gasoline additives, such as tetra-ethyl-lead, required for high compressions in ethanol free fr ee eng engine ines, s, wer weree env envir ironm onment ental al and hea healt lthh haz hazard ards. s. Despite Despi te these apparent risks risks,, the petr petro-der o-derived ived gasol gasoline ine finally came to dominate the transport market after WWII

 biofuels in today’s world is reviewed and discussed, giving insight into some prospects which are still in the research stage but have promising prospects for future production.

(Source:   http://www.ybiofuels.org/ ). (Source:  ). Ethanol as a fuel was revived in the 1970s in Brazil (IEA 2004)) where one of the largest bioethanol industries is located 2004 today.. The bioethanol today bioethanol indus industry try in Brazi Brazill was criticized criticized as environmentally hazardous, as large land area is being used for monocultures. A similar discussion was sparked in North Ameri Am erica ca and Eur Europe ope whe where re sta starch rch pro produc ducti tion on for bio biofue fuell competes over land with the food industry and environmental issues. The sky rocketing price for starch is already hindering the start-up of new bioethanol plants. Like modern crude oil refinery, the bioindustry for biofuels has a dual purpose in the econom economyy, as it is used as a supply of  energy as well as basic chemicals (Zaborsky   1982). 1982). Th Thee upcoming   “ bioref  biorefinery inery”   revit revital aliz izes es th thee ol oldd tr tradi aditi tion on of a

Biofuels in history

Mankind has, for most of its existence, relied on renewable energy resources like wood, windmills, water wheels and animals such as horses and oxen. The development of new energy resources resources was a major drivin drivingg force of the technol technologogical revolution. By the beginning of the twentieth century, up to 30% of arable land was still planted with crops to feed horses and oxen used in transportation. Already, early in the nineteen nine teenth th cent century ury,, alco alcohol holss were repe repeated atedly ly repo reporte rtedd as  biofuels. Even the invent  biofuels. invention ion of ignit ignition ion engine enginess was done with biofuels. Nikolaus August Otto developed his prototype of a spark ignition ignition engine in the 1860s using ethanol and was sponsored by the sugar factory of Eugen Langen who was intere int erested sted in the mas masss prod producti uction on of etha ethanol. nol. Deutz Gas Engine Works designed one third of their heavy locomotives to run on pure ethanol in 1902. Safety and cleanness were contributing factors for that. Ethanol was soon recognised as an antianti-knocki knocking ng addit additive ive for intern internal al combus combustion tion engine enginess and and was added to gasoline between 1925 and 1945. The ethanol  present in the fuel allowed higher piston compression, increasing engine efficiency. Henry Ford was engaged in the chemurgy movement to  bring farm products into production and energy supply supply,, especially during the  “ Great Depression” (Finlay  (Finlay 2004  2004). ). His carr co ca comp mpan anyy ma mark rket eted ed th thee Mo Mode dell T, th thee   “Ti Tinn Lizzy”, running on 100% ethanol (Kovarik 1998 (Kovarik  1998). ). In the Midwest  (USA), blended gasoline made up 25% of Standard Oil ’s

careful caref ul th thri rift ftyy ec econ onom omyy an andd in inte tends nds to ma make ke us usee of al alll energy and carbon stored in biomass, feeding by-products into secondary conversion process or refining them as fuel. Different types of biofuels today Only biod Only biodiese iesell and bioe bioetha thanol nol are presen presently tly prod produced uced as a fuel on an industrial scale (Table  (Table   1). Including ETBE partially made with bioethanol, these fuels make up more than 90% of  the bio biofue fuell mar market ket.. All biof biofuel uelss have to exhi exhibit bit def define inedd chemical and physical properties, meeting the demands of  engine application such as stability and predictable combustion at high pressures as well as the demands of transportation such as safety and energy density. Transportation and distribution

There are gaseous and liquid energy carriers of microbial origin (T (Table able   1). Fue Fuels ls th that at ar aree li liqui quidd at at atmo mosph spher eric ic

Table 1   List of selected biofuels with a potenti potential al microbial production production route, the status of their use and the engine application application

Biofuel

Process

Status

Engine application

Biomethanol Bioethanol Biobutanol ETBE Biomethane Biohydrogen Biodiesel

Thermochemical/microbial Microbial Microbial Chemical/microbial Microbial Microbial Physical/chemical (enzymatic)

Pilot plant Industrial Pilot plant/industrial (until ca 1990) Industrial Industrial Laboratory Industrial (laboratory)

[Pure/blend] MTBE/biodiesel Pure/blend Pure/blend Blend Pure/blend Bioethanol (Syngas)/pure Pure/blend

 

Appl Microbiol Biotechnol (2007) 77:23 – 35

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 pressure and at ambient temperature (e.g. alcohols) can be easily stored, distributed, carried and used as an energy source in cars, trucks, trains and planes. Gases of microbial origin have to be liquidised by cooling or compression to reduce specific volume. As this process is energy intensive, gaseou gas eouss for forms ms of bio biofue fuell are pri primar marily ily use usedd for onon-sit sitee applications in stationary engines. Gaseous fuels such as hydr hy drog ogen en an andd me meth than anee ar aree di diff ffic icul ultt to tr tran ansp spor ortt an andd would require for a new distribution infrastructure to be

reaction, during the anaerobic metabolism of microorganisms (Zeikus 1980 (Zeikus  1980). ). During the process, CO2   is extracted from carbohydrates which have a C/H/O ratio of 1:2:1. Glucose (C6H12O6), a single sugar molecule, would for example be converted conver ted to two molecules CO2 and two molecules ethanol:

developed. Liquid Liq uid biofuel biofuelss hav havee to rem remain ain in a li liqui quidd sta state te and  pumpable at all temperatures encountered. Further requirements on liquid biofuels are a high heat of combustion value to reduce energy losses and costs during transportationn and sta tio stabil bility ity dur during ing sto storag rage. e. Som Somee lon longer ger cha chain in alcohols like butanol have a heat of combustion sufficiently high hi gh to al allo low w fo forr th their eir use in hi high gh th thru rustst-to to-w -wei eigh ght  t  applications such as airplanes (Schwarz and Gapes  2006  2006). ). For saf safee and env enviro ironme nmental ntally ly fri friendl endlyy sto storag rage, e, vap vapour  our   pressure and ignition temperature are a re important factors. The transition from fossil fuels to selected liquid biofuels can be smo smooth oth and sho should uld use pre prefer ferabl ablyy the same, or  slightly sligh tly modi modified, fied, dist distribut ribution ion infr infrastru astructure cture and engin enginee

 produce much less energy from this anaerobic reaction than from fro m oxi oxidat dative ive res respir pirati ation, on, it has to con consum sumee abo about ut ten times the amount of substrate to gain the same amount of  energy; energ y; 2 – 3 ATP compared to 26 – 38 38 ATP in oxida oxidative tive respi re spirat ration ion,, dep depend endin ingg on the or organ ganism ism.. Thi Thiss hig higher  her  turnover turn over of substr substrate ate is an advant advantage age for biotechnology biotechnology.. Thiss ana Thi anaero erobic bic fer fermen mentat tation ion als alsoo hel helps ps to avo avoid id ene energy rgy intensive aeration during industrial production. Most fermentation processes work at mesophilic temperatures atur es (25 – 37°C), 37°C), but therm thermophi ophilic lic proce processes sses (45 – 55°C) 55°C) seem to be more productive and are increasingly researched and developed. A disadvantage of thermophilic processes is a higher sensitivity of the microorganisms to the toxicity of  the solvent products at the higher temperatures. Likewise, a

technology in place for fossil fuels.

higher sensitivity of the fermentation to media parameters like pH or composition of the substrates was noticed in  processes such as thermophilic biomethane fermentations.

