DOI: 10.1111/jpn.

12521

ORIGINAL ARTICLE

The effect of dietary Chlorella vulgaris supplementation on

micro-organism community, enzyme activities and fatty acid
profile in the rumen liquid of goats
E. Tsiplakou1, M. A. M. Abdullah1, D. Skliros2, M. Chatzikonstantinou3, E. Flemetakis2, N. Labrou3 and
G. Zervas1
1 Department of Nutritional Physiology and Feeding, Agricultural University of Athens, Athens, Greece
2 Laboratory of Molecular Biology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens,
Athens, Greece, and
3 Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of
Athens, Athens, Greece

Summary
Microalgae might be considered as an alternative source of fat and/or protein for ruminant’s diets. However,
changes in populations of ruminal micro-organisms associated with biohydrogenation process, methane and
ammonia production in response to microalgae dietary supplementation have not been well characterized. Thus,
16 cross-bred goats were divided into two groups. Each goat of both groups was fed individually with alfalfa hay
and concentrates separately. The concentrates of the control group had no microalgae while those of the treated
group were supplemented with 10 g lyophilized Chlorella vulgaris/kg concentrate (chlor). On the 30th experi-
mental day, samples of rumen fluid were collected for microbial DNA extraction, fatty acid profile and enzyme
activity analyses. The results showed that the chlor diet compared with the control increased significantly the
populations of Methanosphaera stadtmanae, Methanobrevibacter ruminantium and Methanogens bacteria and protozoa
in the rumen of goats. A significant reduction in the cellulase activity and in the abundance of Ruminococcus albus,
and a significant increase in the protease activity and in the abundance of Clostridium sticklandii in the rumen liq-
uid of goats fed with the chlor diet, compared with the control, were found. Chlorella vulgaris supplementation
promoted the formation of trans C18:1, trans-11 C18:1 and monounsaturated fatty acids (MUFA), while the propor-
tions of C18:0 and long-chain fatty acids (LCFA) reduced significantly in the rumen liquid of goats. This shift in
ruminal biohydrogenation pathway was accompanied by a significant increase in Butyrivibrio fibrisolvens trans
C18:1-producing bacteria. In conclusion, the supplementation of diets with microalgae needs further investigation
because it enhances the populations of methane-producing bacteria and protozoa.
Keywords microalgae, Chlorella, rumen, micro-organism, methane

Correspondence E. Tsiplakou, Department of Animal Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
Tel: +30 210 529 4435; Fax: +30 210 529 4413; E-mail: eltsiplakou@aua.gr

Received: 18 December 2015; accepted: 24 March 2016

on milk fatty acid (FA) profile, in response to

Introduction
Microalgae, despite being more popular for biofuel
production (Medipally et al., 2015), have also
gained more attention as a source of biomolecules
such as n-3 polyunsaturated fatty acids (PUFA),
phenols, flavonoids (Goiris et al., 2012; Lum et al.,
2013; Yaakob et al., 2014). Thus, the inclusion of
microalgae in ruminant’s diets has been shown as
an effective nutritional strategy to enrich milk with
PUFA of bovine (Glover et al., 2012) and goats
(Kou
rimska et al., 2014). However, modifications
dietary supplementation with microalgae, may be
accompanied by alterations in ruminal biohydro-
genation (BH) process. Indeed, it has been observed
that alterations in the formation of specific BH
intermediates (FA) in the rumen, after dietary
microalgae supplementation, are associated with
changes in specific population of ruminal bacteria
and protozoa in cattle (Boeckaert et al., 2007,
2008). Nevertheless, no information exists so far on
the effect of microalgae on the abundance of micro-
organisms involved in ruminal BH process.

Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH 1
Chlorella affects rumen microbial population E. Tsiplakou et al.

Furthermore, microalgae with high-fat content of them were offered to the animals twice a day (in
could be, also, considered as possible candidates to two equal parts at 08:00 and 16:00 h). The concen-
reduce methane (CH4) production in ruminants, trates of the control group had no microalgae while
although this has been investigated so far only in vitro those of the treated group (chlor) were supplemented
(Fievez et al., 2007). This may happen because lipids, with 10 g lyophilized C. vulgaris/kg concentrate
and especially PUFA, have antimicrobial effects on (990 g control concentrate + 10 g lyophilized C. vul-
methanogens and protozoa because it can disrupt garis). The concentrate diets were prepared every
their cell membrane (Jenkins et al., 2008; Martin week and were formulated to be isoenergetic and iso-
et al., 2010). While the in vitro results of microalgae proteic (Table 1) because C. vulgaris was added only in
supplementation with high-fat content on CH4 emis- 10 g/kg concentrate and its chemical composition was
sion appear promising, the opposite seems to occur such that did not change the energy and protein con-
with microalgae rich in protein and low in fat content. tent significantly between the two diets (Control vs.
Besides, in a recent in vitro study, Dubois et al. (2013) chlor; Table 1). The quantities of feed offered to the
observed that algae with a very high level of protein animals were adjusted weekly according to their
content increased gas production in the rumen. Thus,
it is clear that in vivo studies are required especially Table 1 The chemical composition of alfalfa hay, Chlorella vulgaris and
using microalgae with high protein content, such as concentrates (Control vs. chlor) and the mean daily nutrients, minerals
Chlorella vulgaris, to determine their impact on metha- and fatty acid intake from forage and concentrates
nogens population in the rumen. This assumption was
Concentrates
further supported by the fact that recently C. vulgaris Chlorella
has been proposed as a protein source in cows diets Alfalfa hay vulgaris Control chlor
(Lodge-Ivey et al., 2014). Additionally, defatted bio-
Chemical composition (g/kg)
mass of microalgae species, derived from biofuel pro- Dry matter 879 927 910 916
duction, has been shown feasible in replacing corn Organic matter 775 848 862 868
and soybean meal in cattle diets (Lopex et al., 2013). Crude protein 145 677 151 155
Thus, the objectives of this study were to determine Ether extract 11.7 10.5 23 24
the effect of dietary C. vulgaris supplementation on (i) Neutral detergent fibre 596 128 246 256
the abundance of micro-organisms involved in rumen Acid detergent fibre 365 42 69 70

