Letters in Applied Microbiology 1990, 10, 183-186 GWG/83

Microbial growth on peptones from fish industrial wastes

SUSAN E. VECHT-LIFSHITZ*, K . A . ALMAst & E. ZOMER Biotechnology Unit, Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91904, Israel and ?Hydro Marine Chemicals, Research and Development Department, P.O. Box 3271, Gronnasen, 9001 Tromso, N o r w a y

Received 13 November 1989 and accepted 11 December 1989

VECHT-LIFSHITZ, S.E., A L M A S , K . A . & ZOMER, E. 1990. Microbial growth on peptones from fish industrial wastes. Letters in Applied Microbiology 10, 183-186.

Industrial fish peptone was an excellent substrate for biomass production in solid and submerged fermentations. The maximal growth rates of several micro- organisms were two to three times higher than those grown on beef (bacto)peptones and the final biomass concentrations were almost twice as great as those grown on beef peptones. Fish peptones did not increase the production of secondary metabo- lites relative to those produced on beef peptones in non-optimized media. Fish peptone has promising potential as a substrate for biomass production.

Growth substrates constitute a major cost in the production of microbial cells and bioproducts, and the nitrogen source tends to be the most expensive medium constituent. At present, com- mercial nitrogen sources come from casein digests, slaughterhouse waste and vegetable extracts. Despite its ubiquity and low price, fish extract has only been used to a minor extent. A recent study (Jassim et al. 1988) showed that fish wastes could be used to produce fast bacterial growth and increased production of staphylo- coccal toxin relative to media containing beef peptone.

Here we report the use of a standardized commercially available fish peptone for pro- duction of biomass and bioproducts. This peptone was prepared by autolysis of viscera (Raa & Gilberg 1976) followed by ultrafiltration and permeate concentration (Gilberg & Almas 1986).

Materials and Methods

MICRO-ORGANISMS

Streptomyces tendae ATCC 31 160, Gibberella jiujikuroi, Bacillus thuringiensis var. israliensis 1884 (B.t.i.) and Bacillus sphaericus 2362 (B.s.)

* Corresponding author.

were provided by the Biotechnology Unit, Life Sciences Institute, Hebrew University of Jerusa- lem, Jerusalem 91904, Israel. The following standard strains were provided by the Depart- ment of Clinical Microbiology, Hadassah Medican Centre, Jerusalem, Israel: Escherichia coli B, Salmonella typhi, Staphylococcus aureus, Serratia marcescens and Proteus vulgaris.

MEDIA

Agar plates were prepared as follows (g/l): beef extract (Difco), 3; agar (Difco), 20; with either bactopeptone (Difco), 5, or 11.7 ml/l fish peptone (Marine Biochemicals, 42% solution).

Media for submerged culture consisted of (g/l): Beef Extract (Difco), 3 ; Yeast Extract (Biolife), 2; and either bactopeptone (Difco), 5, or 11.7 ml/l fish peptone (Marine Biochemicals 42%), made up to the liter with distilled water. The pH values of media for agar plates and for submerged culture were adjusted to 7.0 for all strains except Gibberella fujikuroi (pH 5 5 ) , and Streptomyces tendae (pH 7.5).

PLATE SPREADING METHOD

Experiments were run in duplicate. A flame- sterilized loop was used for streaking in three directions to effect a dilution in the concentra- tion of cells per unit area. The diameters of 10

184 Susan E . Vecht-Lgshitz et al. colonies were measured per plate, using a Fischer-Lilly antibiotic zone reader. Measure- ments were taken at 24,48 and 72 h.

S E R I E S DILUTJONS

Cultures were diluted with sterile distilled water in tubes at a ratio of 1 : 10. Four dilutions were made for colony count and measurements:

and of the initial culture. Aliquots of 0.2 ml from each were spread on agar plates (10 cm diameter), using a sterile glass rod (Dragalsky).

S U B M E R G E D F E R M E N T A T I O N S

Broths from 50 ml overnight shake-flask cul- tures were used as inocula. The volume of the inoculum used was calculated such that the initial A,,, was 005. Duplicate experiments were run in Erlenmeyer flasks containing 100 ml media at 30"C, agitated at 200 rev/min on a gyrotary shaker (New Brunswick Scientific Co.). The growth was monitored by turbidity measurements at 600 nm.

B I O A S S A Y S

Bioassays for lethal doses of B.t.i. and B.s. were performed according to McLaughlin et a!. (1983). Assay for gibberellic acid from G. fujiku-

roi was as described by Theriault et al. (1961). Assay for nikkomycin potency (chitin synthase inhibition) in Streptomyces tendae cultures was performed according to Vecht-Lifshitz et al. (1989).

Results and Discussion

A comparison of the size of colonies on agar plates containing either fish peptone or bacto- peptone indicated that fish peptone enhanced growth up to 221% more than bactopeptone after 24 h, and up to 170% more after 48 h (Table 1). After 72 h there was little significant difference in the biomass concentration of cells grown on the two different peptones (+6% on the fish peptone). These results suggest that this fish peptone greatly enhances growth of colo- nies on agar plates, implying that this peptone could be used as a substrate in clinical micro- biological media where fast growth is required for eflicient diagnoses.