 Engine application

There are There are,, in pri princi nciple ple,, thr three ee str strate ategie giess to uti utilis lisee ene energy rgy from biofuels in engines. The engines have to either be adap ad apte tedd to th thee ex exis isti ting ng fu fuel elss or th thee bi biof ofue uell ha hass to be designed to exhibit, as closely as possible, all important  features of traditional fossil fuels. The third strategy is to use bio biofue fuels ls ble blende ndedd wi with th tra tradit dition ional al fue fuels. ls. Thi Thiss oft often en resul res ults ts in an adv advant antage ageous ous mo modif difica icatio tionn of th thee fue fuell’s  properties, such as an increased octane rating without the use of environmentally harmful additives. Alcohol-b Alcoh ol-based ased biof biofuels uels like meth methanol, anol, ethanol and   n butanol cannot only extend the supply of gasoline and diesel, or even replace them, but are also good additives to existin exis tingg fos fossil sil fuel fuelss as oxi oxigeni genisers sers,, liq liquefi uefiers ers or anti anti-knocking agents (Schwarz et al.  2007  2007). ). However, not all energy ene rgy-r -rich ich pro produc ducts ts of mi micro crobia biall fer fermen mentat tation ionss can be used us ed as fu fuel elss be beca caus usee th they ey ma mayy pr prom omot otee co corr rrosi osion on or  swelling with certain materials or may have other unfavourable characteristics.

redox

C 6 H 12 12 O6   ! 2CO2

þ 2C 2 H 6 O1

Ethanol, with its high (C+H) to O ratio, retains most of  the original energy content in combustion. As a cell can

Biological systems for fuel production

The following description of biofuels and of their microbial  production processes is by no means exhaustive, but will encom enc ompas passs the mos mostt int intere eresti sting ng exi existi sting ng and pos possib sible le  processes where microbial fermentation from bacteria and yeasts is invo involved. lved. All micr microbial obial fermentation fermentation proce processes sses require a source of energy to feed the organisms, which has to come from biomass in the form of sugars (Zeikus  1980  1980). ). Hydrogen

To ex extr tract act as mu much ch en ener ergy gy con conte tent nt as po possi ssibl blee fr from om th thee  biomasss during combustion,  biomas combustion, the transformation transformation into into fuels has

Hydrogen is regarded as an ideal fuel for future transportation because it can be converted to electric energy in fuel cells or burnt and converted to mechanical energy without  obvious production of CO2  (Malhotra  (Malhotra 2007  2007). ). This has lead to the idea that an economy driven by hydrogen fuel is a viable prospect. However, hydrogen production is usually effected by thermal/chemical means and is energy intensive si ve,, so hy hydr drog ogen en pr prod oduc uced ed in su such ch a wa wayy ca cann nnot ot be regarded as a renewable primary energy source. In contrast,  biologica  biolo gicall hydr hydrogen ogen prod productio uctionn from bioma biomass ss would  provide an energy-saving, cost-effective and pollution-free

to reduce the number of oxygen atoms per carbon. This is achieved in a dispro disproportio portionisatio nisationn reactio reaction, n, a balanced redox

alternative should be investigated on these meritsand (Das and Verziroglu 2001 Verziroglu  2001;extensively ; Esper et al. 2006 al.based  2006). ).

Metabolic strategy for production procedures

 

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Hydr drog ogen en can be pr prod oduce ucedd bi biol olog ogica icall llyy by al algal gal an andd Hy cyanobacterial bio-photolysis of water or by photo-fermentation of organic substrates from photosynthetic bacteria. In addition, it can be produced by   “dark”  fermentation from organi org anicc subs substanc tances es by anae anaerob robic ic org organi anisms sms such suchas as acid acidogen ogenic ic  bacteria.  bacter ia. This has the additi additional onal appeal of reduci reducing ng the mass of organic waste (Wu et al. 2005 al. 2005). ). High hydrogen yields can  be achieved by the use of thermophilic thermophilic microorganisms microorganisms such Caldicellulosirupto losiruptorr sacchar saccharolyticus olyticus   or   Thermotoga elfii as   Caldicellu

with fuel cells are technically feasible, VW announced that  hydr hy drog ogen en ca cars rs wi will ll no nott pl play ay a pr prom omin inen entt ro role le in ma mass ss  production until 2020. Initial systems are established on a local lo cal bas basis is to te test st co comp mpon onen ents ts du duri ring ng al alll st stag ages es fr from om  production to utilisation.

(de Vrije et al. 2002 al.  2002;; de Vrije and Claassen 2003 Claassen  2003;; Claassen et  al.   2004). 2004). The These se fer fermen mentati tations ons can be oper operated ated in liq liquid uid  phase with immobilised immobilised cells or by enabling the formation formation of  self-flocculated granular cells or sludge to prevent washout  of the hydrog hydrogen-pro en-producing ducing cells. Moreove Mor eover, r, H2   is a com commo monn pr prod oduct uct in an anaer aerob obic ic  bacterial fermentations and may be an interesting by product in future large-scale industrial fermentation. As an example of this, around 40×106 m3 of H2  and 60×106 m3 of CO2   were were pro produc duced ed ann annual ually ly fro from m the 1960s to the 1980s in a Russian biobutanol plant as by-product, but were not used at the time (Zverlov et al.  2006  2006). ). Microbiological hydrogen production is not yet developed into an economically viable technology, and hydrogen

carbon dioxide from plant biomass, which may come from organi org anicc hou househ sehold old or ind indust ustria riall was waste te or fro from m spe specia cially lly grown energy plants (Yadvika et al. 2004 al.  2004). ). The advantage of the biogas process is the option to use the polysaccharide constituents of plant material to produce energy, such as electrical power and heat, in relatively easy-to-manage and small sma ll ind indust ustri rial al uni units. ts. Alt Altern ernati ativel velyy, th thee gas can be compressed after purification and enrichment and then fed to the gas grid or used as a fuel in combustion engines or  cars. Its great greatest est advant advantage age is the envir environmen onmentall tallyy frie friendly ndly aspect of the technology, which includes the potential for  complete recycling of minerals, nutrients (phosphate etc.) and fibre material (for humification) which come from the fields and return to the soil, playing a functional role by

 product ion is lagging behind expecta  production expectations. tions. Biologic Biological al  production from renewable biomass, which would make it  a sus susta tain inabl ablee pr prim imar aryy ene energ rgyy sou sourc rce, e, st stil illl nee needs ds mo more re research and development. Hydrogen (and also alcohols) cann be us ca used ed in fu fuel el ce cell llss to cr crea eate te el elec ectr tric ic po powe werr fo for  r  transportation. BMW claims that the technology of hydrogen utilisation in combustion engines is mature, while other  companies focus on the use of fuel cells. Although cars

sustainin sustai ningg the soi soill’s vit vitali ality ty for fut future ure pla planta ntatio tion. n. The technology techn ology is curr currently ently mature, mature, but there is plent plentyy of room for opt optim imisa isati tion, on, wh which ich wi will ll res resul ultt in lar large ge hi highgh-tec techh  production plants with integrated utilisation of by-products (biorefinery; Fig. 1 Fig.  1). ). Substrate can be cow manure which is also useful for  inoculation, manure from other farm animals such as pigs, chickens and horses, fat from slaughter waste or frying oil,

