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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, p. 1017-1023 Vol. 46, No.

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0099-2240/83/111017-07$02.00/0
Copyright © 1983, American Society for Microbiology

System Development for Linked-Fermentation Production of
Solvents from Algal Biomass
J. P. NAKAS,* M. SCHAEDLE, C. M. PARKINSON, C. E. COONLEY, AND S. W. TANENBAUM
State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210
Received 10 May 1983/Accepted 25 August 1983

Five species of the genus Dunaliella (D. tertiolecta, D. primolecta, D. parva, D.
bardawil, and D. salina) were examined for glycerol accumulation, growth rate,
cell density, and protein and chlorophyll content. The suitability of each algal
species for use as a fermentation substrate was judged according to glycerol
accumulation and quantities of neutral solvents produced after sequential bacteri-
al fermentations. When grown in 2 M NaCl, with 24 mM NaHCO3 or 3% CO2 at
28°C and with 10,000 to 15,000 Ix of incident light on two sides of a glass aquarium,
four of the five species tested produced ca. 10 to 20 mg of glycerol per liter of
culture. Clostridium pasteurianum was found to convert an algal biomass mixture
supplemented with 4% glycerol to ca. 16 g of mixed solvents (n-butanol, 1,3-
propanediol, and ethanol) per liter. Acetone-was not detected. Additionally, it has
been demonstrated that Dunaliella concentrates of up to 300-fold can be directly
fermented to an identical pattern of mixed solvents. Overall solvent yields were
reduced by >50% when fermentations were performed in the presence of 2%
NaCl. These results are discussed in terms of practical application in tropical
coastal zones.

The bioconversion of renewable resources to Furthermore, these microorganisms accumulate
neutral fuels and solvents represents a potential- high intracellular concentrations of glycerol,
ly important supplement to future energy needs. which presumably serves as an osmotic balance
Although cellulose represents our most abun- in highly saline environments (3, 4, 8). Our
dant natural biopolymer and is therefore an approach to the useful bioconversion of algal
obvious choice as a fermentation substrate, biomass has focused on this genus and its se-
many problems need to be addressed pertaining quentially linked microbial transformation to
to its delignification pretreatment (18) and to- liquid fuels or to commercially important sol-
ward improvements in cellulase efficiency (1, 12, vents. The demonstration of such linked pro-
13, 17, 21). The use of marine algal biomass as a cesses has been reported (16) in preliminary
fermentation substrate presents some advan- form.
tages for the utilization and bioconversion of a MATERIALS AND METHODS
potentially large renewable resource: (i) algae do
not compete with agricultural or forest crops for Microorganisms. A large number of samples were
available land area; (ii) the use of controlled obtained from naturally anaerobic locations. Isolates
culture systems allows for optimization of were selected for their ability to utilize glycerol as a
growth conditions and harvesting techniques; sole carbon source. Three cultures were eventually
and (iii) utilization of cell cultures can supply, selected based on the analytical detection of neutral
solvents in their growth media. These have been
besides carbohydrates, a nutritionally balanced provisionally identified as a Bacillus sp., a Klebsiella
fermentation mixture rich in nitrogen. It is con- strain, and a Clostridium pasteurianum isolate (ob-
ceivable that coastal areas and saline lakes, tained from D. A. Klein, Colorado State University,
especially in tropical regions, may hold promise Fort Collins). Because of high butanol yields, research
for eventually supplying such rapidly growing focused on C. pasteurianum, and all data reported in
energy crops (18). this paper were obtained with this bacterium. The
The genus Dunaliella represents a group of Dunaliella cultures were obtained from the following
halophilic microalgae possessing unique charac- sources: D. tertiolecta from R. R. Guillard of the
Woods Hole Oceanographic Institution, Woods Hole,
teristics which can be exploited in linked fer- Mass.; D. salina, D. primolecta, and D. parva from
mentations (9, 11, 22). Members of this genus R. C. Starr at the University of Texas, Austin; and D.
lack a cell wall and can be lysed in dilute bardawil from the American Type Culture Collection,
medium (25), thereby eliminating elaborate Rockville, Md. (ATCC 30861).
