[1]

Economic liquid growth medium development for high-rate production of

cellular biomass and lactic acid of Lactococcus lactis
M.-P. Zacharof*1,2,3 and R.W. Lovitt1,2,3

Centre for Complex Fluid Processing (CCFP), College of Engineering, Swansea University, Talbot building,
Swansea, SA2 8PP, UK
2
Multidisciplinary Nanotechnology Center (MNC), College of Engineering, Swansea University, Swansea,
Talbot building SA2 8PP, UK
3
Centre for Water Advanced Technologies and Environmental Research (CWATER), College of Engineering,
Talbot building, Swansea University, Swansea, SA2 8PP, UK
*Corresponding author: e-mail:myrtozacharof1981@yahoo.com, Phone: +447413541769

Abstract

L.lactis importance in the alimentary and pharmaceutical industry as a lactic acid and nisin production strain has been well
established. However for its growth, nutrient media such as M17 and Man de Rogosa Sharp (MRS) are used, which are
not suitable for use in large quantities due to the high preparation cost and content of nitrogen sources of beef extract and
peptone from poultry that are not environmentally friendly and have possible health risks. For its intensive propagation
and high rate lactic acid production an economic liquid growth medium was formulated, using Lactococcus lactis NCIMB
700185. To test medium efficiency, the coccus was grown in a 2 L CSTR with continuous temperature and pH control.
The developed medium supported evenly cellular growth offering elevated maximum yields incorporating only the
chemicals compounds needed, resulting in an improvement of the growth rate of the coccus of 70.6%, when compared to
the same strain on MRS, the lactic acid concentration produced was 30.28 g L-1.
Keywords: Lactic acid bacteria, Lactococcus lactis, Nutrient media, Biomass, Fermentation, Lactic acid

1. Introduction
Lactic Acid Bacteria (LAB) are microorganisms, of great industrial importance, related majorly to alimentary
and pharmaceutical industry. Their distinctive ability is lactic acid production, as a major end product of their
metabolism, through fermentation, that is the anaerobic catabolism of carbon compounds existing into several
types of substrates. Of this group, Lactococcus Lactis (L.lactis) graded as a non pathogenic bacterium for
humans (Generally Regarded As Safe, GRAS) [1], has been intensively used in the food industry, as a natural
acidproduction bioreactor. This bacterium, mainly employed as a starter culture, can be inoculated in a
variety of food products, assuring the production of enough lactic acid [2], which will lower the pH to an
acidic point [8] inhibiting the development of other pathogenic microbial strains and helping the preservation
for longer periods of the products. At this point a secondary starter culture of different microbial strains can be
added so as to produce the desirable flavour, taste, texture and odour [3]. Simultaneously with its fermentative
function, the coccus is producing an antimicrobial peptide substance, namely nisin, a 34 amino acid
bacteriocin, of approximately 3000 Daltons molecular weight [4].It has been applied as a food additive
worldwide and has been proven effective against a wide range of food spoiling microorganisms.
Consequently, to expand the potential of this microorganism large quantities of its cellular biomass is
necessary, particularly due to the dependence of nisin and lactic acid production from growth conditions since
both end products are considered primary metabolites [7]. Commercially available media for lactococcoi
include Man De Rogosa medium, (MRS) and M17 which is the most commonly used, Elliker broth,
Lactobacillus -Streptococcus Differential Agar (LS agar) and Al Purpose Tween agar (APT).These media, are
often used for research purposes and do ensure bacterial growth. However they do not support fastidious
growth, or high biomass yields due to the plethora of complex nitrogen sources they contain [5], such as beef
or poultry extract (peptone). In addition, the meat extracts cause environmental (discharged waste) and health
(potential CJD or H1N1 virus) hazards, while the complexity of nutrients leads to highly expensive media
formulation and is unsuitable for economically viable industrial process for culturing lactococcoi. To address
this challenge, for high rates production of cellular mass of L.lactis an economic liquid growth medium was
developed. L.lactis NCIMB 700185were chosen to test the mediums effectiveness in terms of biomass growth

[2]

and lactic acid and production. The medium developed was able to support the growth, in offering high yields
incorporating all the chemicals compounds needed. The cost of materials and optimisation percentages were
also calculated, offering an alternative option to the currently used media.