Fig. Fi g. 1   Schematic Schematic representatio representationn of a twotwo-stage stage biogas plant with  production of electric electric power/heat power/heat and compressed compressed biogas biogas as fuel: 1  slurry storage,  2  waste storage,  3  sanitisation plant,  4  solid substrate storage, 5

desulfurisation (microbial/chemical),  13  gas holder,  14  emergency flare, 15  biogas engine,  16  heat recovery for plant heating,  17  generator,  18 activated carbon filter filter,,  19  compressor,  20  chiller,  21  absorption column, 22  pump,  23  heating,  24  desorption column,  25  air,  26  compressor,  27  compressed compres sed biogas storage,  28  gas station

solid subs substrate trate feed, feed,   6   hydrolysis hydrolysis stage stage,,   7   heating,   8   biofilter,   9 methanogenesis methanoge nesis stage,   10   air for desu desulfur lfurisat isation, ion,   11   condenser,   12

Methane/biogas Biogas plants produce methane gas sustainably along with

 

Appl Microbiol Biotechnol (2007) 77:23 – 35

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organic household or garden waste, municipal solid waste and rott rotten en foods foodstuff tuff.. Even organic waste from hospitals hospitals contai con tainin ningg pap paper er and cot cotton ton,, mun munici icipal pal sew sewage age slu sludge dge,, waste from agric agricultur ulturee or food prod productio uction, n, organ organic-r ic-rich ich indust ind ustri rial al was waste te wat water er etc etc.. can be use usedd as con consum sumabl ablee substrate. Often, energy crops such as maize (whole plant  including the corn), clover, grass, young poplar and willow are esp especi eciall allyy gro grown wn for bio biogas gas pro produc ductio tionn and add added ed  purely or in mixture. To ensure a homogeneous substrate

The German Renewable Energy Act (EEG) and a similar  law in Austria has spurred the construction of 3,500 (G) +600 (A) biogas plants with an average electrical power output of  500 kW (and even more heat) in combined heat and power   plants.. Larger plants  plants plants of 5 MW electri electrical cal power output output are still still rare, but will be constructed in increasing numbers. Their size is limited by land intensive production and the transportation costs for bulky substrate. The large quantity of energy crops required for biogas production provoked discussions among

quality quali ty thr throug oughou houtt the yea year, r, the gre green en pla plant nt mat materi erial al is usually stored as silage, preferably by a process favouring homoferm homo fermenta entativ tivee lact lactobac obacill illii to min minimis imisee carb carbon on loss (Gassen 2005 (Gassen  2005). ). Biogas formation from plant fibres is generally a threestage sta ge pro process cess inv involv olving ing a dif differ ferent ent set of anae anaerob robic ic and facultatively anaerobic microorganisms in each stage:

environmentalists, especially in Germany, about the issues of  monoculture and resulting soil deprivation. However, proper  crop rotation and the recycling of material, such as waste fibre materials, minerals and nutrients, can minimise these effects. Further development of biogas technology is expected to increas inc reasee pro produc ductio tionn eff efficie iciency ncy.. Pres Presentl entlyy, onl onlyy up to a maximum of about 70% of the organic matter in biomass is converted to CH4  and CO2. In order for this to increase, the hydrolysis stage must be enhanced. The separation of the  processes  proc esses for hydrolysis hydrolysis and for acetogenesi acetogenesis/met s/methanoge hanogenesis nesis allows for the application of different optimised conditions in the two stages, such as pH and temperature adjustment. Aside from the trad tradition itional al mesoph mesophilic ilic proce processes, sses, ther thermophi mophilic lic  processes  proc esses are being used more freq frequent uently ly to speed up the

1. hydrolysi hydrolysiss of poly polysaccha saccharides rides (starch, (starch, cellulose, cellulose, hemihemicellulose etc.), proteins and fats into oligosaccharides and sugars, fatty acids and glycerol. This is followed by acidogenesis, the fermentation of these products into mainly mai nly ace acetic tic,, pro propio pionic nic and but butyri yricc aci acid, d, car carbon bon diox di oxide ide an andd hy hydro drogen gen,, al alcoh cohol olss and ot other her mi minor  nor  compounds; 2. aceto acetogenesi genesis: s: the producti production on of acetic acetic acid and carbon carbon diox di oxid ide. e. Du Duee to th thee lo long ng ge gene nera rati tion on ti time me of th thes esee  bacteria this seems to be the limiting process step; 3. met methan hanoge ogenesi nesiss with up to 70% (v / /v  v   ) CH4   and 30% CO2   and and the byby-pro produc ducts ts NH3   and and H2S by sl slow ow-growing archaea, which are sensitive to acidification, ammonia accumulation, low amounts of oxygen and other factors. The bact bacteri erial al com commun munity ity enga engaged ged in thes thesee thr three ee stag stages es may  be similar to those in cows rumen (Einspanier (Einspanier et al. 2004 al.  2004)) or  wastewater treatment plants (Ariesyady et al.   2007). 2007). However, their composition varies depending on the substrate, the type ty pe of fe ferm rmen ente terr an andd th thee pr proc oces esss (e (e.g .g.. me meso soph phil ilic ic or 

reactions reacti ons and esp especi eciall allyy to opt optim imise ise bio biomas masss hyd hydrol rolysi ysis. s. However How ever,, whe whereas reas in man manyy indu industr strial ial bio biogas gas plan plants ts the separation of the hydrolysis stage has already been carried out,, mos out mostt agr agricu icult ltura urall bio biogas gas pla plants nts use the sin singl gle-s e-stag tagee technology. Therefore, several plant components laid out in Fig. 1 Fig.  1 may  may be absent. Dried and desulfurised biogas is usually fuelled without  CO2  separation into stationary block heat and power plants connected to the biogas plants. Utilisation of the excess heat is rarely possible because farms are usually located far  away from residential or industrial areas where it could be used for domestic heating or manufacturing processes. This is not a problem if biogas is compressed like compressed natural gas stored in high-pressure cylinders and used in the

thermophilic; O’Sullivan et al. 2005 al.  2005;; Syutsubo et al. 2005 al.  2005;; Kloc Kl ocke ke et al al..   2007; 2007; Ci Cirn rnee et al al..   2007). 2007). Som Somee ba bact cter eria ia involved have been isolated and characterised, but comprehensiv hen sivee st studi udies es on th thee bi biol olog ogica icall sys system tem in pu pure re pl plan ant t   biomasss fermenting  biomas fermenting plants are still widely widely missing, missing, especially on the hydrolytic and the thermophilic processes. Traditional farm biogas plants are run as a single- or twostage process at around 37°C with an uncontrolled secondary fermentation in large storage tanks. Due to different  optimal opt imal conditions conditions spec specifi ificc for the hydr hydrolyt olytic ic and the methanogenic bacteria, two-stage processes are increasingly applied, particularly in large industrial biogas plants. Most   biogas fermentation tanks are run as liquid fermenters. The v )  biogas fermentation tank may contain more than 12% (w/ v 

Bioethanol fermentation is by far the largest scale microbial  process. State of the art industrial ethanol production uses sugar cane mola molasses sses or enzyma enzymatica tically lly hydr hydrolysed olysed starch (from corn or other grains) and batch fermentation with yeast   Saccharomyces cerevisiae  to create ethanol. Byproducts of this process are CO 2  and low amounts of methanol, glycerol etc. Ethanol does not need to be rectified to high  purity if it is to be used as a fuel. The azeotrop of 95.57 wt  % ethanol with 4.43 wt% water can be used in ignition cars.

dry masss (so mas (so-ca -calle lledd dry fer fermen mentat tation ion)) or les lesss (li (liqui quidd fermentation).

