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IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

TOTAL NITORGEN CONTENT FROM EARTHWORM

(EISENIA FOETIDA) USING THE KJELDAHL METHOD

ZARINA ZAKARIA, ALINA RAHAYU MOHAMED, NOOR HASYIERAH MOHD SALLEH

AND SITI NURSHEELA ABU MANSOR

School of Bioprocess Engineering, University Malaysia Perlis, 02600 Arau, Perlis.

zarinaz@unimap.edu.my

ABSTRACT: In the fish aquaculture management, fish feed is identified as a major

problem. The high cost and scarcity of fishmeal in formulated feeds have led to the use

of other protein sources such as earthworms and animal by-product. Earthworm is an

alternative source of protein to replace the fish meal in the fish feed formulation. In this

study, total nitrogen content in the earthworm powder is determined using the Kjeldahl

method by employing the statistical software, Full Factorial Design (FFD) which could

provide the significant information about the studied parameters. The parameters are, the

digestion time (min) and the volume of sulfuric acid (H2SO4) (ml). From the analysis of

variance (ANOVA), the volume of H2SO4 and the interaction between digestion time

and the volume of H2SO4 are found to be important parameters in the nitrogen

determination process via the Kjeldahl method. The highest nitrogen content obtained

was 12.23%, when using 15 ml H2SO4 and 60 mins of digestion time. The value of R2

is 0.9986, which shows that the selected parameters (the digestion time and the volume

of H2SO4) and its corresponding levels are highly correlated to the percentage nitrogen

content in earthworm powder using the Kjeldahl method.

ABSTRAK: Dalam pengurusan akuakultur ikan, makanan telah dikenalpasti sebagai

masalah utama. Kos yang tinggi serta kekurangan sumber makanan telah menggalakkan

pencarian sumber protein baru seperti cacing tanah dan hasil sampingan sembelihan

haiwan ternakan. Cacing adalah sumber protein alternatif menggantikan ramuan ikan

(fish meal) dalam formulasi makanan ikan. Dalam kajian ini, kandungan jumlah nitrogen

dalam serbuk cacing tanah telah dianalisa menggunakan kaedah Kjeldahl dengan

menggunapakai perisian statistikal iaitu Full Factorial Design (FFD) yang boleh

memberikan maklumat yang penting berkenaan dengan parameter-parameter yang dikaji.

Parameter-parameter tersebut adalah masa pencernaan (min) dan isipadu asid sulfurik

(H2SO4) (ml). Daripada analisa varian (ANOVA), isipadu H2SO4 dan interaksi antara

masa pencernaan dan isipadu H2SO4 telah dikenalpasti sebagai parameter-parameter

utama dalam proses penentuan kandungan nitrogen melalui kaedah Kjeldahl. Kandungan

nitrogen yang tertinggi diperolehi adalah 12.23% bila menggunakan 15 ml H2SO4 dan 60

minit masa pencernaan. Nilai R
2
adalah 0.9986 menunjukkan bahawa parameter-

parameter (masa pencernaan dan isipadu H2SO4) dan aras yang dipilih mempunyai

perkaitan langsung yang tinggi dengan kandungan nitrogen dalam serbuk cacing dengan

menggunakan kaedah Kjeldahl.

KEYWORDS: nitrogen content;, earthworm; Kjeldahl method

1. INTRODUCTION

The aquaculture sub-sector in Malaysia is divided into marine aquaculture and

freshwater aquaculture. For freshwater aquaculture, pond culture is the main system

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

employed [1].Aquaculture is a prominent approach to increase the fish production to

suffice fish available to the populace. It was reported that 1.43 million tonnes of fish

catches last year, not including squids, prawns and oysters, are not enough to feed a

population of 28.5 million [2].

Under the Third National Agriculture Policy, aquaculture production in Malaysia is

targeted to reach 600,000 tonnes annually by the year 2010, which include 200,000 tonnes

freshwater fish. This multifold increase in production from its current level will place an

increasing demand on the supplies of feed (formulated feed and trash feed), since the

aquaculture sector is targeted at high value species such as grouper, snapper and penaeid

prawn. These species require high inputs of fish protein in their feed [1]. The issue of fish

feed is identified as major problem in the fish aquaculture management.

