Bioconversion of Sugar Cane Vinasse into Microbial Biomass by

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Chapter 13

Bioconversion of Sugar Cane Vinasse into Microbial Biomass by Recombinant Strains of Aspergillus nidulans André Ο. S. Lima and Aline A. Pizzirani-Kleiner Department of Genetics, University of São Paulo—ESALQ, Piracicaba-SP, 13418-900, Brazil Sugar cane broth is widely used in Brazil in fermentation to produce alcohol. This process yields a large amount of a toxic byproduct, vinasse. Due to its high organic matter, law prohibits its disposal into rivers. The microbial conversion of the vinasse into Single Cell Protein (SCP)/microbial biomass, as a protein and/or methane (fuel) resource, is being evaluated world wide. The present study illustrates an approach to microbial biomass production based on strains of the fungus Aspergillus nidulans produced through parasexual crossing. Six diploid strains and 23 recombinants were evaluated and compared for their ability to degrade vinasse based on measurement of COD reduction and biomass synthesis. SCP production was based on mycelial dry weight, and indirectly on radial colony growth on Petri dishes. Most of the diploids and certain recombinant strains gave enhanced performance compared to the parental strains.

Sugar Cane Vinasse and Its Application Vinasse Production The Brazilian alcohol project began in 1975 as a result of the world oil crisis. At that time, the Brazilian production of ethanol was 320 million liters per year. Production peaked in 1986 rising to nearly 12 billion liters/year, a 37-fold increase. The increase in production was followed by an increase in production of cars that use hydrated alcohol (alcool) as fuel. By 1992, there was a scarity of alcohol, and the country needed to import ethanol to supply the automotive demand. This was

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© 2000 American Chemical Society In Green Chemical Syntheses and Processes; Anastas, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


161 followed a decline in interest in, and production of, alcohol burning cars as well as a decline in the production of hydrated ethanol. In 1994, federal regulations required that 22 percents of anhydrous alcohol should be added to the commercial gasoline, consequently anhydrous production rose. In 1996-97, Brazilian alcohol production reached 14 billion liters, with one third being anhydrous (Marcus Farhat, personal communication). During the alcohol distillation process, vinasse, a byproduct, is formed at approximately 13 parts vinasse to one of alcohol. In the State of Sâo Paulo alone, the largest producing area in the country, this represents 115 billion liters of vinasse per year. Due to its high organic content, vinasse is prohibited from disposal in rivers. Its composition is variable, being influenced by many factors such as the type of sugar, the composition of the broth, and the distillation process per se. However, some features of this byproduct are standard. It is an acidic liquid, rich in nitrogen, potassium and organic acids such as succinic and lactic (7), and containing amino acids, such as leucine, isoleucine and valine (2). It is poor in sugars with just 10 percent of the total amount of organic matter composed of saccharose and fructose (3). The pollution index of Chemical Oxygen Demand (COD) is 35 to 60 grams of oxygen per liter, and there is also a large Biochemical Oxygen Demand (BOD ) which varies from 7 to 20 grams of oxygen per liter. 5

Vinasse Utilization and Bioconversion There are few practical ways to dispose of vinasse. The most common application is the use of vinasse on agricultural lands as fertilizer (4) providing nitrogen and potassium. The effect of vinasse on the activity of soil microorganisms under a range of conditions shows a temporary increase in their activity (5, 6). Diverse studies have compared the optimization of vinasse as a fertilizer, and there are several official recommendations for vinasse application. Processes aimed at using not just the salts but also the organic content of vinasse are being investigated, for instance, the anaerobic conversion of vinasse to methane (7, 8, 9). The aerobic degradation of vinasse to yield microbial Single Cell Protein (SCP) has also attracted attention. In this case, the biomass produced is used as a protein source for animal feed. Bacteria (70, 2), yeast (77, 72, 73, 14) and filamentous fiingi (14, 15, 16, 17, 18, 19, 20, 21) have been evaluated for this purpose. Others applications, including silaging (22), sugar cane bagasse composting and enrichment (23), or burning and use of the ashes as a soil fertilizer (24), have also been evaluated at experimental and pilot scales. The aerobic conversion of vinasse to microbial biomass provides significant environmental benefit as the vinasse's toxicity (BOD and COD) is reduced and its organic fraction utilized in the production of SCP. In the late 1960's, Brazil had two plants using this process, each producing about 3000 tons of SCP per year. The filamentous fungi gave good yield due in part to the secretion of a wide range of enzymes and easy recovery of SCP by filtration. Certain species of the fungi Aspergillus were identified as especially efficient (75,16,17,18, 20, 21, 25). 5