process development for cell wall breakage. Growth conditions. Algae were grown in a medium
1017

cL xD4. however. especially D. The increase in cell by vigorous aeration. the detector at 235°C.3- propanediol was accomplished using a Finnigan 4000 _ 'mw I- 20 gas chromatograph-mass spectrometer data system :z with a Tekmar accessory (Finnigan/MAT Co. growth rates and the glycerol yield (per which point growth was inhibited. and the culture was mixed low division rate (Fig. the initial pH was ca. which was cultivation. each bacterial strain was transferred several times in Tables 1 and 2 provide a general overview of an algal extract-glycerol broth containing a 10-fold concentration of algae and 2% glycerol.1018 NAKAS ET AL.2 M NaCl. Algae were analyzed for cell count micro. Protein was analyzed by the procedure of NaCl. growth rate. 5. allowing for maxi- mixture (8) supplemented with a 10-fold increase in mum glycerol synthesis and biomass produc- FeEDTA resulting in a final concentration of 15 mM tion.. Dunaliella as potential substrates for subsequent tertiolecta after 5 days of growth. scopically with a standard hemacytometer. it was important to de- mM KH2PO4. with optimum salt concentration for comparison of an initial pH of 7. MICROBIOL. APPL. Mich. The temperature was main.8). 185°C. 0. apparently the result of a from two sides with 10. Norwalk.). 24 mM. 1. 0 Conn.0 within 3 to 5 days and finally reached 9. medium and algal performance are shown in Fig. E 8.5 mM CaCl. .40 quantification was achieved by internal standardiza. 0 _2 LO cl _Z _ sion in distilled water.0 Solvent analyses. with C02-enriched air (3% C02).). Cell densities of the medium contained 2 mM NaHCO3 and was sparged rapidly growing species. salina. The identification of 1. The gas chromatograph was coupled to a Varian CDS-111L chromatography data system. D. some reduction of growth Arnon (2). reached reasonable cell densities only All bacterial cultures were maintained on slants of after 12 days. La San Jose. Analyses were performed on a model 2400 Var- ian gas chromatograph (flame ionization detector) with the column temperature at 190°C. was provided by preliminary data. tion with ethyl acetate. 4-- ous-flow centrifugation at 1. _- O u i. 5 mM MgSO4.). containing 2 M NaCl.5. Bellefonte. 1). essential for rapid growth. Calif. (14). The pH rise was generally proportional to the The more slowly growing species. Inc.500 x g in a Sorvall RC2. the injector at 60 . 2 M NaCI was selected as the the addition of sodium bicarbonate (ca. 12. 0.. From these source. The cultures were therefore tested tryptic soy agar (Difco Laboratories. and . This alga grew adequately in 0.) on days 5 and 12 and harvested after 12 days of except for the C. the glycerol content was carried out in a 100-liter aquarium illuminated per cell increased. as modified by Potty (19). the volume) were optimal.2 to 1. a- raphy with a stainless steel column (1.. Detroit. and a micronutrient termine growth conditions. Effect of NaCl concentration on (A) cell Algal growth studies. Here. Since the purpose of this population density (0) and glycerol content per cell project was to compare five species of the genus (x) and (B) total glycerol production per liter by D.6 M phyll content was determined by the procedure of NaCl. at tration. L 1 2 3 4 RESULTS NoaC Concentration (M) FIG. with. tertiolecta and D. The rapidly growing species main- kept as a spore inoculum over sterile soil. Alternately. 5 mM KNO3. cIV L. Inoculations some characteristics of the five species exam- were adjusted to a final volume of 10% of the fermen- tation mixtures.000 to 15.0 and reached pH 8..82 m by 0. Enrichment with a carbon size was observed microscopically. pasteurianum culture. Pa.000 lx on each side (measured at the surface). ENVIRON.1 bacterial fermentations. Algae were harvested by continu. which in turn was related to a tained between 27 and 30°C. Inc. For D. were near maximum after 5 days. Before tained their maximum cell numbers over the 12- inoculation into unsupplemented algal concentrates. The pH of the solution increased the five Dunaliella species. At this NaCl concen- to 9. whereas the protein content remained Lowry et al. The glycerol content were measured by the method of Ben-Amotz and (per cell) increased linearly between 0. and the carrier gas at 30 ml/min. tertiolecta. and concentrates were prepared by suspen.0 at the end of primolecta.2 and 2 M Avron (4). larger cell size. This increased concentration was relationship between NaCl concentration in the found to be necessary for the nonmarine species of Dunaliella. details of the Fe (Dun medium). unchanged. 1.31 cm) packed with Porapak Q (Supelco Inc. day period. Solvent determinations were performed on culture supernatants by gas chromatog. In this treatment. All fermentations were conducted in 125-ml Erlenmeyer flasks (100-ml volume) for 5 days at 30°C. Chloro. B refrigerated centrifuge (Ivan Sorvall. Glycerol content and glycerol utilization at a 2 M concentration. These relationships shifted dramati- Laboratory-scale mass culture of all algal species cally at 3. 12 days.