The high cost and scarcity of fishmeal in formulated feeds has led to the use of other

protein sources such as earthworms, insects, snail, mussels, maggot, frog, and plants in

fish feeds [3].However, there are some problems related to the quality of protein sources

for fish feed. The occurrence of leaching of total protein content and lipid which was low

(<15%) in all fish silage pellets such as silage-poultry by-product meal was reported [4].

Some small scale keli (Clarias spp) or catfish breeders in Malaysia prefer to use the

animal by-product such as chicken gut as the fish meal to the catfish due to its low in price

and easily available. However, this leads to the odour pollution around the fish pond and

its nearby area. Moreover, the tissue strength of fish meat was found to be low that it is

only suitable for local use only.

Eisenia feotida or earthworm is viewed as possible alternative of protein source in

fish meal. Moreover, earthworms are being used as fish bait and this practice is common

in fishing activity throughout Malaysia. Owing to their high reproductive rate, low feeding

costs and ease of breeding in captivity, earthworms constitute an extremely interesting

protein source for fish feed. Mostly, earthworms with high protein are also used to feed

chickens, pigs and as a dietary supplement for ornamental fish [5, 6]. The earthworm in

dried or dehydrated form has protein content higher than other protein source such as fish

meal, meat and bone meal, and soybean meal [7].Moreover, Eisenia feotida proteins were

not toxic to a human cell line at low concentrations [8].

Determination of protein content is an important measurement especially in

biochemical analysis such as electrophoresis, immuno-analysis, molecular biology as well

as other research applications [9]. The protein concentration can be determined by using

different methods such as Kjeldahl method, combustion method, Bradford method, Biuret

method, Lowry method, UV-visible spectroscopic method [9-13]. The Kjeldahl method

provides several advantages: it could work under appropriate conditions with analysis

simultaneously. Moreover, the distillation process is very fast and efficient [11]. On the

other hand, Biuret method is impractical because of the detection limit and large volumes

of sample requirement [9].To the authors best knowledge, there has yet any papers

published on the use of statistical tools (Design Expert software) in the determination of

protein content in earthworm powder by employing the Kjeldahl method of analysis.

Utilization of statistical tools like Design Expert software offers many advantages.

Design of experiment (DOE) is an efficient tool in such a way that it could eliminate

the time consuming phase which could not be achieved when using the conventional

OFAT (one-factor-at-a-time) method [14]. Furthermore, the OFAT method is inefficient,

requires more experimentation than a factorial and unable to detect interactions between

the parameters or factors [15]. Factorial experiments through 2
k
study are the only way to

discover interactions between parameters [15]. A 2
k
design is useful prior to response

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

surface study in order to conduct screening experiments to identify the important

parameters that had effects on the process [16].

Therefore, in consideration of this, the objectives of this paper are to screen the active

parameters which are H2SO4 volume (15-25 ml) and digestion time (40-60 min) that play

significant roles in protein content determination in earthworm powder. These parameters

were chosen since for standard operating procedure of Kjeldahl, the minimum conditions

are 25 ml and 60 min for H2SO4 volume and digestion time respectively. Full Factorial

Design (FFD) was used to achieve the objective and consequently, the process conditions

that would give the highest protein content in earthworm powder using the Kjeldahl

protein analysis could be obtained.

2. MATERIALS AND METHODS

2.1 Materials

Earthworm powder was purchased from local company. Sodium hydroxide, H2SO4,

boric acid and the Kjeldahl tablets were purchased from Merck.

2.2 Preparation for Scrubber

Five liters of sodium hydroxide (8%) was prepared. The pH indicator (0.5 g

bromothymol blue was dissolved in 500 ml ethanol (95%) and 500 ml distilled water was

added to the solution) was made and added into the 8% of NaOH. Activated charcoal in

granular form was installed at the scrubber unit (Buchi Scrubber B-414, Switzerland).

2.3 Preparation for Digestion

One gram of the earthworm powder was weighed on a nitrogen free paper before it

was placed in a digestion tube. Two Kjeldahl tablets (10 g) were added to increase the

speed of reaction in digestion process. H2SO4 (98%) with varying volume was added and

the sample was suspended by gently swirling the tube. Additional blanks (without

earthworm) were prepared. The digest system K-437 (Buchi, Switzerland) unit was

preheated at 300 °C for 30 min. The digestion process was started from 300
o
C up to

420°C with varying digestion time. After the sample was completely digested, it was

cooled down to ambient temperature prior for distillation.