BH process (Ruminococcus flavefaciens, Butyrivibrio fibri- Control diet chlor diet

solvens, Ruminococcus albus, Fibrobacter succinogenes Pro-
Nutrients intake (g/animal/day)
tozoa, Fungi), CH4 (Methanosphaera stadtmanae,
Dry matter 2323 2330
Methanobrevibacter ruminantium, Methanogens) and Organic matter 2120 2126
ammonia (Clostridium sticklandii, Peptostreptococcus Crude protein 384 389
anaerobius) production, (ii) rumen enzyme (cellulase, Ether extract 44 45
protease, lipase and a-amylase) activities and (iii) Neutral detergent fibre 1129 1142
rumen FA profile of goats. Acid detergent fibre 594 595
Minerals intake (mg/animal/day)
Se 0.32 0.35
Materials and methods Zn 144.00 130.00
Cu 18.40 18.10
Sixteen cross-bred dairy goats, at 90–98 days in milk, Fe 714.00 702
of comparable age (3–4 years old) and body weight Mn 150.00 152.00
(BW: 43.0
2.3 kg) were maintained at the Agricul- Mo 3.80 3.70
tural University of Athens. Housing and care of ani- As 0.27 0.23
mals conformed to Ethical Committee guidelines of Co 1.28 1.30
Ni 6.53 6.50
Faculty of Animal Science. The animals were divided
Sb 0.32 0.41
into two homogeneous subgroups, each (n = 8) bal- Pb 0.74 0.73
anced by their BW and milk yield. Throughout the Fatty acid intake (g/animal/day)
experimental period, each animal of each group was C16:0 9.67 10.09
fed individually according to its requirements. The C18:0 1.83 1.84
goats of both groups were fed, after 2-week adaptation cis-9 C18:1 9.78 10.23
period, with a ration consisted of alfalfa hay and con- C18:2n-6c 19.31 19.36
C18:3n-3 0.83 1.10
centrates (forage/concentrate = 53/47). The forages
C20:5n-3 – 0.29
provided separately from the concentrates while both

2 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
E. Tsiplakou et al.
individual requirements based on their BW and
fat-corrected milk yield. The average daily intake of
alfalfa hay and concentrates was 1.4 and 1.2 kg/ani-
mal, and the average daily intake of nutrients is
shown in Table 1. The milk yield of the goats was 2.50
and 2.60 kg/day in average for the control and chlor
groups, respectively, while the milk fat and protein
content (%) were 3.45 vs. 3.57 and 2.84 vs. 2.80
respectively. Diet selectivity did not occur, and no orts
were left from forages and/or concentrates after each
feeding. The concentrate (g/kg as fed) consisted of
maize grain, 340; barley grain, 380; soybean meal,
150; wheat middlings, 110; calcium phosphate, 15;
salt, 3; mineral and vitamin premix, 2. The mineral
and vitamin premix contained (per kg as mixed)
150 g Ca, 100 g P, 100 g Na, 100 mg Co, 300 mg I,
5000 mg Fe, 10 000 mg Mn, 20 000 mg Zn,
100 000 mg Se, 5 000 000 IU retinol, 500 000 IU
cholecalciferol and 15 000 mg a-tocopherol. The
whole experimental period lasted for 30 days. All ani-
mals had free access to fresh water.
Rumen samples collection
On day 30 of the experiment, rumen fluid was col-
lected from each goat using a stomach tube before
feeding. Taken into account that the stomach tube
when is inserted in to the cranial dorsal (atrium), and
not in the central rumen, may affect ruminal fermen-
tation parameters, due to the presence of saliva in this
site (Shen et al., 2012), and in order to reduce saliva
contamination and increase the representativeness of
ruminal fluid collected from the goats, the stomach
tube was inserted into a depth of approximately
120–150 cm and then the first 20 ml of ruminal fluid
was discarded (Ramos-Morales et al., 2014). Immedi-
ately after collection, the rumen fluid was filtered
with cheese cloth to separate the solid particles, and
three subsamples (approximately of 50 ml each) were
frozen at 80 °C until microbial community, enzyme
activities and FA analysis.
Microbial community analysis
Extraction of microbial DNA from total rumen contents
DNA extraction took place using a modified protocol
of QIAGEN’s (QIAGEN, Redwood City, CA, USA)
plant DNeasy mini kit including a typical
cetyltrimethylammonium bromide (CTAB) extraction
step (Sharma et al., 2003). Specifically, 2 g of rumen
digesta was grinded to fine powder using liquid nitro-
gen. This powder was then transferred into a CTAB-
based extraction buffer (2% CTAB, 100 mM Tris-HCl
Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
Chlorella affects rumen microbial population
pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2-mercaptoethanol
and 100 lg/ml of proteinase K) and incubated at
65 °C for 2 h. RNase A (10 mg/ml) was added to the
sample afterwards and incubated for 30 min at 37 °C,
in order to remove RNA contamination. A three-time
extraction with an equal volume of chloroform: isoa-
myl alcohol 24:1 followed at 4 °C before precipitation
with isopropanol overnight at 20 °C. DNA extrac-
tion from the pellet took place using QIAGEN’s kit
according to manufacturer protocol. The quality of
extracted DNA was estimated by ND-1000 spectropho-
tometer (NanoDrop, Wilmington, DE, USA) and a
0.70% agarose gel.
Determination of bacterial load by real-time qPCR
Target DNAs were amplified using the following list of
primers (Table 2), which have been previously
described (Sharma et al., 2003). Quantitative real-
time PCRs were performed on the Stratagene
MX3005P using 29 Fast Start SYBR-GREEN Master
ROX (Bio-Rad, Hercules, CA, USA), primers at a final
concentration of 0.2 lM each, and 1 ll of DNA
(40 ng/ll) as template. The conditions for carrying
out the polymerase chain reaction were depending on
the size of the amplicon (Table 3). Primer specificity
and formation of primer dimers were monitored by
dissociation curve analysis. Relative genomic concen-
tration of the target species was calculated as a ratio of
the genomic concentration of the total bacteria genes,
as (1 + PCR efficiency)(Ct targetCt total bacteria), where
PCR efficiency was calculated employing the linear
regression method on the Log (Fluorescence) per cycle
number data, using the LINREGPCR software (Ramakers
et al., 2003). This method calculated the ratio of each
DNA micro-organism relative to the benchmark,
which was the total bacterial DNA. The qPCRs were
performed on three biological replicates.
Rumen enzyme activities
Cellulase activity was measured according to a pub-
lished method and based on spectrophotometric stop
rate determination (Ghose, 1987). Protease activity
was determined using casein as substrate according to
Folin and Ciocalteau (1929) and Anson (1938). a-
Amylase assays were performed according to Bernfeld
(1955). Lipase activity was determined spectrophoto-
metrically using p-nitrophenyl laurate as the substrate
(Abd-Elhakeem et al., 2013).
Rumen FA determination
The rumen fluid samples were freeze-dried before
analysed for FA according to the method of O’Fallon
et al. (2007). For the determination of FA
3
Chlorella affects rumen microbial population E. Tsiplakou et al.