77 0.8 0.63 D. lecta due to an elevated cell density. trates of D. glycerol. a greater yield of potentially fer. D. salina 0. protein. tertio.0% ture medium. However.VOL.8 D.99 10.4% total solvents after fermentation.1 0. D.6 15. densities were slightly higher. Such concentrates are viscous as the both media.00 4. result of high polymer content but were found to The data presented in Table 2 lists the glycer.53 6. and this substrate level was poorly in the C02-sparged medium and pro.0%) but produced the highest distribution into medium. tertiolecta and D. 300-fold concentrates of duced the lowest glycerol yield of all cultures in the algae.30 1.45 2. biomass resulted in less total solvent production When 3% CO2 was bubbled through the growth (1. Although D. and the resultant total glycerol The fermentation of D. mately twice the protein when compared with plied with 3% CO2 was more rapid. be acceptable for flask fermentation. salina increased butanol. with butanol comprising approximately mentable carbon was obtained from D. Composition of 300-fold concentrates of five Dunaliella species grown in Dun medium with 24 mM NaHCO3 or 3% CO2 as a carbon source Composition per ml of concentratea Species Glycerol (mg) Protein (mg) Chlorophyll (mg) NaHCO3 CO2 air NaHCO3 CO2 air NaHCO3 CO2 air D.5 12.94 7.2 D.74 5. In the first series of experi- yields of glycerol per milliliter of culture medium ments.5 3.86 8.13 9. salina cell ucts. parva 2.6 7. salina 1. When grown with 3% be comparable to those obtained with carbohy- TABLE 2.80 0.8 9. D. parva yield was only half the level of the other algae. salina. pasteurianum was studied as a function of The highest cell density was exhibited by D.58 2.90 4. Except for D. by almost fourfold. bardawil 0. D. tertiolecta was used in most later yielded 1% total solvents in the fermentation fermentation linkage feasibility and optimization broth.98 1. primolecta 2. In bicarbonate medium. ined in 24 mM bicarbonate or in 3% C02.2 3.3 22.5 1.2 2.79 19.6 0.10 D.7 21.30 D. but the total glycerol yield Solvent yields were substantial at a 1% glycer- increased by only 20%.9 12. overall Fermentations.0 0.9 D.6 D.23 21.77 4. 46.83 5. growth in media sup. the cell density of D. by C. respectively. 1983 PRODUCTION OF SOLVENTS FROM ALGAL BIOMASS 1019 TABLE 1. D. however.96 5.77 2. The solvent yield var- tertiolecta in both 3% CO2 medium and the ied both with algal species and with glycerol bicarbonate medium (Table 1).9 4. tertiolecta produced equivalent (>16 g/liter) was obtained using 10-fold concen- amounts of glycerol per milliliter of either cul. the algae grown with concentration of the cellular photosynthate to bicarbonate produced comparable amounts of achieve subsequent solvent yields which might all three components.08 21.17 3. and final cell other Dunaliella species. parva as the fermentable substrate reason.6% bardawil and D. For this using D.36 8.75 and 0.38 1.53 6. tertiolecta 5. species tested.57 1. Also. C02. parva 4. The highest solvent yield of 1.5 a 5 Cell densities were obtained after days of growth (8 days for D. salina) in 100-liter aquaria.30 1. 0.26 1.70 1. the fermentation of Dunaliella biomass were almost identical.43 3. salina both yielded in counts were the lowest of all five Dunaliella excess of 1.06 4. parva grew ol supplementation.43 3.28 6.55 D. tertiolecta 2. Comparison of five Dunaliella species grown in Dun medium with 24 mM NaHCO3 or 3% CO2 as a carbon source Cell no a (106/ml) Glycerol (pg/cell) Chlorophyll (pg/cell) Glycerol (mg/liter of Species ___________ __________ __________ culture medium) NaHCO3 CO2 air NaHCO3 CO2 air NaHCO3 CO2 air NaHCO3 CO2 air D.25 13. bardawil 5. added glycerol (Table 3). half of the fermentation products. . found to occur in Ca.6 0.36 18. and chlorophyll contents of algal The use of algal biomass and accumulated concentrates grown with bicarbonate versus 3% glycerol as fermentation substrate necessitates CO2. bardawil supplemented with 4. However.52 19. concentration.61 5. with butanol comprising 93% of the prod- studies.1 1.24 4.15 4. primolecta and D. primolecta 5.58 a Normalized to a 300-fold concentrate of the original culture. Generally.94%.94 6.18 3. bardawil produced approxi- respectively. ol.60 27.