This is known as AEHC in Brazil (álcool etílico hidratado combustível). However, higher water content causes prob-

engines of urban co-generation plants. In addition, direct  use in car combustion engines is possible (Fig. 1 (Fig.  1). ). Ethanol

 

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Appl Microbiol Biotechnol (2007) 77:23 – 35 35

gasoline mixtures partly because it leads to lems in ethanol – gasoline  phase separation between the water and gasoline. Yeast fermentation of glucose syrups to ethanol has been welll pro wel progre gressed ssed in rece recent nt year years. s. Inh Inhibi ibitor tor sens sensiti itivit vityy,  productt tolera  produc tolerance, nce, ethano ethanoll yield and speci specific fic ethan ethanol ol  productivity have been improved in modern industrial strains to the degree that up to 20% ( v / /v  v   ) of ethanol are  produced  produc ed in presen present-day t-day indust industrial rial yeast fermen fermentation tation vessels from starch-derived glucose.

 boxylase and alcohol dehydrogenase from Z. mobilis  (Ohta et al. 1991 al.  1991). ). The residual slurry from the mild acid hydrolysis is, after  neutralisation, fermented in an SSF process in the presence of industrial cellulases to hydrolyse the cellulose to glucose with a derivative of   Klebsiella oxytoca strain M5A1, which was similarly engineered to contain the  Z. mobilis  ethanol  producing pathway (strain P2). M5A1 also harbours a natur nat ural al cel cello lobio biose se upt uptak akee sys system tem and tw twoo ad addit dition ional al

The future will see a techn technical ical process using lignocellulignocellulosic hyd losic hydrol rolysa ysates tes (Gr (Gray ay et al.   2006). 2006). Ho Howe weve verr, as th thee enzyma enz ymatic tic hyd hydrol rolysi ysiss rea reacti ction on of cel cellul lulose ose is abo about ut tw twoo orders ord ers of mag magnit nitude ude slo slower wer tha thann the ave averag ragee eth ethano anoll fermentation rate with yeast, there is a theoretical gap in simultaneo simu ltaneous us saccha saccharifi rificatio cationn of cellu cellulosic losic biom biomass ass and ethanol fermentation (SSF). This must be addressed if total  biomass is to be fermented, not only glucose syrups, for  example from starch (Hahn-Hägerdahl et al.   2007). 2007). This method would considerably widen the substrates available for use in ethanol production. Improvement of substrate  plants are reviewed in Torney et al. (2007 2007). ). Subst Substrate rate consists of glucose (hexose) and xylose (pentose) in mass  proportions depending on the type of plant material.

cellulase genes from   Erwinia chrysanthemi  which reduces the amount of added fungal cellulase dramatically (Brooks and Ingram   1995; 1995; Zhou et al.   2001). 2001). Although bacterial fermentations do not yield such high ethanol concentrations as yeast fermentations, with  E. coli  LY165, up to 9% (v / /v  v   ) of ethanol could be reached (L. Ingram, personal communication; Golias et al. 2002 al.  2002). ). Pentose sugars released from the hemicellulose in lignocellul cel lulosi osicc bio biomas masss pre presen sentt a pro proble blem. m. The mai maize ze pla plant  nt  (without the corn) contains about 24% (of dry mass) xylose and 2% arabinose (in comparison with 45% glucose) which cannot be neglected if the yield of solvents and complete substra subs trate te uti utilis lisati ation on are an issu issue. e. Thes Thesee valu values es for sin single gle sugars suga rs vary vary,, but gene general ral hard hardwoo woods ds and agr agricu icultu ltural ral raw

However, the fermentation of pentose sugar with industrial yeast strains is a difficult task and still under development  (Hahn-Hägerdahl et al.   2007), 2007), although some pilot plants are already running. The fermentation of all sugars in cellulosic biomass is a task easily performed by most bacteria, although there may  be problems with catabolite repression. Hence, bacterial ethanol fermentation is still discussed.   Zymomonas mobilis is considered the work horse of bacterial ethanol fermentati ta tion on and th thee sou sourc rcee of th thee enz enzym ymes es fo forr me meta tabol bolic ic engineering of other bacteria towards ethanol production (Alterthum and Ingram 1989 Ingram  1989). ).  Z. mobilis  is restricted to the substrates sucrose, glucose and fructose which it catabolises through the Entner  – Doudoroff Doudoroff pathway. It also, however,

material materi al cont contain ain a lar larger ger pro propor portion tion of   D-xy -xylose lose and L-arabinose, while softwood is less rich in pentoses (Hayn et al.  al.   1993). 1993). Many of the industrial yeast strains lack the xylose utilisation pathway consisting of xylose reductase and xylitol xylit ol dehydr dehydrogenase. ogenase. The state of metabo metabolic lic engineering engineering to yield pentose-fermenting yeast strains is reviewed in HahnHägerdahl et al. (2007 (2007). ). These strains will play an important  role in the efficient conversion of plant biomass to ethanol and are already being used in pilot projects. Further research will concentrate on osmotolerance, inhibitor sensitivity and ethanol yield of new yeast strains. Chee Ch eese se wh whey ey ha hass al also so be been en us used ed as a su subs bstr trat atee fo for  r  ethanol fermentation. Several industrial processes were run worldwid worl dwidee using   Streptococcus Streptococcus fragili fragiliss   as in the Dansk

 produces considerable amounts of the by-products sorbitol, acetoin, glycerol and acetic acid as well as the extracellular   polymer levan. This bacterium was used for continuous ethanol fermentation in a fluidised bed reactor (WeusterBotz 1993 Botz  1993). ). On a different microbiological platform, L. O. Ingram developed two strains of Enterobacteriaceae to circumvent  the inherent problems with yeast and   Z. mobilis  (Ingram and Doran 1995 Doran  1995). ). A new bioethanol plant, located in the  bay of Osaka, Japan, will be in operation later in 2007 using a combination of mechanical pretreatment and mild acid hydrolysis of waste wood to yield a pentose syrup which is to be fermented with an advanced recombinant   Escherichia coli   B str strain ain,, LY16 Y1655 (M (Moni oniruz ruzzam zaman an et al.