Table 2 Primers used for qPCR and the amplicon size

Amplification target Primer Amplicon (nt) References

Total bacteria
Forward 50 -CGGCAACGAGCGCAACCC-30 130 Zhou et al. (2012)
Reverse 50 -CCATTGTAGCACGTGTGTAGCC-30 130
Methanogen bacteria
Forward 50 -TTCGGTGGATCDCARAGRGC-30 145 Zhou et al. (2012)
Reverse 50 -GBARGTCGWAWCCGTAGAATCC-30 145
Butyrivibrio fibrisolvens
Forward 50 -GAGGAAGTAAAAGTCGTAACAAGGTTTC-30 1200 Zhou et al. (2012)
Reverse 50 -CGGTCTCTGTATGTTATGAGGTATTACC-30 1200
Ruminococcus flavefaciens
Forward 50 -CGAACGGAGATAATTTGAGTTTACTTAGG-30 132 Zhou et al. (2012)
Reverse 50 -CGGTCTCTGTATGTTATGAGGTATTACC-30 132
Fibrobacter succinogenes
Forward 50 -GGT ATG GGA TGA GCT TGC-30 121 Zhou et al. (2012)
Reverse 50 -GCC TGC CCC TGA ACT ATC-30 121
Protozoa
Forward 50 -GCTTTCGWTGGTAGTGTATT-30 321 Zhou et al. (2012)
Reverse 50 -CTTGCCCTCYAATCGTWCT-30 321
Ruminococcus albus
Forward 50 -CGGCAACGAGCGCAACCC-30 175 Zhou et al. (2012)
Reverse 50 -CCATTGTAGCACGTGTGTAGCC-30 175
Total fungi
Forward 50 -GAGGAAGTAAAAGTCGTAACAAGGTTTC-30 120 Zhou et al. (2012)
Reverse 50 -CAAATTCACAAAGGGTAGGATGATT-30 120
Clostridium sticklandii
Forward 50 -TATCCTAAAATTACAATAGATATT-30 1000 Fonknechten et al. (2009)
Reverse 50 -TTAAAGAAGTTCTTTTTCAATATAT-30 1000
Peptostreptococcus anaerobius
Forward 50 -CGT CTW ATT TNA TGC TTG CA-30 943 Riggion and Lennon (2002)
Reverse 50 -AGC CCC GAA GGG AAG GTG TG-30 943
Methanosphaera stadtmanae
Forward 50 -CTTAACTATAAGAATTGCTGGAG-30 150 Zhou et al. (2009)
Reverse 50 -TTCGTTACTCACCGTCAAGATC-30 150
Methanobrevibacter ruminantium
Forward 50 -AATATTGCAGCAGCTTACAGTGAA-30 336 Ufnar et al. (2007)
Reverse 50 -TGAAAATCCTCCGCAGACC-30 336

concentration, an Agilent 6890 N gas chromatograph
equipped (Agilent Technologies, Wilmington, DE,
USA) with an HP-88 capillary column
(60 m 9 0.25 mm i.d. with 0.20-lm film thickness,
Agilent) was used. A flame ionization detector tem-
perature was set at 260 °C, and the chromatographic
analysis involved a temperature programmed run
starting at 120 °C and held for 1 min. Then, the anal-
ysis followed by two step-up ramp, one of 1.25 °C/
min to 230 °C and another of 10 °C/min to 240 °C,
and held for 3 min. Helium was used as the carrier gas
with a linear velocity set at 30 cm/s.
Feed samples analyses
The concentrates were analysed for organic matter
(Official Method 7.009), dry matter (Official Method
7.007), crude protein (CP; Official Method 7.016) and
ether extract (EE; Official Method 7.060) according to
AOAC (1984) and for neutral detergent fibre assayed
without a heat-stable amylase and acid detergent fibre
expressed exclusive of residual ash according to Van
Soest et al. (1991; Table 1). The trace elements in
feeds were determined using inductively coupled
plasma mass spectrometry, ICP-MS (Elan 9000; Perkin
Elmer Life and Analytical Sciences Inc., Waltham,
MA, USA). Additionally, the main FAs of feeds were
analysed according to the method of O’Fallon et al.
(2007) using GC-FID as previously described.
Statistical analysis
Treatment effect (Control vs. chlor) on rumen micro-
bial community, enzyme activities and FA analysis
was test by one-way ANOVA, using SPSS statistical