21 1.3-propanediol.01 0.45 4.0 2. Klebsiella sp.01 0.12 1.80 1. a concentration shown).14 0.76 6.02 0. ENVIRON.22 0.5 0.81 0.3-Propanediol Ethanol Acetate (gAliter) D.78 0.0 6.04 D. bardawil biomass.04 0.15 1.52 2.01 0.01 0.06 0.01 0.0 4. providing 6. pasteurianum is shown ber of the genus Bacillus. late fermentation.36 0.5 g/liter.42 4. In each case.01 0.62 0. converts glycerol- in Table 4. bardawil never reached high inhibition at 3% NaCl.33 0.0 0.09 0.5 g of mixed solvent per liter at a 300-fold concentration.O1b 0. Considerable variation in cell pasteurianum is shown in Table 5.71 1.09 0.01 0.71 0.01 0.18 0.0 0. This can most likely be attributed However. drates derived from more traditional biomass vided a fermentable biomass substrate with salt resources. MICROBIOL.01 0. salina despite an demonstrate a severe inhibition (>50%) of sol.37 0.28 0.0 0.0 4.15 0. Unsupplemented algal biomass was amended algal mixtures into ethanol to a final harvested and concentrated 10-.0 4.12 0. concentration of 7.18 14.01 0.0 9.14 0.5 1. tertiolecta 158.5 0.28 6.0 0. parva 104.17 0.25 1. verts the algal biomass-glycerol supplement tration) ranged from 2 to 6.01 0.5 0.38 0.1020 NAKAS ET AL. Standard errors were always less than ±5%.21 14.0 4.15 D.19 0. and 300.62 2. phically grown biomass to neutral solvent end A soil isolate.90 0.01 0. Comparison of solvent production: C.02 10.78 0.42 0. with the solely to 1.01 0.01 0.08 0.24 0.30 0.9 . bardawil served as the best unamended substrate. attempt to optimize media and culture condi- vent formation at 2% NaCl and almost total tions.68 0.01 0.15 0.um) which reflected harvesting procedures used in this research pro.08 0. bardawil 176.2 to 5.0 1.0 7.28 1.47 0.47 2.06 0.42 0.0 1. increased glycerol concentrations per cell vol- . Solvent concentrations cell densities under any of the stated conditions. isolated from sewage sludge con- Total solvents produced (at a 300-fold concen.79 16.04 10.80 1.01 0. These alternate transformations of the of at least 150-fold algae was required to stimu.20 0.11 0.5 0.45 0.21 0.36 4.81 0. erol at 2 M NaCl. actually increased to peak levels (12 g of total the yield of glycerol per volume of culture solvents per liter) at 1% NaCl and declined medium was comparable to that of other Duna- sharply at salt concentrations approaching 2%.53 0. tentatively classified as a mem- product formation by C.3-propanediol (trimethylene glycol) at major products distributed between butanol and a final concentration of 4. These data yield was obtained with D. primolecta 108.33 2.36 1.36 0.19 D.94 0.0 to 9. salina 65. The actual linkage of photolithotro. D.87 1.37a 4. b Limit of detection.0 1. Additionally.03 D.02 0. APPL.35 2. pasteurianum with each species of Dunaliella (10-fold concentrate) with added glycerol Algal source Protein content % Added Product distribution (g/liter) Total solvent production (mg/iter) glycerol n-Butanol 1. liella species.0 7.02 0.0 O. 150-.64 a Values represent the mean of three replicates. it should be emphasized that the to a larger cell size (11 ± 1. Although D. DISCUSSION These data coincided with elevated protein and All five species of the genus Dunaliella grew glycerol values for D.3 g/liter (data not 1.16 0. TABLE 3.0 4.01 1.09 0.0 0.0 6.26 0. well and produced near-maximum levels of glyc- The effect of salt on solvent formation by C. algal photosynthate are under investigation. a fold and served as the fermentable substrate.70 1. concentrations of below 1%.25 0.06 1.71 0.6 g/liter.