In Green Chemical Syntheses and Processes; Anastas, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

162


The focus of this chapter is the aerobic conversion of vinasse by Aspergillus into SCP. Specifically, parasexual recombination is evaluated as a genetic tool for improving Aspergillus ' ability to consume vinasse. The development work followed three steps. First, the optimal growth medium was established; second, the Aspergillus species were compared; and third, using A. nidulans as the model system, diploids and recombinant strains were constructed and evaluated for vinasse conversion.

Material Methods Species Aspergillus niger NRRL 337; A. oryzae ATCC 7252; A. sulphureus IZ (USP) A186; A. nidulans strains: Master Strain Ε (MSE) [suAl adeE20yA2 adeE20; wA3; galAl; pyroA4; facA303; sB3; nicB8; riboB2] (26)\ kbnc \proAl pabaA6 Τ (I-II); bncA\\ PPY IproAl pabaA6yA]\ B M [biAl; methGl]; INO [inoB]\ V E [veAl] (27). All strains were stocks from ESALQ,-USPfrommineral oil or Si0 . 2

Medium The strains were maintained on Complete Medium (CM) for A. nidulans (28) as modified in (29). The vinasse medium was established in this work; its pH was adjusted to 6.8 with 4N NaOH. For plate assays, 1.5% agar was added. Sugar Cane Molasse and Vinasse The sugar cane molasses was donated by a distillery from the Companhia Industrial e Agricola Ometto, and the vinasse was obtained at the pilot distillery of the Dept. Ciência e Tecnologia Agroindustrial (ESALQAJSP). Both were stored at 1°C. NPK solution The commercial fertilizer NPK (10:10:10) was crushed, solubilized (1%) in distilled water,filtered(Whatman #1), autoclaved (1 atm, 20min.) and stored at 4°C. Radial Growth The strains were point inoculated on Petri plates containing the appropriate medium for each assay and the colony diameter was measured with the help of a light box. Dry Biomass Production The dry biomass production was measured after growing the strain in liquid medium under defined growth conditions - see individual experiments. After cultivation, the mycelium was filtered through nylon screen (95 mesh), washed and dried at 70°C until constant weight.


163 Pollution Indexes The COD as well the BOD were performed as described in (30). The percent decrease was calculated as a function of the control, uninoculated sample.


5

Parasexual Crosses, Diploid and Recombinant strains The parasexual crosses (a process that results in the formation of recombinant nuclei) were conducted as described in (28), in which haploid spores from two genetically contrasting strains (different autotrophs) were cultivated in minimal medium. By this method, diploid cells containing genetic material of both parents were isolated. Recombinant haploid strains were then obtained by the exposure of the diploid to benomyl (benomyl is a agricultural antifungal agent that disrupts the formation of spindle microtubules, inhibiting mitosis and inducing non-disjunction). The diploid condition was confirmed by the ability to grow in minimal medium, by the spore size (data not shown) that is bigger, and by the capacity for the haploid to segregate from the diploid during growth on the plate.