4 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
E. Tsiplakou et al.
Table 3 qPCR conditions according to the amplicon size
For replicons from 100 to 200 bp were used the following conditions:
• Step 1: Initial denaturation at 94 °C for 4 min
• Step 2: Denaturation at 94 °C for 1 min
• Step 3: Hybridization of primers at 58 °C for 1 min
• Repeat steps 2–4 for 41 cycles
For replicons between 300 and 600 bp were used the following
conditions:
• Step 1: Initial denaturation at 94 °C for 3 min
• Step 2: Denaturation at 94 °C for 30 s
• Step 3: Hybridization of primers at 63 °C for 1 min
• Step 4: Elongation at 72 °C for 1 min
• Repeat steps 2–4 for 35 cycles
For replicons close to 1000 bp were used the following conditions:
• Step 1: Initial denaturation at 95 °C for 10 min
• Step 2: Denaturation at 95 °C for 1 min
• Step 3: Hybridization of primers at 50 °C for 1 min
• Step 4: Elongation at 72 °C for 1 min
• Repeat steps 2–4 for 46 cycles
package (version 16.0; SPSS Inc., Chicago, IL, USA).
Post hoc tests were performed using Duncan’s multiple
range test, and significance was set at p < 0.05.
Discussion
Up to now, the microalgae have been considered as
possible candidates to reduce CH4 emissions due to its
high eicosapentaenoic and docosahexaenoic (DHA)
content, because it has been shown that dietary fat
supplements, especially those containing unsaturated
FAs, reduce CH4 production (Beauchemin et al.,
2008; Martin et al., 2010; Moate et al., 2011). This
assumption has been verified by an in vitro study
where a reduction in CH4 production (up to 80%) was
found by the addition of a DHA-rich supplement (Fie-
vez et al., 2007). However, in this study, the relative
proportions of M. stadtmanae, M. ruminantium and
Methanogens to the total bacteria population, respec-
tively, increased significantly in the rumen liquid of
goats fed with the chlor diet compared with the con-
trol (Fig. 1). An increase on gas production in the
rumen by algae, with very high protein content, was
also observed in a recent in vitro study (Dubois et al.,
2013). Up to now, no information exists about the
impact of dietary supplementation with microalgae on
rumen micro-organisms and CH4 production in vivo.
However, some existing data are related to anaerobic
digestion of microalgae for biodiesel and, conse-
quently, CH4 production. Indeed, C. vulgaris, com-
pared with Dunaliella tertiolecta, when was used as
feedstock for biofuel production (under anaerobic
conditions at 37 °C), gave 12-fold higher CH4 emis-
sion, results which were attributed to its higher
Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
Chlorella affects rumen microbial population
protein content (36% vs. 15% on a dry weight basis;
Lakaniemi et al., 2011). Moreover, Sialve et al.
(2009) concluded that the conversion of microalgae
after lipid extraction into CH4 is a process that can
recover more energy than that from the cell lipids.
Although the higher lipid content of the cell resulted
in higher theoretical potential CH4 yield, the lipid
hydrolysis is considered to be slower than that of pro-
teins. Thus, Pavlostathis and Giraldo-Gomez (1991),
when calculated the minimum values of limiting gen-
eration time for anaerobic treatment, found values of
0.43 and 3.2 days for proteins and lipids respectively.
However, it should be pointed out here that the high
protein content of C. vulgaris (Table 1), which was
used as feed supplement in this study, was not the rea-
son for the increase in the Methanogens population of
goats fed with the chlor diet, compare with the con-
trols because no great differences on CP content
existed between the two dietary treatments (Table 1).
Besides, when some authors related CH4 yield from
microalgae, during biofuel production, to their com-
position, especially with the lipids, proteins and carbo-
hydrates content (Sialve et al., 2009; Mairet et al.,
2011; Gonz alez-Fern
andez et al., 2012), they found
no strong correlations among each macronutrient
with CH4 yield (Torres et al., 2013). These facts clearly
show that the ratio between various macromolecules
is not the most important parameter determining the
actual CH4 yield from microalgae. The cell wall disrup-
tion of microalgae plays more important role
(Gonz alez-Fernandez et al., 2012). Indeed, some pre-
liminary studies have shown that proteases, which
exist in the rumen, may be a useful biological catalyst
for C. vulgaris cell wall disruption in order to double
CH4 production compared to the raw biomass (Mahdy
et al., 2014a,b). Moreover, Chakraborty et al. (2013)
when characterized the composition of C. vulgaris
found that 89.8% of it was glucose, mostly as a-cellu-
lase. It has been shown recently that the cellulolytic
pre-treatment before anaerobic digestion increases
performance and biogas production efficiency (L€ u
et al., 2013; Mu~ noz et al., 2014), because it allows the
methanogenic bacteria to gain access to the intracellu-
lar content of microalgae. This process in the rumen
can be carried out by the action of cellulolytic bacteria
(L€u et al., 2013).
A significant reduction in the cellulase activity
(Table 4) and in the abundance of R. albus population
in the rumen liquid of goats fed with the chlor diet,
compared with the control, was observed, while the
relative proportions of R. flavefaciens and F. succinogenes
remained unchanged among the two dietary treat-
ments (Fig. 1). The reduction in R. albus, one of the
5
Chlorella affects rumen microbial population E. Tsiplakou et al.