2.4 Preparation for Distillation

One liter of boric acid (4%) with pH 4.65, 1 liter H2SO4 (0.25 M) and 500 ml NaOH

(10%) were prepared. The cold sample was distilled using Auto Kjeldahl Unit K-370

(Buchi, Switzerland).

2.5 Preparation for Titration

Once the distillation process was done, the distilled sample was titrated with 0.25 M

sulfuric acid from KjelFlex (Buchi, Switzerland) and stop once the colour became slight

purple or pH 4.65. The volume of titrant used was recorded.

2.6 Calculation of % Nitrogen:

For 0.25 M H2SO4:

%� �
����
�� – ������� �� � �.�� � ��.��

������ ! "#�$�� ���
(1)

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

2.7 Calculation of % Protein:

% Protein = % Nitrogen x empirical protein factor.

A conversion factor (F) is used to convert the measured nitrogen concentration to a

protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram

of protein) is used for many applications, however, this is only an average value, and each

protein has a different conversion factor depending on its amino-acid composition [17].

3. RESULTS AND DISCUSSION

A 2 level–two-factor-design 2
2
with digestion time, A (40-60 min) and volume of

H2SO4, B (15-25 ml) was employed (Table 1). These 2 factors are used to view its

correlation with the response (R1) which is the percentage of nitrogen content (%

Nitrogen). 2
2

With 7 centre points corresponds to 17 experimental runs were suggested.

The results are obtained and tabulated in Table 2.

Table 1: Parameters under investigation.

Range and levels

Independent variables Units Symbols -1 0 +1

Digestion time mins A 40 50 60

Volume of H2SO4 ml B 15 20 25

Table 2: Experimental results for earthworm protein.

Std Run

Factor 1

A:time digestion

(minute)

Factor 2

B:sulfuric acid

(ml)

Response 1

RI (%

Nitrogen)

13 1 50.00 20.00 12.21

12 2 60.00 25.00 0.49

11 3 60.00 25.00 1.12

1 4 40.00 15.00 11.41

17 5 50.00 20.00 11.98

3 6 40.00 15.00 11.82

8 7 40.00 25.00 1.17

4 8 60.00 15.00 12.07

6 9 60.00 15.00 12.23

14 10 50.00 20.00 11.95

5 11 60.00 15.00 12.15

15 12 50.00 20.00 11.91

10 13 60.00 25.00 0.80

7 14 40.00 25.00 0.79

16 15 50.00 20.00 11.81

9 16 40.00 25.00 0.74

2 17 40.00 15.00 11.74

From the result, it clearly showed that the highest nitrogen content could be achieved

at the centre point level for both of the involved parameters (run no 1, 5,10,12,15 in Table

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

2). The lowest nitrogen contents were obtained at low and high level of digestion time

against high volume of H2SO4 (Run no. 2, 3, 13, 14 and 16 in Table 2). Therefore, this

indicated that at high level of H2SO4, it could destroy the nitrogen content in the

earthworm regardless of the digestion time.

From the analysis of variance (ANOVA) in Table 3, the Model F-value obtained is

2918.04 which imply that the model is statistically significant. The p-value < 0.0500

indicates that the model terms are significant. The p-value for Factor A is 0.1154 which

shows that the Factor A is not significant; however the p-values for Factor B and AB are

0.0001 and 0.0274 respectively which indicate that the Factors B and AB are significant.

Moreover, the value of F-values for volume of H2SO4 is the highest compared to other

parameters. It can be inferred that the volume of H2SO4 play an important role in the

nitrogen determination process.

The curvature F-value of 2637.10 implies that there is significant curvature (as

measured by difference between the average of the center points and the average of the

factorial points) in the design space. The results from ANOVA was further supported by

the R
2
value shown in Table 4. The R

2
value of 0.9986 shows that the selected factors

which are the digestion time and the volume of H2SO4 and its corresponding levels are

highly correlated to the percentage nitrogen content in earthworm powder using the

Kjeldahl method.

Table 3: ANOVA for earthworm protein.

Source
Sum of

Squares

Degrees of

freedom (df)

Mean

Square

Value

p-value

Prob> F

Model 14332.62 3 4777.54 2918.04 < 0.0001 significant

A-digestion time 4.72 1 4.72 2.88 0.1154 Not significant

B-sulfuric acid

volume
14317.59 1 14317.59 8744.95 < 0.0001 Significant

AB 10.31 1 10.31 6.30 0.0274 Significant

Curvature 4317.56 1 4317.56 2637.10 < 0.0001 Significant

Pure Error 19.65 12 1.64 -- -- --

Cor Total 18669.83 16 -- -- -- --

Table 4: Analysis of protein content in earthworm.