thus differing bardawil might be more suitable. D.20 a Values represent the mean of three replicates. Standard errors were always less than ±+5%.24 0.28 2.9 0. The fermentation of algal biomass was ra To develop an operational fermentation sys- and efficient based upon glycerol as the ma tem based on Dunaliella species.5 1. able solvents.01 0. 46. mentation substrate. Acetone was not detected.01 0.34 1. and glycerol accumulations.81 0. D.01 0. With 300-fold algal concen- The selection of an alga for mass cultivat trates. be possible species of cho stration of a direct linkage between a halophilic for biomass production. The possibil markedly from the classic butanol-acetone-etha- of commercial cultivation of Dunaliella spec nol fermentation that uses a number of well- as energy crops in marginal. 23).40 97.8 0. and ethanol was biomass.34 D.13a 0. slower-growing in butanol and 1.90 96.17 150 21.05 0.10 0.10 0. bardawil 300 111.15 10 2.26 3.10 D.54 96.50 0.VOL.15 0.7 0. In an intensive system wh species.07 0. produces a solvent mixture rich opment is not critical.40 98. These efficiencies may be ex- D.01 0.57 150 54. In these studies.17 0.77 0.06 1.4 0.06 0.1 0.01 0.50 95.01 0.35 97. D.66 93.35 95. utilizing algal concentrates Fold Initial glycerol % Glycerol Alga Product distribution (g/liter) 1EhnlAeae Total solvent production concn ga A concn (Lmol/ml) (>mol/m utilization utilization n-Butanol 1.01b 0.2 0. a rapidly divid The fermentation of Dunaliella biomass and species such as D.90 6.74 95.73 2.80 93.02 0.43 0.02 0. parable amounts of glycerol per cell. Solvent production by C. appeared to be well-suited plained by the high protein and general nutrient biomass production after acclimation to hi.94 10 1. primolecta a menting bacteria.15 0.07 0. bardawil biomass will be to a large extent determined by the t) produced approximately twice as much n-buta- of cultivation system used and the quality nol as did substratum from other Dunaliella water available. tertiolecta. ume.67 1. 16.26 150 25. tertiolecta.16 150 31.05 0. D.3 0. brackish envir described clostridia and easily fermentable sub- ments was given impetus by the data in Tabl strates such as molasses or corn steep liquor which demonstrated comparable growth rat (20). the fermentation of D.11 10 1.6 1.06 1.66 0. The two marine species.01 0.01 1. In similar amount of glycerol'in comparison w experiments designed to test direct bioconver- D. did produci classic butanol fermentation (15.01 0. 80 to 100% of the accumula tance. However.16 0.20 0.11 0. tertiolecta would be advan accumulated glycerol (Tables 3 and 4) with a geous.62 0.73 97.87 0.9 1. bardawil biomass provided the best fermentation si again demonstrated superior potential as a fer- strate.23 150 23.29 0.54 0.20 2.3-propanediol. With 300- salt conditions. salt tolerance fermentation component. b Limit of detection.08 0. content of the available algal biomass.22 3. solvent production was relatively easy to cultivate under both bicarb4 roughly equivalent to the yield from a 1% glycer- ate and forced-air conditions and provided cc ol-supplemented medium (Table 2). growth rate is important.0 0. pasteurianum.3-Propanediol Ethanol Acetate (g/liter) D.19 D.46 0.30 0. parva 300 61.20 94.1 0. TI The novelty of this research lies in the demon- would. Since washing of the osmotically sensi- glycerol was converted to solvents by the I tive algae after collection appears to be impracti- .3 0.20 2. bardav and photolithotrophic biomass resource and the despite lower final cell densities. primolecta 300 49.12 0. tertiolecta 300 50.01 0.08 0.00 94.16 D.5 1.1 2.23 10 1.02 0.89 0. These two species proved to fold algal concentrates.05 0.61 0. using usually present at less than 10% of the recover- carbonate versus CO2 as the carbon source.25 95.17 10 3. but if the time required for culture dev Clostridium sp.30 96. therefore. ca. 1983 PRODUCTION OF SOLVENTS FROM ALGAL BIOMASS 1021 TABLE 4. barda sion of algal biomass (Table 4). When calculated o by the bacterial fermenters is of crucial impor- molar basis.01 0.02 0.97 0. salina 300 47.