Fig. 1 Relative proportions of Methanosphaera stadtmanae, Methanobrevibacter ruminantium, Methanogens, Protozoa, Fungi, Ruminococcus flave-
faciens, Butyrivibrio fibrisolvens, Peptostreptococcus anaerobius, Ruminococcus albus, Fibrobacter succinogenes and Clostridium sticklandii to the
total bacterial population in the rumen fluid of goats of both groups.

Table 4 Rumen enzyme activity (units/ml) of goats of both groups the chlor-fed goats. Besides, as previously mentioned,
Enzymes Control chlor p the cell wall of C. vulgaris is a good substrate for pro-
teases action (Mahdy et al., 2014a,b). Moreover,
a-Amylase 7.08
2.46 8.30
1.26 NS although the differences in the CP content were not
Cellulase 8.11
2.17 4.40
0.98 *
significant among the two dietary treatments (Control
Protease 3.04
0.08 7.54
0.64 **
Lipase 3.86
0.38 5.05
0.91 NS
vs. chlor) in this study, the CP content of C. vulgaris is
rich in non-protein nitrogen consisted of nucleic acids,
*P < 0.05, **P < 0.01. nitrogen-containing cell walls and amines (Becker,
2007) which needs attention when C. vulgaris partici-
main cellulolytic bacteria in the rumen, may be due to pates in higher levels in animals diets. A significant
amino acids limitation required for its growth. It is release of ammonia, during microalgae fermentation
considered that the hyperammonia bacteria are obli- for biofuel production, has been already found
gatory amino acids fermenters that rapidly reduce the (Sialve et al., 2009). Additionally, in a recent study,
availability of amino acids to stimulate growth of cel- Lodge-Ivey et al. (2014) observed greater ammonia
lulolytic, hemicellulolytic or amylolytic bacteria. concentration in the rumen of cows fed a forage diet
Besides, a computer model suggests a direct antago- supplemented with lipid extracted C. vulgaris. It
nism by carbohydrate-fermenting bacteria against should be mentioned also here that in this study, the
hyperammonia population (Rychlik and Russell, goats fed diets with relatively high forage-to-concen-
2000). Indeed, in this study, a significant increase in trate ratio. Moreover, the same EE and starch content
the relative proportions of C. sticklandii (Fig. 1), one of among the two dietary treatments may explain no sig-
the most important hyperammonia bacteria, and in nificant differences on lipase and a-amylase activity in
protease activity (Table 4) in the rumen fluid of goats the rumen liquid of goats which was found in this
fed with the chlor diet, compared with the control, study (Table 2).
was observed. This enhances the above assumption, Furthermore, the abundance of B. fibrisolvens
although the abundance of P. anaerobius remained increased significantly in the rumen liquid of goats fed
unchanged among the two dietary treatments (Fig. 1). with the chlor diet compared with the control (Fig. 1).
The cell wall of C. vulgaris may again explains the sig- The cell wall of C. vulgaris may again explain these
nificantly higher abundance of C. sticklandii (Fig. 1) findings. The fact that B. fibrisolvens, further to its cel-
and protease activity (Table 4) which were found in lulolytic, has also proteolytic activity (Cotta and

6 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
E. Tsiplakou et al. Chlorella affects rumen microbial population

Hespell, 1986) makes the cell wall of C. vulgaris good Table 5 The mean individual fatty acids (FA; % of total FA), FA groups
substrate for its development (L€ u et al., 2013; Mahdy and S/U ratio in the rumen fluid of goats of both groups
et al., 2014a,b; Mu~ noz et al., 2014). Thus, the fact FA Control chlor p
that C. vulgaris affected positively these bacteria spe-
cies is further confirmed by the FA profile in the C4:0 1.14
0.12 1.21
0.05 NS
C6:0 0.83
0.06 0.80
0.06 NS
rumen liquid of goats. More specifically, the propor-
C8:0 0.12
0.02 0.23
0.05 NS
tions of trans C18:1, trans-11 C18:1 (vaccenic acid) and C10:0 0.15
0.02 0.29
0.07 NS
MUFA increased while those of C18:0, LCFA and the C11:0 0.18
0.09 0.35
0.02 NS
ratio of saturated-to-unsaturated FAs decreased in the C12:0 5.12
0.55 13.08
3.45 NS
rumen liquid of goats fed with the chlor diet compared C14:0 8.23
0.11 9.23
0.13 NS
with the control (Table 5). The isomerization and C14:1 1.44
0.29 1.33
0.21 NS
hydrogenation of the cis-D-9 bond of feeds FAs in the C15:0 1.08a
0.22 0.81b
0.09 NS
C15:1 0.22
0.01 0.34
0.02 **
rumen during BH is carried out by bacteria collectively
C16:0 15.60
1.38 13.53
0.27 NS
known as group A (Harfoot and Hazelwood, 1988; C16:1 0.42
0.09 0.24
0.08 NS
Jenkins et al., 2008; Alves and Bessa, 2014). Many of C17:1 0.72a
0.06 0.51b
0.01 *
the Butyrivibrio spp. belong to group A bacteria, as C18:0 50.61a
1.83 38.40b
2.67 *
they are unable to hydrogenate octadecenoic acids trans C18:1 0.32a
0.04 0.75b
0.12 *
(trans C18:1, trans-11 C18:1) and thus are incapable of trans-11 C18:1* 1.09a
0.25 2.86b
0.43 *
completing BH (C18:0 production). cis-9 C18:1 7.39
0.41 7.85
0.17 NS
C18:2n6t 0.12
0.02 0.46
0.16 NS
Chlorella vulgaris may also affect methanogenesis in
cis-9, 2.06a
0.21 3.34b
0.54 *
the rumen through a direct effect on H2 production. trans-11 C18:2
Besides, L€ u et al. (2013) found that bioaugmentation C18:2n6c 4.43
0.49 4.68
0.15 NS
with cellulolytic and hydrogenic bacteria improved C18:3n6 0.09
0.05 0.29
0.08 NS
the degradation of C. vulgaris biomass producing C18:3n3 1.04a
0.08 1.36b
0.02 *
higher levels of H2. Moreover, Carver et al. (2011) C20:0 0.61a
0.02 0.44b
0.03 *
reported H2 production from C. vulgaris even under C20:1 1.78
0.19 2.90
0.44 NS
C21:0 0.24
0.03 0.56
0.15 NS
dark fermentation at 60 °C. Additionally, H2 use by
C20:2 1.20
0.02 1.33
0.07 NS
microalgae cells under dark and anaerobic conditions C20:3n6 0.31
0.04 0.49
0.10 NS
has been also reported by other researchers (Gfeller C20:3n3 0.87a
0.02 0.68b
0.03 **
and Gibbs, 1984; Miura et al., 1986; Ueno et al., C22:2 0.26
0.04 0.21
0.02 NS
1998). Hydrogen is also one of the major products of C20:5n-3 0.08a
0.01 0.24b
0.02 **
fermentation by protozoa in the rumen (Moss et al., C24:0 0.46
0.14 0.60
0.01 NS
2000). Thus, the significant increase in the abundance C24:1 0.08
0.01 0.12
0.07 NS
SCFA 2.43a
0.10 2.88b
0.10 *
of protozoa population in the rumen liquid of goats,
ΜCFA 22.64
1.27 28.34
3.68 NS
fed with the chlor diet compared with the control LCFA 51.92
1.98 40.00
5.62 NS
(Fig. 1), which was observed, proves their close rela- ΜUFA 13.45a
0.23 16.89b
1.12 *
tionship with methanogens. Besides, it is already PUFA 7.51
0.49 8.54
0.24 NS
known that methanogens found both attached and S 76.98a
0.67 71.22b
1.85 *
inside ciliate protozoal cells contribute between 9% U 20.96a
0.46 25.43b
1.35 *
and 37% of the rumen CH4 production (Finlay et al., S/U 3.68a
0.11 2.83b
0.23 *