Gaerings industry process and  Kluyveromyces fragilis in the Milbrew Mil brew proce process, ss, wher whereas eas   K. fra fragili giliss   is us used ed in mo most  st  commercial plants (Pesta et al.   2006; 2006; Siso   1996). 1996). Whey  permeate was fermented for decades in New Zealand (Gapes and Gapes, 2007 Gapes,  2007). ). The Canbery process currently in operation in Ireland uses ultrafiltrated whey with yeast  fermentat ferm entation. ion. In futu future, re, chees cheesee whey substrate substrate will gain importanc impo rtancee becaus becausee of incre increasing asing cheese prod productio uctionn and  problems during disposal resulting from the high organic matter content. Ethanol fermentation from cellulosic biomass has been  proposed using cellulolytic clostridia (Zeikus 1980 (Zeikus  1980;; Lynd et  al. 2002 al.  2002;; Demain et al. 2005 al.  2005). ). The thermophilic bacterium Clostridium thermocellum  will readily hydrolyse cellulosic

1996). This strain is devoid of the genes responsible for  1996). side product formation formation and contains the pyruv pyruvate ate decar decar--

 biom ass and will degr  biomass degrade ade hemi hemicell cellulos ulosee as well as cellulose, but uses only the cellodextrins (not the glucose)

 

Appl Microbiol Biotechnol (2007) 77:23 – 35

29

2002). However, the derived from cellulose (Lynd et al.   2002). strains are sensitive to higher ethanol concentrations and  produce a range of less favourable by-products (T (Tailliez ailliez et  al. 1989 al.  1989). ). Metabolic engineering of  C.   C. thermocellum  seems to be fea feasib sible le now now,, and str strain ain dev develo elopme pment nt for ethanol ethanol  production is a promising target. The production of bioethanol by fermentation of Syngas (a CO/H2 mixture from gasified biomass) is conceivable. A number num ber of bact bacteri eriaa for formin mingg org organic anic com compoun pounds ds lik likee

methanol (Kildiran et al. 1996 al.  1996). ). Even a mixture of alcohols characteri chara cteristic stic of aceto acetone ne –  butanol fermentation could be used (Nimcevic et al.  2000  2000). ). Eventually, enzymatic transesterification can be utilised (see Microdiesel below). At present, the microbiology of biodiesel degradation is a concern because of bacterial oxidation etc. during storage as we well ll as th thee un unav avoi oida dabl blee wa wate terr co cont nten entt le lead adin ingg to corrosion problems. Another problem is the high energy input inp ut in RME pro produc ductio tionn whi which ch dim dimini inishe shess the ove overal ralll

acetate, ethanol and butanol have been isolated including Clostridium   ssp.,   Moorella   ssp.   Carboxydocella   ssp. An industrial process for the fermentation of Syngas to ethanol  based on  C. ljungdahlii  has already been developed. During this process, municipal waste is gasified and cooled down to fermentation temperature (while using the waste heat to  produce electricity) and is blown into the fermenter fermenter.. The ethanol produced by the organisms is then separated by distillati disti llation. on. The proce process ss has already been run at pilo pilott plan plant t  scale. A commercial plant is expected (Henstra et al. 2007 al.  2007;; http://www.brienergy.com). http://www.brienergy.com ). Biologica Biol ogicall ethan ethanol ol ferm fermentat entation ion from mola molasses sses and starch is basically a mature technology. The utilisation of  non-food nonfood subs substra trates tes such as cel cellul luloseose-cont containi aining ng wast wastee

energy gain (see Table 4 Table  4). ). The glycerol produced during transesterification creates a deposit problem in some areas. It could be fermented, e.g. to 1, 1,3-p 3-prop ropan anedi ediol ol and pos possib sibly ly to oth other er pr produ oduct ctss by metabolically engineered bacteria (Cheng et al.   2007) 2007) or  to methane in biogas plants where it can be added in low concentrations as co-substrate. A potential future fuel completely produced by bacteria is Microdiesel (Kalscheuer et al. 2006 al.  2006). ).   E. coli  cells were metabo met abolic licall allyy eng engine ineere eredd by int introd roduci ucing ng the pyr pyruva uvate te decarboxylase and alcohol dehydrogenase genes   pdc   and adhB, respectively, from  Zymomonas mobilis  for abundant  ethano eth anoll pro produc ductio tion. n. The gen genee   atfA   for an unsp unspeci ecific fic Acinetobacter baylyi  was introduced acyltransferase from   Acinetobacter

material is in the pilot stage. A lot of experience with the use of bioethanol in engines currently exists in the field. Present-day spark ignition engines have to be modified for  mixtures of more than 5% ethanol to gasoline, especially for the use of E85 or E100. However, the technology exists and is installed in so-called flex fuel vehicles (FFV). Most  car companies are now offering FFVs, e.g. as   “Totalflex” (VW)) or   “Flexpower ”   (Chevrolet/O (VW (Chevrolet/Opel/ pel/GM). GM). Ford Ford,, Saab and Volvo were the likely pioneers of this technology. In Bras Br asil il,, 3 mi mill llio ionn FF FFVs Vs ha have ve be been en so sold ld to da date te,, an andd in Sweden, 15,000 FFVs were sold in 2005 alone. Bioethanol can be run in diesel engines, but additives are necessary to  prevent phase separation (Lapuerta et al. al. 2007  2007). ).

to esterify ethanol with the acyl moieties of CoA thioesters of fatty acids. If the cells are grown aerobically in the  presence of glucose as an energy and carbon source and of  oleic acid, ethyl oleate was the major product. However, de novo no vo sy synt nthe hesi sise sedd fa fatt ttyy ac acid idss we were re no nott us used ed by th thee acyltransferase, which made the external addition of fatty acids necess necessary ary.. This indi indicates cates that consi considerab derable le furt further  her  development is needed (O’Connell Connell 2006  2006). ). However, a new concept conce pt of the micr microbiol obiologica ogicall produ production ction of biodi biodiesel esel has  been shown with these experiments. Conv Co nver ersi sion on of pl plan antt oi oill to bi biod odie iese sell is a ma matu ture re technology. However, microbial contribution to the production ti on pr proce ocess ss is cl close ose to ze zero ro at pr prese esent nt.. In Incl clus usio ionn of   biologically fermented ethanol and butanol will not pose

Biodiesel

technical techni cal pro proble blems. ms. The use of enz enzyme ymess or bio biolog logica icall systems in transesterification is to be developed. Most diesel cars are now licensed to use a biodiesel –  v   ). dies di esel el bl blen endd of up to 5% (v / /v  ) . Th Thee co conv nver ersi sion on of a conventional diesel engine for pure biodiesel use is offered  by many companies and costs in Germany up to 1,500 € per  car. The modified engine, however, requires more frequent  engine oil changes. Whether microbially produced biodiesel will run without problems and which engine modifications will have to be installed are yet to be shown.

Biodiesel is a monoalkyl ester of fatty acids from vegetable oil and is presently produced by catalytical transesterification with petr petrochem ochemically ically derived methanol, also calle calledd alcoholysis (e.g. rape seed oil methyl ester, RME; Ma and Hanna   1999). 1999). Instead of using vegetable oil, microalgae could be grown in photobioreactors for the production of a suitab sui table le oil oil.. Bec Becaus ausee of the their ir hig highh oil pro produc ductiv tivity ity,, the specific demand of land area needed is strongly reduced by this concept in contrast to oil from plants (Yusuf   (Yusuf   2007). 2007). Although bacterial processes are presently not involved in itss pr it prod oduct uctio ion, n, bi biod odie iese sell is me ment ntio ione nedd he here re du duee to it itss  potential for 100% biological production in the future. As a first step, alcohols from microbial fermentations such as etha et hano nol, l, pr prop opan anol ol an andd bu buta tano noll ca cann be us used ed in inst stea eadd of 

n-Butanol and acetone

Acetone was produced up to WWI from wood. The supply of  wood bec wood becam amee ins insuf uffic ficien ientt at the sta start rt of the war because because acetone demand increased in line with the manufacture of 

 

30

Appl Microbiol Biotechnol (2007) 77:23 – 35 35

cordite, a cartridge and shell propellant in which acetone was an essential ingredient. The Russian chemist C. Weizmann, later Israeli President, President, devel developed oped the ABE ferm fermentat entation ion  processs at Manch  proces Manchester ester Univ Universit ersityy. He ident identifie ifiedd a produ producing cing  bacterium  bacter ium which was later known as   C. acetob acetobutylicu utylicum m. Facilities were built in the UK and in France using maize starch as a substrate, while rice starch was used at facilities in India (Jones and Woods   1986). 1986). In this process, acetone is  produced  prod uced toget together her with butan butanol ol and ethan ethanol ol (ABE 3:6: 3:6:1). 1).