Results and Discussion

Medium Establishment Nutrient supplementation of vinasse has been evaluated by other researchers to assess enhancement of biomass productivity (77, 72, 16, 20, 25, 31, 32). In our initial trials we attempted to optimize the culture media by using a randomly picked strain of A. nidulans (BM) and evaluating its radial growth using vinasse plus different added compounds. Molasses The effects of the addition of sugar cane molasses in increasing concentrations were evaluated in terms of growth and cost of molasses. According to the results (Table 1), optimal relation between radial growth and the cost of the supplement was obtained with the addition of two percent molasses per liter of vinasse (w:v). NPK The addition of a commercial fertilizer with salts of nitrogen, phosphorous and potassium (10:10:10) was evaluated. The vinasse appears to have adequate amounts of these components for microbial development, since a decline in their radial growth was observed when these salts were added to the media (Table 2), even when reduced (1/5) doses were evaluated (data not shown). As a control, fertilizer was replaced with the NPK salts from A. nidulans complete medium. Thus in further assays, vinasse medium was standardized as vinasse supplemented with two percent molasses (w:v).


164 Table 1. The Effect on the Radial Growth of A. nidulans by the Addition of Sugar Cane Molasses to Vinasse 1

Colony Diameter (mm) 24 h 48 h

2


Molasses 0 1 2 3 4 5

3

41 51 54 58 59 60

a b be c c c

079 a 112 b 129 c 134 c 142 d 144 d

'Experimental outline: completely random, split-plot in time (24 and 48 hours), 10 repetitions. Cultivation at 37°C. Molasses (g/lOOmL of vinasse). Letters represent the groups formed by the Tukey test (5% confidence); the same letters = no significant variation. 2

3

Table 2. The Effect on the Radial Growth of A. nidulans by the Addition of NPK solution 1

Colony Diameter (mm) 67 hours 43 hours

NPK(%) 0 2 4 6 8 10 12 CM

103 a 72 b 65 c 51 d 49 d 47 d 50 d 74 b

162 a 137 b 126 b 104 c 107 c 102 cd 093 d 152 a

3

1

Cultivation conditions: 37°C, medium vinasse + 2% molasses (w:v). NPK % (v:v). CM = 6 g NaN0 + 1.5 g KH P0 + 0.5 g KC1 * L* . Experimental outline: completely random, split-plot in time (43 and 67 hours), 16 repetitions. Letters represent the groups formed by the Tukey test (5% confidence), same letters represent no significant variation. 1

3

2

2

4

3

Vinasse Biodégradation by Aspergillus species The next stage was to compare the model A. nidulans (Abnc) with other Aspergillus species that had been assessed to be efficient in vinasse degradation. These species were A niger (9,12,7), A oryzae (4, 9, 12, 24) and A sulphureus (Dr. Choit Kiyan, personal communication).


165 Temperature The first trial was to determine a common temperature for growth of all 4 species. For that purpose we measured the radial growth of the species on solid vinasse medium at three different temperatures (27, 32 and 37°C) twice ( after 35 and 70 hours of cultivation). All the species grew well at 32°C (Table 3). This temperature was subsequently used.


Table 3. Effect of temperature on the radial growth of Aspergillus species

1

Colony Diameter (cm) A. nidulans

Cultivation (h)

°C

35

27

0.63 c

32

0.95 b

70

2

A. oryzae

A. sulphureus

1.56 c

1.70 b

0.49 c

2.40 b

2.46 a

0.77 a 0.64 b

A. niger

37

1.10 a

2.53 a

0.65 c

27

1.96 c

4.27 c

4.19 b

1.24 b

32

2.90 b

6.17 a

5.63 a

1.34 a

37

3.23 a

5.95 b

1.97 c

1.26 ab

'Experimental outline: completely random factorial (temperature X species), two measurements (35 and 70 hours of incubation), 6 repetitions. Cultivation on vinasse medium. Letters represent the groups formed by the Tukey test (5% confidence), same letters represent no significant variation. 2