1994; Newbold et al., 1995; Hook et al., 2010).
Conclusions
The increase in Methanobacteria and protozoa popula-
tion in the rumen liquid of goats, fed forage-based
diet supplemented with C. vulgaris, should be taken
into account when microalgae are incorporated
to ruminants’ diets. Dietary supplementation with
C. vulgaris enhances the abundance of C. sticklandii
and consequently ammonia production in the rumen
trans-11 C18:1*: This value is not included in the trans C18:1 content; SCFA:
Short-chain fatty acids = C4:0 + C6:0 + C8:0 + C10:0 +C11:0; MCFA: Medium-
chain fatty acids = C12:0 + C14:0 + C15:0 + C16:0; LCFA: Long-chain fatty
acids = C18:0 + C20:0 + C21:0 + C22:0 + C23:0 + C24:0; MUFA: Monounsatu-
rated fatty acids = C14:1 + C15:1 + C16:1 + C17:1 + trans C18:1 + trans-11
C18:1 + cis-9 C18:1 + C20:1 + C24:1; PUFA: Polyunsaturated fatty acids = cis-
9, trans-11 C18:2 + C18:2n-6c + C18:2n-6t + C18:3n6 + C18:3n3 + C20:3n6 +
C20:3n3 + C20:2 + C22:2 + C20:5n-3; S: Saturated fatty acids = SCFA +
MCFA + LCFA; U: Unsaturated fatty acids = MUFA + PUFA. Means with
different superscript letters in each row (between dietary treatments) for
each fatty acid differ significantly (p ≤ 0.05).
*P < 0.05, **P < 0.01.

Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH 7
Chlorella affects rumen microbial population E. Tsiplakou et al.

of goats, which is effectively utilized. Moreover,
C. vulgaris inclusion in the goat diet promoted a shift
in ruminal BH pathway towards the formation of
trans-C18:1 FA, an effect that was associated with
changes in the B. fibrisolvens population in their
rumen liquid. Additionally, changes in some cellu-
lolytic and proteolytic bacteria in the rumen liquid of
goats were observed accompanied by modifications in
the respective rumen enzyme activities (cellulase, pro-
tease). Finally, an antagonism may be take place
among some cellulolytic (R. albus) and proteolytic
(C. sticklandii) bacteria, but this needs further investi-
gation.