There is much interest in bacterial butanol fermentation, especially after the announcement by BP and DuPont to finance fin ance dev develo elopme pment nt of a mod modern ernise isedd pro produc ductio tionn pla plant  nt  supported by research and development. This could include the introduction introduction by recom recombinan binantt gene technology technology of the  butanol  butan ol metab metabolic olic pathw pathway ay from one of the solve solvent  nt   producing clostridia to another bacterium which is more tolerant of the products. The production of the byproducts could cou ld be red reduce ucedd or ter termin minate ated, d, and the process process cou could ld

Butanol had no value at the time (Schwarz et al. 2007 al.  2007). ). The ABE fermentation was the largest scale bioindustry ever run second to ethanol fermentation. By 1927, butanol was incr increasi easingly nglyused usedfor for the pro produc ductio tionn of the lacq lacquer uer solv solvent  ent   butylacetate  butylac etate and for the synthetic rubber industry industry.. Japan, and  possiblyy other combat  possibl combatants, ants, used butano butanoll as an aviati aviation on fuel during WWII when they exhausted their fossil fuel supply. Japan’s converted sugar refineries on the island of Formosa were therefore a strategic target for allied bombers. Acetone and butanol production from fossil fuel became competitive in the early 1960s, eventually forcing the ABE fermentation industry out of production except in the USSR  and in South Africa. The South African commercial ABE fermentation industry continued into the 1980s. During the

greatly benefit from new and cheaper substrates derived by hydrolysis from lignocellulosic biomass, as well as from advanced sterile fermentation and downstream processing technology. A biogas plant connected to the plant would enhance energy conservation considerably. It was demonstrated in June 2006 that   n-butanol can be used either 100% in unmodified 4-cycle ignition engines or   blended up to at least 30% (70% diesel) in a diesel compression engine or to 20% (80% kerosene) in a jet  turbine engine (Schwarz et al. 2006 al.  2006). ). However, biobutanol  production has only recently started again after decades of  inactivity. The production of biobutanol from lignocellulosic biomass is promising and is on the agenda for a number of companies.

1960s and 1970s, more than 100,000 tonnes per annum of   biobutanol were produced by fermentation in the USSR. Substrates included hydrolysates of lignocellulosics from agricu agr icultu ltural ral was waste te mat materi erials als (Zv (Zverl erlov ov et al.   2006). 2006). Th Thee  process was constantly improved and run in continual mode, generally unknown to producers in the West. As the USSR disintegrated in the early 1990s, production stopped. Despite the lack of a viable biobutanol economy, research and development in universities of Europe, South Africa, New Zealand and the USA continued. A continuous fermentation  pilott plant operating  pilo operating in Aust Austria ria in the 1990s introduced introduced new technologies and proved economical feasibility with agricultural waste potatoes potatoes (Nimcevic (Nimcevic and Gapes Gapes 2000  2000;; Gapes 2000 Gapes 2000). ). The Austrian plant helped bridge the skill gap between the

Methyl-tert -butylether -butylether (MTBE) is produced by acid catalysiss fr ysi from om iso isobut buten en and met methan hanol, ol, bot bothh com coming ing fro from m v   ) is added to  petrochemical sources. As much as 15% (v / /v  gasoline as anti-knocking agent, but became discredited due to its car carcino cinogen genici icity ty and gro ground und wat water er cont contami aminati nation on ability. Recently, some production sites were converted to  produce the ethyl ether instead (ETBE) which introduces  biologically produced ethanol into the fuel additive (e.g. at  Degus De gussa) sa).. The use of mi micro crobi biall allyy pro produc duced ed   n-butanol instead of ethanol is possible and would increase the mass  percentage of biologically produced sustainable fuel. The

termination of US, USSR and South African production and the recently reported renewal of production (Schwarz et al. 2007). 2007 ). Howe However ver,, the trad tradition itional al clos clostridi tridial al ferm fermentat entation ion of   butanol  butan ol and aceto acetone ne is suffe suffering ring from the diff difficult iculties ies of  switching the acidogenic fermentation stage to the solventogenic stage, and thus, a discontinuous production mode, from common phage infections, the rising substrate costs and the effort required for downstream processing. Often Of ten mis mis-de -descr scribe ibedd as a   “new”   fuel, fuel, biobu biobutanol tanol has  been in almost continuous production since 1916, and most  of the time as a solvent as well as a basic chemical. Today, new uses for butanol are emerging, e.g. as a diesel and kerosene replacement, as silage preserver, biocide and C4 compoun com poundd for chem chemica icall ind indust ustry ry..   n-Bu -Butan tanol ol has man manyy

resulting BTBE has favourable characteristics. Metha Me thanol nol was waswi widel delyy use usedd as fu fuel el in Ger Germa many ny dur durin ingg WWI WWIII (Demirbas 2007 (Demirbas  2007). ). It can be used as pure fuel, as a blend or in thee fo th form rm of MT MTBE BE.. Th Thee tr tradi aditi tion onal al pr proc ocess ess pr prod oduc uced ed renewab rene wable le meth methanol anol by the ther thermoch mochemi emical cal conv conversi ersion on of  wood (wo (wood od alco alcohol) hol).. A biom biomassass-to-b to-biome iomethan thanol ol proc process ess usin us ingg ba bact cter eria ia is ba base sedd on su suga garr be beet et pu pulp lp as su subs bstr trat atee (Atlantic Biomass Conversions). Dry sugar beet pulp contains about 60% (w/ w) pectin. The methylated carboxyl groups of  galacturonic acid in pectin are de-esterified by a pectin methyl esterase (PME) to yield methanol. Therefore, the well-studied   Erwinia chrysanthemi  B374 was transferred into   E. PME of  Erwinia coli   to achi achieve eve a high higher er tem tempera perature ture tole toleranc rance. e. Met Methano hanoll conver con versio sionn fr from om sug sugar ar bee beett pul pulpp was studied studied as was th thee

advantageou advanta geouss char characte acteris ristic ticss whi which ch mak makee it a supe superio rior  r  gasoline replacement (Schwarz and Gapes 2006 Gapes  2006). ).

separati separ ation on of the met methan hanol. ol. An ind indust ustria riall pro proces cesss was designed but not yet realised (Wang 2006 (Wang 2006;; Anonymo Anonymous us 2004  2004). ).