Biomass Production and Pollution Reduction After establishing the optimal medium and the growth temperature, the 4 species were evaluated for biomass production and COD and BOD reduction. The species were cultured in liquid vinasse medium at 32°C with agitation. The results varied significantly between species (Table 4). Two groups were separated by the Tukey test for biomass production and BOD reduction. As radial growth measurements had low correlation to the dry biomass data, they could not be used as an indicator for others assays in liquid medium; however, the correlation of 0.7 between COD and BOD reduction, enabled the estimation of BOD reduction by that of COD. With the highest ratio between dry biomass produced and the COD/BOD consumed (conversion index [%]), A. nidulans was identified as the best converter (Table 4). However, the aim of the process was not only to produce biomass, but also to consume or degrade vinasse. Thus, a good converter may not be the best producer or dégrader. As an example, with A niger and A. sulphureus, the first showed more productivity and reduction of vinasse than the second, which had better conversion indexes (Table 4). 5

5

5

5

5


166 Table 4. Biomass Production and Pollution Reduction of Vinasse by Aspergillus species 1

COD

BOD

5

Dry Productivity Reduction Conversion Reduction Conversion Biomass ( /uh ) (o/ ) Index(%) (%) Index (%/ (mg) 6.58 55.85 ab 3.06 0.267 A. nidulans 507 a 31.12 c 5.78 61.88 a 1.92 A. niger 0.260 48.27 a 493 a 2

g

our

3

A

0


5

369 b

0.194

A. sulphureus 407 b

0.215

A. oryzae

43.42 b

1.60

57.96 ab

4.61

34.73 c

2.21

50.51 b

5.85

6

1 Incubation Conditions: 7x10 spores, 50 mL vinasse medium (Erlenmeyer 250mL), 32°C, 40 hours, agitation of 120 rpm, 3 replicates. Averagefrom2 digestionsfroma compost sample constituted by the 3 replicates. Conversion index: biomass produced (g) / COD or BOD5 consumed (g) * 100. Averagefrom4 digestions (2 by each dilution)froma compost sample constituted by the 3 replicates. Letters represent the groups formed by the Tukey test (5% confidence), same letters represent no significant variation. 2

3

4

5

Vinasse Conversion by Recombinant Strains of A. nidulans The main aspect of this study was the development and evaluation of some diploid and recombinant strains of A. nidulans for vinasse utilization. Diploids, Recombinants and Genealogy The diploid strains were constructed by parasexual crosses using four genetically contrasting strains as parents, namely M (MSE), Ρ (PPY), Β (BM) and I (INO). The combination of these strains resulted in 6 diploid strains: M//P, M//B, M//I, P//B, P//I and B//I. Four recombinants were isolated by haploidization from each diploid, for example Rec MPI, Rec MP2, etc. The prototrophic haploid strain V E was used as a control. Growth Kinetics Next, a determination was made of the growth kinetics and the time required for the culture to attain the stationary phase of development. We evaluated biomass production (Figure 1) and radial growth (data not shown) of five strains (Μ, Ρ, B, M//P, M//B). The results were analyzed by logistic regression (data not shown). The growth on solid medium was similar to the that observed on liquid vinasse. In both cases, the results suggest that the prototrophic diploid strains started their log phase of growth earlier than the auxotrophic haploids. The control VE was not assayed. No correlation was observed between the strain performance and spore viability (data not shown). The time yielding an average of 90% of the maximum biomass


167


production for all strains was 43 hours. This culture period was used for further cultivation.