References
Abd-Elhakeem, M. A.; Elsayed, A. M.;
Alkhulaqi, T. A., 2013: New colorimetric
method for lipases activity assay in
microbial media. American Journal of
Analytical Chemistry 4, 442–444.
Alves, S. P.; Bessa, R. J. B., 2014: The
trans-10; cis-15 18:2: a missing interme-
diate of trans-10 shifted rumen biohy-
drogenation pathway? Lipids 49,
527–541.
Anson, M. L., 1938: The estimation of pep-
sin, trypsin, papain and cathepsin with
hemoglobin. Journal of General Physiology
22, 79–89.
Association of Official Analytical Chemists
International, 1984: Official Methods of
Analysis, 14th edn. AOACI, Arlington,
VA.
Beauchemin, K. A.; Kreuzer, M.; O’Mara,
F.; McAllister, T. A., 2008: Nutritional
management for enteric methane abate-
ment: A review. Australian Journal of
Experimental Agriculture 48, 21–27.
Becker, E. W., 2007: Micro-algae as a
source of protein. Biotechnology Advances
25, 207–210.
Bernfeld, P., 1955: Amylases, a and b.
Methods in Enzymology 1, 149–158.
Boeckaert, C.; Fievez, V.; Van Hecke, D.;
Verstraete, W.; Boon, N., 2007: Changes
in rumen biohydrogenation intermedi-
ates and ciliate protozoa diversity after
algae supplementation to dairy cattle
European. Journal of Lipid Science and
Technology 109, 767–777.
Boeckaert, C.; Vlaemink, B.; Dijkstra, J.;
Issa-Zacharia, A.; Van Nespen, T.; Van
Straalen, W.; Fievez, V., 2008: Effect of
dietary starch or micro algae supple-
mentation on rumen fermentation and
milk fatty acid composition of dairy
cows. Journal of Dairy Science 91, 4714–
4727.
Carver, S. M.; Hulatt, C. J.; Thomas, D. N.;
Tuovinen, O. H., 2011: Thermophilic;
anaerobic co-digestion of microalgal
biomass and cellulose for H2 production.
Biodegradation 22, 805–814.
Chakraborty, M.; McDonald, A. G.;
Nindo, C.; Chen, S., 2013: An a-glu-
can isolated as a co-product of biofuel
by hydrothermal liquefaction of Chlor-
ella sorokiniana biomass. Algal Research
2, 230–236.
Cotta, M. A.; Hespell, R. B., 1986: Prote-
olytic activity of the ruminal bacterium
Butyrivibrio fibrisolvens. Applied and Envi-
ronmental Microbiology 52, 51–58.
Dubois, B.; Tomkins, N. W.; Kinley, R. D.;
Bai, M.; Seymour, S.; Paul, N. A.; de
Nys, R., 2013: Effect of tropical algae as
additives on rumen in vitro gas produc-
tion and fermentation characteristics.
American Journal of Plant Sciences 4,
34–43.
Fievez, V.; Boeckaert, C.; Vlaemink, B.;
Mestdagh, J.; Demeyer, D., 2007: In vitro
examination of DHA-edible micro-algae.
2. Effect on rumen methane production
and apparent degradability of hay. Ani-
mal Feed Science and Technology 136,
80–95.
Finlay, B. J.; Esteban, G.; Clarke, K. J.;
Williams, A. G.; Embley, T. M.; Hirt, R.
P., 1994: Some rumen ciliates have
endosymbiotic methanogens. FEMS
Microbiology Letters 117, 157–162.
Folin, O.; Ciocalteau, V., 1929: Enzymatic
assay of protease using casein as a sub-
strate. Journal of Biological Chemistry 73,
627.
Fonknechten, N.; Perret, A.; Perchat, N.;
Tricot, S.; Lechaplais, C.; Vallenet, D.;
Vergne, C.; Zaparucha, A.; Le Paslier,
D.; Weissenbach, J.; Marcel Salanoubat,
M., 2009: A conserved gene cluster rules
anaerobic oxidative degradation of L-
ornithine. Journal of Bacteriology 191,
3162–3167.
Gfeller, R. P.; Gibbs, M., 1984: Fermenta-
tive metabolism of Chlamydomonas rein-
hardtii. Plant Physiology 75, 212–218.
Ghose, T. K., 1987: Measurement of cellu-
lase activities. Pure and Applied Chemistry
59, 257–268.
Glover, K. E.; Budge, S.; Rose, M.; Rupas-
inghe, H. P.; Maclaren, L.; Green-John-
son, J.; Fredeen, A. H., 2012: Effect of
feeding fresh forage and marine algae
on the fatty acid composition and oxida-
tion of milk and butter. Journal of Dairy
Science 95, 2797–2809.
Goiris, K.; Muylaert, K.; Fraeye, I.; Fou-
bert, I.; De Brabanter, J.; De Cooman,
L., 2012: Antioxidant potential of
microalgae in relation to their phenolic
and carotenoid content. Journal of
Applied Phycology 24, 1477–1486.
Gonz alez-Fern andez, C.; Sialve, B.; Ber-
net, N.; Steyer, J. P., 2012: Impact of
microalgae characteristics on their con-
version to biofuel. Part II: Focus on bio-
methane production. Biofuels Bioproducts
and Biorefining 6, 205–218.
Harfoot, C. G.; Hazelwood, G. P., 1988:
The rumen microbial ecosystem. In:
Lipid Metabolism in the Rumen. Elsevier
Science Publishing, London,
pp. 285–322.
Hook, S. E.; Wright, A.-D. G.; McBride, B.
W., 2010: Methanogens: methane pro-
ducers of the rumen and mitigation
strategies. Archaea 2010, 945785.
doi:10.1155/2010/945785.
Jenkins, T. C.; Wallace, R. J.; Moate, P. J.;
Mosley, E. E., 2008: Recent advances in
biohydrogenation of unsaturated fatty
acids within the rumen microbial
ecosystem. Journal of Animal Science 86,
397–412.
Kou
rimsk a, L.; Vondra
ckov
a, E.;
Fantov a, M.; Novy, P.; Nohejlov a, L.;
Michnov a, K., 2014: Effect of feeding
with algae on fatty acid profile of goat’s
milk. Scientia Agriculturae Bohemica 45,
162–169.
Lakaniemi, A. M.; Hulatt, C. J.; Thomas,
D. N.; Tuovinen, O. H.; Puhakka, J. A.,
2011: Biogenic hydrogen and methane
production from Chlorella vulgaris and
Dunaliella tertiolecta biomass. Biotechnol-
ogy for Biofuels 4, 34.
Lodge-Ivey, S. L.; Tracey, L. N.; Salazar,
A., 2014: Ruminant Nutrition Sympo-
sium: the utility of lipid extracted algae
as a protein source in forage or starch-
based ruminant diets. Journal of Animal
Science 92, 1331–1342.