In submerged culture, little difference was observed in the first three hours of growth between cultures grown on fish peptone media and bactopeptone media. During the exponen- tial growth phase (3-8 h approximately), the maximal growth rate on fish peptone was up to 5.5 times as great as that on bactopeptone

Table 1. Comparison of colony size on agar plates containing either fish peptone or bactopeptone

Colony size (mm)

24 h 48 h 72 h

Fish Bacto- Fish Bacto- Fish Bacto- Micro-organism peptone peptone peptone peptone peptone peptone

a b 0.87 0.57 0.82 0.64 a Salmonella typhi 1.00 0.41 1.53 1.45 b 1.35 0.88 1.72 1.39

Staphylococcus aureus a - - 0.22 0.17 b 0.21 0.19 0.62 0.23 a Serratia marcescens 1.12 0.59 b 1.15 0.65 1.41 1.10 a Proteus oulgaris - - 0.46 0.30 b 063 0.19 1.82 1.04

var israliensis b 6.07 2.95 6.10 4.46 Bacillus sphaericus a 0.48 0.47 1.15 0.84

b 1.07 0.49 2.32 0.97 a b - - 0.12 0.10 3.60 3.41 a b - - 2.78 2.15 4.10 3.88

- - - - - - Escherichia coli - - - - - - - - - -

- ~ - - - - - - -

- - - - Bacillus thuringiensis a 2.82 1.12 - - - - - -

Gibberella fujikuroi - - - - - -

Streptomyces tendae - - - - - -

Size given as an average of 10 colonies. a, agar plates inoculated by plate spreading method; b, agar plates inoculated using series dilution method.

Fish peptones in fermentation media 185

media and the total growth was up to 3.2 times as great (Table 2 and Fig. 1). There was a sig- nificant increase in biomass concentration in all cultures grown on fish peptone media relative to that of those on bactopeptone medium. These results indicate that this fish peptone may be used as a substrate for growth in industrial fer- mentations, particularly where fast growth and dense biomass concentrations are required.

The study reported by Jassim e t al. (1988) showed that their fish extract yielded similar growth rates of Staphylococcus aureus in fish and beef peptones in submerged cultures. In contrast, it was observed here that the growth rate of this species was about double on fish peptone (Table 2). This discrepancy was prob- ably due to differences in the fish peptone com- position and different production methodology.

Neither the fish peptone medium nor the bac- topeptone medium proved to be optimal for production of secondary metabolites and there was little significant difference between them. This could be explained by there being a surplus of nitrogen which inhibited the onset of second- ary metabolism. Nikkomycin production was 1 W O times greater in optimized media (Vecht- Lifshitz e t al. 1989kup to 4 g/1 compared with only 0.1-0.2 g/l nikkomycins on fish and bacto- peptone media.

Jassim et al. (1988) demonstrated that toxin production was enhanced synergistically with addition of fish peptone and proteose peptone to the culture medium. Whereas, gibberellic acid production on either fish or bactopeptone media was low (25-40 mg/ml) compared to 50 mg/ml on standard sucrose media (as described by Borrow et al. 1964). High concen- trations of biomass of G. fujikuroi were quickly

fiL

Time ( h )

Fig. 1. Growth curves of micro-organisms in shdke- flask culture on fish peptone media (W) and on bacto- peptone media (0). (a) Bacillus sphaericus; (b) Escherichia cufi; (c) Streptumyces tendue.

Table 2. Comparison of growth rates and total microbial growth in submerged culture in fish peptone and on bactoDeDtone

Micro-organism

Escherichia coli Salmonella typhi Staphylococcus aureus Serratia marcescens Proteus vulgaris Bacillus thuringiensis

var israliensis Bacillus sphaericus Gibberella fujikuroi Streotomvces tendae

Maximal growth rate (6A,,,/h)

Fish peptone Bactopeptone

0.84 0.21 0.58 0.25 0.79 0.40 0.94 0.57 0.44 0.39 1.01 0.23

1.09 0.19 1.24 0.84 1.26 0.24

Total growth (48 h) (AGs0)

Fish peptone Bactopeptone

5.95 2.61 4.20 2.15 6.50 2.04 3.31 1.94 3.12 1.70 5.21 3.18

5.81 2.53 0.39 0.35 0.54 1.25

186 Susan E. Vecht-Lifhitz et al. obtained (1.11 g/1 in 48 h, Table 2) on fish peptone. This suggests that the fish peptone could be used in the first stage of a two-step production process, where the first stage would be for biomass production and the second stage for gibberellic acid production.

Bioassays of toxin production by B.t.i. and B.s. showed that there was no significant differ- ence between the toxicity of samples from fish peptone media and from bactopeptone media. These media have to be modified and optimised for production of secondary metabolites.

The fish peptone has other advantages which should allow its use in industrial fermentations. Firstly, it is relatively cheap as it is a waste product of the fish industry. Secondly, it is sterile and has high clarity, having been filtered through a membrane system with a 10 kdal cutoff (Clausen et al. 1985), and is highly soluble in water. These properties may also reduce the number of stages in downstream processing required.

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