Others

 

Appl Microbiol Biotechnol (2007) 77:23 – 35

31

Future biofuels will be produced by a combination of  chemical –   –   physical treatments and biological fermentation, e.g. chemical ETBE synthesis from bioethanol or the acid  pretreatment of lignocellulosics followed by enzymatic hydrol hyd rolysi ysiss and fer fermen mentat tation ion.. The mas masss pro produc ductio tionn of  indust ind ustri rial al eth ethano anoll has com comee und under er pub public lic scr scruti utiny ny and criticism, as it has become evident that increased demand for starch substrate derived from corn has pushed up prices (Odling-Smee 2007 (Odling-Smee  2007). ). Alternative substrates such as ligno-

Table 2   The fut future ure goa goals ls for the imp implem lement entatio ationn of biof biofuels uels in

cellulosics have yet to be exploited (Zaldivar et al.  2001  2001;; Lynd et al.   2002; 2002; Demain et al.   2005). 2005). Development is ongoing, and many companies and publicly funded institutions are involved in the development of feasible processes.

Germany Japan Spain

Biofuels in different countries and regions

 Numbers are percent (energy) of fuel consumption.

Some bi Some biof ofue uels ls ar aree pr pres esen entl tlyy on th thee ed edge ge of ec econo onomi micc viability viabi lity,, but comm commercia erciall intr introducti oduction on needs furt further her subsidati sid ation on unt until il via viabil bility ity is rea reache chedd (Ga (Gapes pes   2000). 2000). Mo Most  st  countries, recognising the hydrocarbon reduction benefits, allow tax exemptions on biofuels (Gapes and Gapes 2007 Gapes  2007). ). Europe The European Union intends that biofuels will account for  5.75% (energy value) of automotive fuels by 2010 and 10%  by 2020. To achieve this goal, a tax exemption of up to 100 % on biofuels was implemented by an EU directive in 2003. The development of biofuels production in the EU, Germany and Spain is shown in Fig. 2 Fig.  2 and  and the future goals for some industrialised countries in Table 2 Table  2.. The Eur Europe opean an cou countr ntryy mos mostt ded dedica icated ted to eth ethano anoll utilis uti lisati ation on is Swe Sweden den,, whi which ch has foc focuse usedd on eth ethano anoll  production from crops, sugar cane and wood waste. FFVs are on the market since 2001. The fuel E85 for these cars is sold at 140 public gas stations around the country. Already, in 2005, Sweden achieved its 2009 target (Table 2 (Table  2). ).

various countries (total production) Percentage of   biofuels Australia Austria Canada China France

Sweden UK

2007 2 00 008

2009 2 01 010

2015 2 02 020

1.00 4.30

5.75 3.50

3.00

10.00 5.75

3.00

10.00

7.00 6.25

6.75

8.00 8.

9.00 –  12.00 5.00

100.00 5.00

The largest single ethanol production facility in Europe is loc locate atedd in Zei Zeizz (Ge (Germ rmany) any) where where whe wheat, at, bar barley ley and 3 tritic tri ticale ale sta starch rch are fer fermen mented ted and 260 260,00 ,0000 m /annum etha et hano noll is pr prod oduc uced ed in ad addi diti tion on to DD DDGS GS (s (sol oldd as “Protigrain®”). An extension for sugar beet fermentation is under construction, which will increase total production to 360,000 m3/annum. In Ge Germ rman anyy al alon one, e, 2. 2.55 MT of bi biod odie iese sell ha have ve be been en  produced in 2005 from rapeseed oil grown on 1.3 million hectares of land area and are sold on 1,900 gas stations. Germany allowed tax exemption on biofuels, triggering a high increase in biodiesel production. Recently, the tax exemption, an effective instrument for promoting biofuels, has been replaced in Germany by a law forcing the addition of biofuels to conventional fuels. A new law starts taxation on biodiesel and vegetable oil (as fuel) from 2006 on which will gradually increase up to 0.45   €  per litre in 2012. This should be compared with the taxation of 0.4857   €/l on fossil diesel fuel. The tax exemption for bioethanol still applies because thewas market is not yet regarded mature. Tax exemption not implemented in theasUK, which allows other incentives for biofuels. Consequently, the goal for biofuel biofuelss imp implem lement entati ation on in th thee UK fo forr 202 20200 is comparably low (Table   2). In contrast, Spain and France have made endeavours to promote biofuels and produce  bioethanol as well as biodiesel. Australia The performance of the biofuels market in Australia is poor. Less than 0.1% of the gasoline market was substituted with  bioethanol in 2005. Biodiesel is sold to an even lesser  extent. The reasons for this were the absence of a consumer 

Fig. 2  Increase in production of biofuels for transportation in EU25

countries, Germany and Spain

de dema mand nd fo for r bi biof ofue uels ls in becaus beca usee of Current high hi gh pr pric ices es an andd po poor  or  consumer confidence biofuels. endeavours by

 

32

Appl Microbiol Biotechnol (2007) 77:23 – 35 35

government nment and indu industri stries es aim to reach a 1.0% implemenimplemengover tation tati on of biofuels in 2010. Africa The bio biofue fuell ind indust ustry ry in Af Afric ricaa is mar margin ginal, al, whe wherea reass the  production potential is enormous. According to information from the operating company, a bioethanol plant is presently  being built in South Africa (Bothaville).  North and South America In Brazil, a mixture of ethanol and gasoline is sold as Gasoho Gas oholl or   “gas gasoli oline ne typ typee C”   which which con contai tains ns 21 – 23% 23% EtOH. EtO H. Fif Fiftee teenn bil billio lionn lit litres res of bio bioeth ethano anoll hav havee bee beenn  produced together with bagasse and vinasse (dried distillers grains and solubles) as feed for cattle. However, bagasse from sugar cane has a low nutrition value and is generally used in biogas production to produce additional energy to run the plants. Brazil also plans to achieve a production of 2  billion litres of biodiesel per year by 2020. The USA has surpassed Brazilian production in June 2006 with an installed annual capacity of 18 billion litres bioethanol  productionn and an addi  productio addition tional al 8 bill billion ion litre litress plann planned ed for the near future. The Energy Tax Act (1978) was the primary moti mo tive ve fo forr ta taxx ex exem empt ptio ions ns on bi biof ofuel uelss in th thee US USA. A. In 20 2005 05,, th thee Bush administration announced a strategy to increase biofuel  productio  prod uctionn from 14.8 to 27.8 billion billion litres litres within within 10 years. Only small volumes of biofuel are sold in Canada. The government gover nment began an invest investment ment programme programme to achie achieve ve 3.5% biofuel use by 2010. Specifically, it may support the construction of a large-scale plant for the production of  cellulosic ethanol in the coming years. Asia Because it lacks agricultural acreage, Japan must import its  biofuels. However, a demonstration plant producing ethanol from fr om was waste te woo woodd is und under er con constr struct uction ion.. A bi bilat latera erall cooperation agreement to import bioethanol from Brazil is to be implemented. China, India and Thailand are emerging as significant fuel ethanol producers. In China, the average etha et hano noll pl plan antt si size ze is al alre ready ady 0. 0.38×10 38×103 m3/ann /annum, um, the 3 3 larges lar gestt pla plant nt pro produc ducing ing 1.1×10 m /annu /annum. m. Biodi Biodiesel esel  production is on the agenda of some countries.