2. Materials and Methods

2.1. Bacterial strains
Lactococcus lactis NCIMB 700185 was provided in a lyophilised form by National Collection of Industrial
Food and Marine bacteria (NCIMB), Aberdeen, Scotland, United Kingdom. The coccus was revived twice by
inoculating the selected strains into 50 ml serum vials containing basal medium (de Man Rogosa Sharp broth,
MRS) and were statically incubated at 30C (Thermo Scientific Series 6000 Incubator, USA) for 24 hours.
Stock culture solutions of each strain were made through cryopreservation method [1]. For constant use, the
coccus was regularly reinoculated (on a weekly basis) into 30 ml serum vials containing basal medium and were
preserved at 2 C.
2.3. Growth on basal medium, Man de Rogosa, Sharp (MRS) broth
The specified quantities of powdered materials, namely, glucose 20 g L-1, yeast extract 15 g L-1,bacteriological
peptone (from poultry) 10 g L-1, sodium acetate 5 g L-1, tri-sodium citrate 2 g L-1, potassium hydrogen
phosphate 2 g L-1,magnesium sulphate 0.2 g L-1, manganese sulphate 0.05 g L-1, were weighted into an
electronic balance (Sartorius, CP4202S, JENCONS-PLS, Germany) and they are added into an Erlenmeyer flask
containing 1L of distilled water. Once mixed the medium (pH 6.2) was decanted into 10 ml aliquots were placed
into pressure tubes. Once cooled, the head tubes went under gaseous flow of nitrogen [1] and then sealed with
rubber stoppers and aluminium Wheaton seals. The sealed tubes were secured and were autoclaved at 121 C for
15 min. No glucose was added prior to autoclave in the medium as to avoid caramelization effect, but added to
the required concentrations to test the effect on the growth. The tubes were gently mixed in a vortex and
inoculated with 1 ml inoculum size, and statically incubated (Thermo Scientific Incubator, UK) at 30C. The
medium was dispensed into the pressure tubes under the presence of gaseous nitrogen flow to achieve complete
anaerobic conditions
2.4. Development of optimised medium broth
The influence of carbohydrate sources (glucose), nitrogen sources (peptone, yeast extract), pH buffering agents
(sodium acetate) and metal ions (manganese and magnese salts), on the coccus growth were investigated to
optimise the medium and compared with MRS This was done by varying the concentration of a component
while other components were constant. The effect of each component, prepared separately, was tested within a
concentration range between 0 g L-1, to 40 g L-1, and introduced to the medium via a sterile syringe in the
desired amounts. Each concentration was triplicated to obtain the average data, including positive and negative
controls. After the introduction in the tubes of the desired amounts of each component, the tubes were gently
mixed into a vortex mixer and then inoculated. Complete anaerobic conditions, were achieved by dispersing the
media into the pressure tubes under the presence of gaseous nitrogen flow. The growth rate and the amount of
growth were measured by spectrophotometry. After the determination of the optimum concentration for growth
of every component, all the components were combined resulting into the manufacture of an optimised medium
[4]. The optimised medium had a composition of 20 g L-1 glucose, yeast extract 10 g L-1, peptone 4 g L-1,
sodium chloride 10 g L-1, potassium hydrogen phosphate 5.5 g L-1, (pH 6.5) and the coccus was grown at 30C.
2.5. Measurement of cellular growth and biomass
The cellular growth was measured by placing the pressure tubes into a spectrophotometer fitted with a test tube
holder (PU 8625 UV/VIS Philips, France) at 660 nm. The tube had a 1.8 cm. light path. Maximum specific
growth rate (max, h-1) [7] and final biomass (g L-1) concentration of the microbial strain were determined into
basal medium under a 10 hour circle of static incubation into 32C (Thermo Scientific Series 6000 Incubator,
USA). To convert the optical density (O.D.) measurements into dry weight units (g L-1) a dry weight

[3]

determination assay was performed, [1] resulting into of linear equation (two variables) of an intercept-slope
form of for dry weight determination where x stands for optical density units. The equation for, L.lactis is the
following respectively, Y = 0.21405 X +0.00347
2.6. L.lactis growth on a ph controlled batch reactor -investigation of the effect of ph lactic acid and cellular
biomass productivity
The pH range tested was between 4.0 to 7.0 in a 0.5 point step at 30C. A 2L capacity continuously stirring tank
(CSTR) Pyrex glass reactor, analytically described elsewhere, [6] (Fig.1 [a, b, c]) was used for the procedure.
The sampling was performed on an hourly basis on a 10 hours circle via the sampling port with a 10 ml sterile
syringe and all the samples were measured for biomass concentration, lactic acid production and bacteriocin
production.
[ a]