Chen. use of 10-fold algal concentrates supplemented 7. it (%) nol panediol nol (g/liter) tate would be conceivable to begin to examine cost 0. Evaluation of substrates for butanol production. Gay. Econ. 4% glycerol.49 7. Process development substrate has routinely produced 10 to 20 g of and evaluation for algal glycerol production. Soc. Ser. tained from more traditional biomass resources 14. The algal concentrate contains 16 38:176-188. M. 1979. the use of food crops as fermentation substrates cal. This wide range of values reflects differences in pelleted cell densi. I.5 algal system. pasteurianum and 10-fold concentrates of medium by the application of suitable membrane D. Can.93 0.42 1. this linked algal system needs fur.. Monot. The use of cum strains producing butanol and acetone.. The potential of systems integration in version of highly proteinaceous biomass to fatty microbial photosynthesis. J.0 3. the Bioeng. molasses or pure glucose as a fermentation 6. In our investigation. Isolation of Clostridium acetobutyli- Clostridium acetobutylicum (20). 8. 4.22 linked-fermentation process. Biochem. Appi. Plant Physiol. and the food-versus-fuel relatively high NaCl concentrations. (5.53 0.22 comparisons. Protein measurement with the Folin phenol solvents can be obtained with the addition of 3 to reagent. Environ. B. and J. Ben-Amotz. L. Trans.0% and total inhibition at 3. acetobutylicum in a synthetic medium. Sakajoh. especially in tropical coastal ar- 0. Microbiol. E. concentrations in the actual fermentation medi. sion in Escherichia coli. Biol. A. 10. which can be utilized for cell growth as tion. 23:1267-1287. 11. 181:25-53. 1978. p. Environ.1 5.). in less-developed countries. 12. The LITERATURE CITED data presented in Table 5 indicate an increase in 1. Brown. Hanselmann. and erol) can be increased to 3 to 4%. W. Lett. Humphrey. to evaluate the economic feasibility of this 0. Glycerol as a Solvent yields might be further enhanced by photosynthetic product in Dunaliella tertiolecta. Griffith.83 1. and C. 2:111-116. Acetone-butyl alcohol fermenta- trate.0%. Biotechnol. Chi. Lignochemicals. 1969. 51:875-878. grant 800 1204). Effects of NaCl on solvent formation.07 0. which is well within the tolerance of the fermenting bacteria used. TABLE 5.37 2. ACKNOWLEDGMENT ty during harvesting of the algae.94 0. A. If the level of rials to useful products.23 2. A. In this regard. J. Chem. dures for optimal biomass yield.74 10. 1982. Demain. Also.88a 0.0 0. 24:1-15. Although similar levels of total Randall. S.43 1. G. The highest This research was supported by a grant from the U. the now classical butanol fermentation with 5. J. stocks (6. 1973. A..92 9. L. D. McLachlan. Chem.0 7. 21). 24). Environ.Ace. the biocon. and M. Molecular solvent production to 1.95 eas.81 0. 43:1125-1132. The role of glycerol The feasibility of the described fermentation in the osmotic regulation of the halophilic alga Dunaliella process is best evaluated by comparison with parva. Biochemi- salt removal or the isolation of solvent-produc. Biotechnol. um reached 0.0% salt. 1982.2 M. 1979.0 4. 15. 9. The hydrolysis of cellulosic mate- acids is worthy of further study. C. Cragie. Dev. and R. H.04 0.56 12. the final algal biomass will always contain has questionable merit. Microbiol.85 2. F. mixed solvents per liter. Armentrout. the recovery of glycerol from the extracellular using C. and W. Microbiol. Etha.-R.. with 4% glycerol resulted in 14 to 16 g of mixed 20:509-517. Halliwell. N. APPL. 191-209.5 7. Ben-Amotz. H. ENVIRON. H. trates. Experientia lytic activity.59 1. 7. At this point. the solvent A. M NaCl after concentration. H. solvents per liter. fermentable substrates (algal biomass and glyc. salt-tolerant clostridia. T. E.. L.. thermocellum. A. Academic Press. 