Figure 1. Growth Kinetics ofA. nidulans strains cultivated on liquid vinasse medium. Approximately 9 χ JO spores (3 replications) were grow on WOmL of vinasse medium at 37*0 with agitation of 150 rpm. Samples were taken every 12 hours for 60 hours. The results were evaluated by logistic regression analysis. 6

COD Reduction and Biomass Production To compare SCP production and COD reduction four kinds of A. nidulans strains: parental, diploid, recombinant and a control, were grown in liquid vinasse medium. Figure 2, a dispersion graph, shows SCP production and COD reduction for the four strains. While recombinants showed a wide range of performance; nearly all diploid strains, and a few recombinants, showed activity over that of the parents. Analyzing the results, using the Student Newman Keuls test (a=0.05), in which a different letter is assigned for each statistically different group gave the following results. For COD reduction, diploids (group A) were superior, the parentals and the control (group B) were less active, and the recombinants (group C) showed the least activity. For biomass production, the diploids (group A) were again the most active, followed by parentals (group A, B), control (group B) and recombinants (group C). Analyzing some specific strains, Parent I had the best performance of all parent strains; its diploids also seem to be superior to all other diploids (not statistically tested). In the different crosses, however, the activity of their recombinants (Rec M//I, Rec Β/Λ, Rec P//I) varied; for example: the cross between M and I yielded the best recombinants for biomass and COD (Student-Newman-Keuls test, oc=0.05) with an intermediate performance in relation to that of the parents, while the cross between Β and I produced recombinants for biomass and COD (Student-NewmanKeuls test, a=0.05) that were less efficient than the other recombinants and less 1



168 efficient than the parents. The greatest increase was detected in the diploid MP, which had a 50% increase in SCP production as compared to parent M, and a 30% increase in COD reduction as compared with parent P. We conclude that the parent sets used in the crosses have a greater influence on the recombinants than on the diploids as the diploids' overall performance was constant, and they were generally the most efficient. The same was not observed for the recombinants which presented a much greater amplitude of variation. This suggests that enhancement of both measured parameters is more easily obtained by the construction of diploids rather than by recombinants. However, it is important to note that diploids are unstable and tend to segregate to the haploid, undesirable in an industrial process. This condition can be avoided by the use of a recessive lethal mutation, a mutation that when present on a single allele (haploid state) kills the cell and prevents unwanted changes in the fermentation culture.

ί 0

0 φ-Parentals

0 0

φ-Diploids

0

0 - Recombinants φ - Control 10 2.0

3.0

4.0

5.0

6.0

7.0

Dry Biomass (gl) Figure 2. Dry Biomass Production and COD Reduction by A. nidulans strains. Approximately 9 χ J Ο spores (4 replicates) were cultivated in WOmL of vinasse medium at37°C with agitation at 150 rpm for 43 hours. 6

A correlation of 0.87 between dry biomass production and COD reduction was found from the results presented in Figure 2. To determine if the increased


169 performance of the diploids over that of the recombinants was due to the presence of multiple auxotrophic markers in the recombinants, we made a genetic comparison of the recombinants. No correlation was detected between the quantity of genetic markers and their performance (data not shown).


Final Consideration Utilization of alcohol industry by-products such as sugarcane bagasse, straw, yeast filter cake and vinasse, has the potential to reduce both the cost of alcohol production, and the cost to the environment due to their disposal. Due to the enormous volume produced per year, vinasse can be used in different systems of conversion. This study indicates that A. nidulans can be constructed to more optimally convert vinasse to biomass. Using parasexual recombination, it was possible to increase the indices of pollution reduction, and of SCP production by the construction of diploid and recombinant strains. In so doing, we have demonstrated that A nidulans can be used as a genetic model for the study of vinasse degradation. This approach to the synthesis of microbial biomass from vinasse makes possible the transformation of a known pollutant into a benign resource. By reusing the carbon and energy inherent in vinasse via an environmentally friendly biological process, A. nidulans bioconversion produces a useful, non-toxic agricultural feedstock.

Acknowledgments Thanks to Dra. Dejanira F. de Angelis (UNESP- Brazil) for the laboratory facilities, to CAPES (Brazil) for the scholarship and to Dr. Douglas E . Eveleigh (Rutgers University - USA), Gavin Swiatek and James K. McCarthy for the English review.

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Bioconversion of Sugar Cane Vinasse into Microbial Biomass by

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