Std. Dev. 1.28 R-Squared 0.9986

Mean 50.14 Adj R-Squared 0.9983

C.V. % 2.55 Pred R-Squared 0.9971

PRESS 41.88 Adeq Precision 102.225

Figure 1 shows the graph of half normal plot. Point AB, A and B indicates the

positive and negative effect for the factors. Parameters that fall along the straight line are

considered as the noise or error from replicates. Factor B is the farthest from the noise

with p-value< 0.0001, while factor AB with p-value of 0.0274 which is < 0.05 indicates

that these 2 factors are significant.

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

Fig. 1: Graph of half normal plot.

Fig. 2: Graph of slight interaction;●: 15 ml of H2SO4;●:25 ml of H2SO4.

The interaction graph shows in Fig. 2 is the relationship between the digestion time

and the volume of H2SO4 with its nitrogen yields. It shows the yield of nitrogen content

against high level of H2SO4 (25 ml) and low level of H2SO4 (15 ml) together with the

digestion time (40-60 min). For 15 ml H2SO4, at 60 min then nitrogen yield is higher than

the other one since a sufficient amount of H2SO4 was used in digestion. However, the

percentage of nitrogen content when using 25 ml H2SO4 was lower because the excess

amount of H2SO4 results in the loss of nitrogen compound and caused excessive foaming

that occurred during the digestion solution.

IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

Naturally, protein digests by pepsin into amino acid. Besides enzyme, acid can be

used to digest protein but in different manner. H2SO4 originally used by the founder of this

method and still being used for its efficacy.

H2SO4 is an oxidizing agent that reduced nitrogen in food into ammonia and other

organic matter. As the organic material is oxidized the carbon content is converted to

carbon dioxide and the hydrogen is converted into water. Digestion is the most time

consuming step and one of disadvantage of using Kjeldahl method. However, this

procedure has enabled the obtainment of nitrogen higher yield at shorter time and this is

the advantage of this analysis.

Complete digestion was not achieved by using H2SO4 alone. The combination of

H2SO4 and high temperature has produced better results. The use of concentrated H2SO4 at

high temperatures poses a considerable hazard.

4. CONCLUSION

The nitrogen content in the earthworm powder was statistically analysed using FFD

through Kjeldahl method. The statistical tool (FFD) employed in the study has shown that

the volume of H2SO4 (B) and the interaction between the digestion time and the volume of

H2SO4 (AB) are important parameters in the nitrogen determination process. The highest

nitrogen content obtained was 12.23% when using 15 ml H2SO4 and 60 mins of digestion

time. The conversion of total nitrogen (protein and non-protein) will give a total of

76.465% crude protein. It was presume only small percentage of non-protein nitrogen

existed in earthworm powder (1.5 – 2%) and still the value was higher than previously

reported protein content in earthworm, 64.4–72.9% [18] and 62% [8].

Therefore, it can be concluded that the determination of nitrogen content using the

Kjeldahl method and the statistical software (FFD) enabled the obtainment of the highest

crude protein content in earthworm. This indicated that the Kjeldahl method is a robust

and fast technique to measure nitrogen content and the application of FFD could enable

the appropriate conditions for each parameters involved. Consequently, this could save

time, chemicals and raw materials. Since, the protein content of earthworm obtained was

high, therefore earthworm proteins could be considered for use as a supplement in animal

diets especially for fish cultivation.

Digestion time does not provide much effect to the experiment but yet need to be

considered since the longer the digestion time, the temperature will increase. At higher

temperature, nitrogen compounds are decomposed to elemental nitrogen, leading to

nitrogen loss thus lowering the yield of protein content in earthworm.

ACKNOWLEDGEMENT

The authors gratefully acknowledge Ministry of Agriculture for the Science Fund (9006-

0006) and School of Bioprocess, University Malaysia Perlis for their financial support and

facilities respectively.

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IIUM Engineering Journal, Vol. 14, No. 1, 2013 Zakaria et al.

NOMENCLATURE

H2SO4 Sulfuric acid

FFD Full Factorial Design

UV-Visible Ultra-Violet visible

DoE Design of Experiment

OFAT One-Factor-At-A-Time

NaOH Sodium Hydroxide

R1 Response 1

ANOVA Analysis of Variance