Table 3   Bioetha Bioethanol nol production in various countries countries (data from RFA RFA

2007) 2004 (106 m3)

2005 (106 m3)

2006 (106 m3)

Brazil USA China India France Russia

15.09 13.37 3.65 1.75 0.83 0.75

15.99 16.13 3.80 1.70 0.91 0.75

16.99 18.37 3.85 1.90 0.95 0.65

South Africa UK Saudi Arabia Spain Thailand Germany Others Total

0.42 0.40 0.30 0.30 0.28 0.27 3.34 40.75

0.39 0.35 0.12 0.35 0.30 0.43 4.75 45.97

0.39 0.28 0.20 0.46 0.35 0.76 3.55 48.70

Ranking by production in 2004

72.6% was produced in Brazil and the USA. The USA multiplied its output in the past decade and is now the world’s most potent ethanol producer. Table 3 Table  3  lists the ten most important producers of bioethanol, who combined, are responsible for over 91% of the world ’s production. An example of the rapid development of the bioethanol industry is the USA (Fig. 3 (Fig.  3). ). The general tendency to build larger plants can be observed in all countries allowing tax exem ex empt ptio ions ns fo forr bi biof ofuel uels. s. It is an und undisp isput uted ed fa fact ct th that  at   biofuels are presently on the edge of economic viability viability,, and their introduction requires subsidisation to make the industry capable of sustaining itself economically. A ra rapi pidl dlyy ex expa pand ndin ingg ma mark rket et fo forr bi biof ofue uels ls can be  predicted for the near future. The demand of the EU25 coun co untr trie iess fo forr fu fuel el et etha hano noll is es esti tima mate tedd to re reac achh 11. 1.4× 4× 106 m3/annum in 2010, causing a large gap between actual  production and estimated demand based on 5% displacement of conve convention ntional al fuel (on energ energyy basis; IEA 2004 IEA 2004). ). Howev Ho wever er,, the str strong ong dem demand and fo forr bio biofue fuell sub substr strate atess causes competition with food production. Its early effects

Global microbial biofuel production

Biodiesel and bioethanol are the only biofuels produced worldwide in substantial amounts, bioethanol the only fuel 6

3

 by microbial fermentation. About 48.7× 10 m /annum of   bioethanol were produced worldwide in 2005, of which,

Fig. Fi g. 3   Developm Development ent of bio bioeth ethanol anol pro produc duction tion in the USA (RFA (RFA

2007)) 2007

 

Appl Microbiol Biotechnol (2007) 77:23 – 35

33

Table 4   Yields of biofuel per hectare

Country Plant oil Biodiesel

US EU Germany Tanzania Bioethanol USA EU EU Brazil/India/  Tanzania Germany  New Zealand Zeal and  –     –     –     –     –    Biomethane Germany Germany Germany Germany BTL-Fuel

 

– 

 

Crop

GJ fuel/   (ha annum)

Rapeseed Soybean Sunflower/Barley Rapeseed Palm Oil Corn Wheat Sugar Beet Sug ugar ar Ca Cane ne

50.8 16.3 32.5/35.8 50.4 186.0 66.0 52.9 116.3 137. 13 7.5/ 5/1112 12.1 .1/  /  173.0 54.1 36.2 84.6 23.2 – 46.5 46.5 154.4 65.5 – 160.7 160.7 78.2 – 126.9 126.9 71.9 163.4 29.9 217.3

Wheat Corn Stover Switchgrass Corn Stover Miscanthus Switchgrass Poplar Wheat Corn silage Grassland crop energy crops/   residues Ene nerrgy cr crop opss

115 15.3 .3 – 139.7 139.7

The energy yield from microbially produced biofuels based on hectare and year in different countries and on biofuels yields per tonne on crop input. For comparison, some non-microbially produced renewable biofuels are included. Projected data for bioethanol production from projected lignocellulosic lignocellulosic substrates are added (calculation (calculation on data of IEA 2004 IEA  2004,, GTZ 2005 GTZ 2005,, FNR  2007,  2007, Sanderson 2006 Sanderson 2006,, Judd 2003 Judd 2003,, Malhotra 2007 Malhotra  2007 and  and own data).

have been observed in Mexico, causing a starch price too high for people to pay. To minimise such negative effects, a high yield of biofuels must be targeted. In Table 4 Table  4,, different  yields yie lds of bio biofue fuels ls pro produc ductio tionn per hec hectar taree are com compar pared. ed.

Table 5   Further development needs for biofuel development 

 Needs for development  Breeding of climate adapted energy plants for each area Utilisation Utilis ation of cellulo cellulose se and hemic hemicellulos ellulosee (whol (wholee plant) as substr substrate ate Simultaneous fermentation of pentoses and hexoses  New strains strain s or enzymes for biofuel fermentation, e.g. strains with higher fermentation temperature and better product tolerance Improvement Impro vement in energ energyy balanc balancee includi including ng all releva relevant nt factor factors: s: substrate, growth, refinery, product separation, waste management  and disposal, distribution etc.; effect on CO2  balance Standardisation of new types of biofuels and fuel mixes and adaptation to present day engines Development of new engines for the optimised use of biofuels

These data are based on real figures available for common crops with realised industrial processes and prognosis for  new crops and technolo technologie gies. s. The fi figur gures es are based on  biofuel yields per tonne on crop input. To evaluate total greenhouse-gas savings or other environmental consequences, a detailed accounting of life cycles and net energy  balances of the biofuels have to be determined (e.g. made in Hill Hi ll et al al..   2006). 2006). Ho Howev wever er,, dif diffe fere rent nt stu studi dies es yi yield eld in strongly stro ngly diff differing ering resul results ts (Sand (Sanderson erson   2006; 2006; FNR   FNR   2007). 2007). Anyhow, it can be seen that depending on the country of   production, an average fermentative process can be more acreage acrea ge effec effective tive than biod biodiesel iesel prod productio uctionn (FNR   2007; 2007; GTZ   2005; 2005; Hi Hill ll et al.   2006; 2006; Ju Judd dd   2003; 2003; RF RFA A   2007; 2007; Sanderson 2006 Sanderson  2006). ). However, for all these processes, substrate production methods which do not interfere with the production of food must be developed, and further research needs are numerous (Table 5 (Table  5). ).

Conclusion

Biofuels produced from renewable biomass are the sustainable energy resource with the greatest potential for CO 2 neutral neut ral pro product duction ion.. They can easi easily ly be imp implem lemente entedd gradually to supplement fossil fuels by processes such as  blending. Biofuels can be produced from biomass by thermochemical means (e.g. BTL) or by fermentation with microbes. Both seem to be about equally land-efficient. Production maturity will be reached more rapidly by new and (at least   partially) microbially produced biofuels. Biotechnology for future biofuels will include microbial as well as chemical/technical production methods and often a mixture of both. Bioethanol blending to gasoline is only the beginning of these technologies. Biofuel production will have to include the biomass of  entire plants to reach higher yield. This will also reduce competition with food production and nature conservation. Microbial biofuels have great development potential in  process steps such as pretreatment, fermentation, substrate separation, energy coupling and others. Biolo Bi ologi gical cal re resea searc rchh wi will ll nee needd to con contri tribu bute te to an improved biofuel production by breeding of energy plants, enzymatic hydrolysis, specialised fermentation strains and waste treatment. Acknowledgement   We than thankk the Ger German man Res Resear earch ch Fou Foundat ndation ion

(DFG) and the German Agency of Renewable Resources (FNR) for  support to WHS and VVZ. The advice and support of M. Slattery, J. R. Gapes, W. Hiegl, R. Igelspacher and A. Schwarz in preparing, reading and commenting on the text is gratefully acknowledged.

 

34

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