[b]

[c]

Fig. 1.[a,b,c] [a]CSTR Fermenter on a stirring plate,[b] pH controller, pump, scale, alkali/acid solution feed [c]pH Waterbath
for temperature control

2.7. Determination of end products through high performance liquid chromatography

L.lactis was reported to produce mainly lactic acid and a very small amount of acetic acid during the exponential
growth phase of its metabolism. In order to quantify the amount of the produced lactic acid and also to confirm
the fact that the strain was homofermentative all the samples were analysed by the High Performance Liquid
Chromatography (HPLC) method. The HPLC system is in detail described elsewhere [6].
2.8. Numerical Analysis of the Experimental Data
Each differential parameter was triplicated to obtain the average data. The data were statistically analysed for
accuracy and precision, calculating standard deviation, standard error, experimental error, regression factor and
reading error (Microsoft Excel software Version 2003). All the numerical data were proven to be highly
accurate and reproducible, having standard deviation of mean below 5% and experimental error below 5%,
offering highly significant results.

3. Results and Discussion

3.1. Growth performance on basal medium, MRS
In order to evaluate the max and growth yield (X, g L-1) L.lactis was primarily grown on MRS medium and had
a max of 0.20 h-1. The final biomass concentration after a 10 h static incubation achieved was 1.50 g L-1
suggesting slow growth. The growth medium was therefore reformulated and simplified. A step towards
simplification is the evaluation of the remaining components on the growth performance of the strain. Each
component was tested in a range of concentrations, while all the remaining parameters concentration was kept
constant, equal to the initial concentration of the MRS medium.

[4]

3.2. Evaluation of the effect of yeast extract and peptone concentrations on growth
Several researchers [4] have introduced the idea of partial dependence of biomass development and metabolites
production of L.lactis; on the amount of nitrogen sources like yeast extract within defined growth media. Yeast
extract and peptone serves as the carbon, nitrogen and vitamin source needed to satisfy the growth requirements
of the microorganisms. The effect of yeast extract and peptone on growth was studied on a range of
concentrations between 0 g L-1 to 25 g L-1 with max of the bacteria increases simultaneously with the
concentration of the component. L.lactis, a concentration of 20 g L-1 and 4 g L-1 yeast extract and peptone
respectively the highest max 0.23 h-1 , with biomass concentrations of 1.8 g L-1. Further increase in biomass was
not observed most probably due to the exhaustion of carbohydrate source.
3.3. Evaluation of the effect of glucose concentrations on growth
As the yield of biomass in all anaerobic bacteria was strongly dependent on carbohydrate feed [7], glucose as
energy source. The effect of glucose concentration was studied, in a range of concentrations between 0 g L-1 to
40g L-1.Growth of strains tested was stimulated by the addition of glucose in both yield and growth rate, up to a
concentration of 20 g L-1, with L.lactis having a max of 0.20 h-1 and final biomass concentration of 2.00 g L1
.Above 20 g L-1 the organisms growth was inhibited. Glucose is a carbon as well as energy source for
microbial growth, has been mainly correlated with cellular biomass production, while in the case of Lactic Acid
Bacteria (LAB) glucose has been considered to be converted to lactic or acetic acid, carbon for structure,
depending on the bacterial strain used as well as the medium composition and physicochemical culturing
conditions [8,9] .Numerous studies regarding LAB growth and acids production ,have been conducted in other
carbohydrate sources such as fructose, mannose, lactose, xylose and starch,[8,9] however optimum results have
been achieved when glucose is used as the major carbon source.
3.4. Growth on optimum medium and investigation of the pH effect over the growth of L.lactis
Combining all the optimised growth parameters in the desired quantities formulated a simplified liquid medium,
of which numerous components included in the basal medium were omitted. Manganese and magnesium salts
and sodium acetate has been found not having a significant effect on growth, thus were omitted. To ensure
buffering capacity though, sodium chloride was introduced in the medium. This developed medium served the
aim of enhancing the cellular productivity, ensuring high growth (Table 1). When comparing the growth of the
coccus on MRS and the formulated media it was clearly demonstrated that the optimised medium improves
growth with a significant high increase in the maximum specific growth rate and on the final cell concentration,
with L.lactis having a max of 0.29 h-1 and a final biomass concentration of 2.5 g L-1. The optimised medium was
then used for further investigative studies including pH. The pH effect testing was performed on a 2L CSTR
operated batchwise, using as substrate the formulated medium. For L.lactis the maxwas enhanced when the
culture was controlled pH 7.0 (max0.72 h-1), with pH 6.5 also supported good growth (max0.66 h-1). These
experiments gave higher biomass yields and max as compared to the uncontrolled pH growth systems. It can be
also observed that on acidic pH values of 4 and 4.5, the growth was completely inhibited. In this experimental
set up, the amount of organic acid (mainly lactic acid) produced by the bacilli is equal to the amount of NaOH
used for pH maintenance. Over the 10 h fermentation the pH 5, 5.5 and 6 the organisms were still growing
although they had slower maximum specific growth rates and long lag periods prior to growth. It was clear
though that the coccus growth was quite sensitive in alkali as well as to extreme acidic points.
3.5. Determination of lactic acid production
The higher amount of lactic acid production was observed on pH 7.0 for the selected L.lactis. As being a
homolactic strain it is assumed that the amount of glucose utilised for growth is directly converted to lactic acid.
During the exponential growth phase all were producing high amounts of lactic acid when grown on the
optimised medium. The production of lactic acid was investigated in a 2L capacity batchwise operated CSTR,
using the optimised formulated medium. Harvesting of samples for biomass and lactic acid was performed in an
hourly basis. For L.lactis 30.28 g L-1 of lactic acid were produced (Table 1). Similar results in lactic acid
production by these organisms are found in the literature [8] these however are related to the growth the bacteria
on lactose based media and food sources, suggesting that the developed medium can serve as a strong
alternative solution.