1.45 12. Acetone and butanot production by Clostridium ther research to facilitate the processing proce. 1982. 13. J. In A. 1951.S. 7:94-98. San Pietro (ed. well as for solvent formation. Copper enzymes in isolated chloro- plasts.39 2. Adv. Avron. C. Compere. isolating related bacteria with elevated proteo.42 0. Farr. J. Bacteriol. was observed in our studies with algal concen.. 1981.98 solvent-producing bacteria adds a new dimen- 3. Department of Agriculture Science and Education Adminis- tration (Alcohol Fuels Research Division. Ind. 0.69 sion to the concept of using renewable resources as petroleum substitutes and chemical feed- a Data represent the mean of duplicate analyses. 10. Calam. ing. Heden. the success of large-scale algal cultivation 3.14 3. . 2. and R. Rosebrough. W. production to 3 to 4% at low energy input. Appl. 1949. 44:1318-1324. A. Madia. Hastings. Glycerol production in the alga linked to bacterial fermentations may depend on Dunaliella.08 0. tertiolecta or other separation techniques (18) could in- Product distribution (g/liter) Total solvent crease the fermentable substrate concentration NaCl Buta. Although no salt inhibition of solvent production 41:1355-1362. J.1. cal and photosynthetic aspects of energy production. Microbiol. J. L. 4:31-45. MICROBIOL.1022 NAKAS ET AL. New York. Johnson. 1979. K. Arnon. to 21 mg of protein per ml of 350-fold concen. Saccharification of complex cellulos- ic substrates by the cellulase system from Clostridium yields would then be comparable to results ob. R. 1980. and R. 1980. Microbiol. Also. 1981.01 0. with partial cloning of genes for cellobiose utilization and their expres- inhibition at 2. Lowry. A. 193:265-275.. Martin. J.06 The use of algal biomass as a substrate for 2. Plant Physiol. Our algal argument need not be addressed in this type of biomass generally contained between 0. Appl. G. 42:777-778.3-Pro.. D. J. Inc. Petitdemange.1 and 0.

Production of Hardy. J. tion from halophilic. 188-209. In H. Environ. W. J. 1978. 1982. Applied Derepression of an NAD-linked dehydrogenase that Science Publishers. 1. and D. Heden. Ng. K. C. Envi. 29:535-539. E. 1959. Microbiol. 152:1001-1007. G. T... C. Martin.. 41:1337-1343. Bioeng. 10:125-134. L. New York. Kuhn.. 3rd ed. Woods. 1969. Appl. 19. Zeikus. Perlman (ed. H. C. Bacteriol. E. A. p. M. and J. L. and D. photolithotrophically grown algae by Bioeng. C. 2nd ed. Appl. Parkinson. linked fermentations.VOL. 1981. O'Neil.. St.. and C. Walton. Solar bioconversion systems based on algal ology. Energy from biomass. vol. In A. Microbiol. 22. Prescott. 8:115- 21. 1980. R. A. K. Science butanol-acetone by fermentation.-T. production by thermophilic bacteria: fermentation of cel. um acetobutylicum. and J. D. W. Ng. Industrial microbi. and S. McDonald. 25. glycerol production. L.. Dunn. Nakas. T. Bery. Foo. 1983. lulosic substrates by cocultures of Clostridium thermocel. Ethanol J. A. S. fication of cellulose by acid hydrolyses. Schleser (ed. Production of feedstock chemicals. Chartier. 1983. I. and C. Williams.). 1983 PRODUCTION OF SOLVENTS FROM ALGAL BIOMASS 1023 16. and R. 46. C. C. 1979. Symp. K. T. 20. Busche.). McGraw-Hill Book Co. Jones. Roberts. 24. Schaedle. Academic Press. J. Coon. Tang. Symp. and E.. Foo. and D. S. p. Determination of proteins in the pres. 1982. 298-302. Biochem. Biotechnol. Martin. M. . C. Van Der Westhuizen. 44:1277- ron. R. J. Strub. K. 1281. Colcord. Tanenbaum. M.. T. Process optimization for sacchari- ley. G. M. Lin. and G. Anal. 23. Sondhi. R. Biotechnol. Potty. 219:733-739. Peppler. Neutral solvent produc. New York. C. 18. ence of phenols and pectins. P. V. New York. M. D. J. serves an Escherichia coli mutant for growth on glycerol. A. P. E. A. Microbial technology. S... Ben-Bassat. Autolytic activity and butanol tolerance of Clostridi- lum and Clostridium thermohydrosulfuricum. Inc. G. R. 130. 17.