[5]
Table 1: Comparative table and optimisation percentages of metabolic products of L.lactis produced on all the three media
Bacterium

Media

max,
h-1

max
optimisation
percentage
(%)

Final Biomass
Concentration
(g L-1)

Final Biomass
Concentration
Optimisation
percentage
(%)

Total
glucose
consumption
(g L-1)

Total
lactic acid
production
(g L-1)

L.lactis

Basal
Optimised
Optimised
(pH
controlled
culture)

0.20
0.29
0.72

70.6

1.5
2.3
4.2

53

2.98
13.53
16.53

1.87
9.67
30.28

260

180

Total
Lactic acid
production
Optimisation
percentage
(%)
417
1519

1,2,3

Optimisation percentages are calculate as percentage increase based on the increase achieved comparing with the results to the basal
medium

3.6. Costs
Although the medium is simplified, offering high yield of cellular biomass and lactic acid, as well as cost
effective comparing to the basal medium (Table 2) its adaptation will be strongly depend as well on the cost
effectiveness of recovering the end products. Calculating the overall cost of the nutrient medium in industrial
scale is a complex task as a variety of costs including operating costs i.e. energy consumption, cleaning, labour
and maintenance; and capital costs, such as equipment and scale of operations. However a brief calculation has
been done mainly focusing on the cost of the raw materials.
Table 2: Comparative table of costs of the basal; and optimised media
Basal Medium Components
Costs[10](/kg) Optimised Medium Components
Glucose
24.20
Glucose
Yeast extract
254.00
Yeast extract
Peptone (from poultry)
184.00
Peptone(from poultry)
Manganese sulphate
72.40
Sodium Chloride
Magnesium sulphate
71.10
Potassium dihydrogen orthophosphate
Potassium dihydrogen orthophosphate 71.80
Sodium acetate
53.00
Tri-sodium citrate
40.10
Total (8 components)
770.60
Total (5 components)

Costs[10](/kg)
24.20
254.00
184.00
51.90
71.80

585.90

4.0 Conclusions
It can be concluded easily from the experimental results, that the basal medium used cannot support fastidious
growth, and is possibly unsuitable for use in industrial scale due to its high cost of synthesis and content in
nitrogen sources, while the optimised medium can offer highly improved biomass and lactic acid yields.

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