8 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
E. Tsiplakou et al.
Lopex, N. M.; Pereira, R. A. N.; Pereira, M.
N., 2013: Intake; milk yield; and blood
acid–base balance of cows in response to
marine algae meal [Abstract]. Journal of
Animal Science 91(Suppl. 2), 29.
L€
u, F.; Ji, J.; Shao, L.; He, P., 2013: Bacterial
bioaugmentation for improving methane
and hydrogen production from microal-
gae. Biotechnology for Biofuels 6, 92.
Lum, K. K.; Kim, J.; Lei, X. G., 2013: Dual
potential of microalgae as a sustainable
biofuel feedstock and animal feed. Jour-
nal of Animal Science and Biotechnology 4,
53.
Mahdy, A.; Mendez, L.; Blanco, S.; Balles-
teros, M.; Gonz alez-Fern andez, C.,
2014a: Protease cell wall degradation of
Chlorella vulgaris: effect on methane
production. Bioresource Technology 171,
421–427.
Mahdy, A.; Mendez, L.; Ballesteros, M.;
Gonzalez-Fernandez, C., 2014b:
Enhanced methane production of Chlor-
ella vulgaris and Chlamydomonas rein-
hardtii by hydrolytic enzymes addition.
Energy Conversion and Management 85,
551–557.
Mairet, F.; Bernard, O.; Ras, M.; Lardon,
L.; Steyer, J. P., 2011: Modeling
anaerobic digestion of microalgae using
ADM1. Bioresource Technology 102, 6823–
6829.
Martin, C.; Morgavi, D. P.; Doreau, M.,
2010: Methane mitigation in ruminants:
from microbe to the farm scale. Animal
4, 351–365.
Medipally, S. R.; Yusoff, F. Md.; Banerjee,
S.; Shariff, M., 2015: Microalgae as sus-
tainable renewable energy feedstock for
biofuel production. BioMed Research
International 2015, 519513.
Miura, Y.; Ohta, S.; Mano, M.; Miyamoto,
K., 1986: Isolation and characterization
of a unicellular marine green alga
exhibiting high activity in dark hydro-
gen production. Agricultural and Biologi-
cal Chemistry 50, 2837–2844.
Moate, P. J.; Williams, S. R. O.; Grainger,
C.; Hannah, M. C.; Ponnampalam, E.
N.; Eckard, R. J., 2011: Influence of
cold-pressed canola; brewers grains and
hominy meal as dietary supplements
suitable for reducing enteric methane
emissions from lactating dairy cows.
Animal Feed Science and Technology
166–167, 254–264.
Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
Moss, A. R.; Jouany, J. P.; Newbold, J.,
2000: Methane production by rumi-
nants: its contribution to global
warming. Annales De Zootechnie 49,
231–253.
Mu~ noz, C.; Hidalgo, C.; Zapata, M.; Jeison,
D.; Riquelme, C.; Rivasa, M., 2014: Use
of cellulolytic marine bacteria for enzy-
matic pretreatment in microalgal biogas
production. Applied Environmental
Microbiology 80, 4199–4206.
Newbold, C. J.; Lassalas, B.; Jouany, J. P.,
1995: The importance of methanogens
associated with ciliate protozoa in rumi-
nal methane production in vitro. Letters
in Applied Microbiology 21, 230–234.
O’Fallon, J. V.; Busboom, J. R.; Nelson, M.
L.; Gaskins, C. T., 2007: A direct method
for fatty acids methyl ester synthesis:
application to wet meat tissues, oils and
feedstuffs. Journal of Animal Science 85,
1511–1521.
Pavlostathis, S. G.; Giraldo-Gomez, E.,
1991: Kinetics of anaerobic treatment: a
critical review. Critical Reviews in Envi-
ronmental Control 21, 411–490.
Ramakers, C.; Ruijter, J. M.; Deprez, R. H.;
Moorman, A. F., 2003: Assumption-free
analysis of quantitative real-time poly-
merase chain reaction (PCR) data. Neu-
roscience Letters 339, 62–66.
Ramos-Morales, E.; Arco-P erez, A.;
Martın-Garcıaa, A. I.; Y anez-Ruiz, D. R.;
Frutos, P.; Herv as, G., 2014: Use of
stomach tubing as an alternative to
rumen cannulation to study ruminal
fermentation and microbiota in sheep
and goats. Animal Feed Science and Tech-
nology 198, 57–66.
Riggion, M. R.; Lennon, A., 2002:
Development of a PCR assay specific
for Peptostreptococcus anaerobius.
Journal of Medical Microbiology 51,
1097–1101.
Rychlik, J. L.; Russell, J. B., 2000: Mathe-
matical estimations of hyper-ammonia
producing ruminal bacteria and evi-
dence for bacterial antagonism that
decreases ruminal ammonia production
(1). FEMS Microbiology Ecology 32,
121–128.
Sharma, R.; John, S. J.; Damgaard, M.;
McAllister, T. A., 2003: Extraction of
PCR-quality plant and microbial DNA
from total rumen contents. BioTech-
niques 34, 92–94, 96–7.
Chlorella affects rumen microbial population
Shen, J. S.; Chai, Z.; Song, L. J.; Liu, J. X.;
Wu, Y. M., 2012: Insertion depth of oral
stomach tubes may affect the fermenta-
tion parameters of ruminal fluid col-
lected in dairy cows. Journal of Dairy
Science 95, 5978–5984.
Sialve, B.; Bernet, N.; Bernard, O., 2009:
Anaerobic digestion of microalgae as a
necessary step to make microalgal bio-
diesel sustainable. Biotechnology Advances
27, 409–416.
Torres, A.; Fermoso, F. G.; Rinc on, B.; Bar-
tacek, J.; Borja, R.; Jeison, D., 2013.
Challenges for cost-effective microalgae
anaerobic digestion. In: R. Chamy (ed.),
Biodegradation – Engineering and Technol-
ogy. ISBN: 978-953-51-1153-5. InTech,
pp, 139–160. Available from: http://
www.intechopen.com/books/biodegra-
dation-engineering-and-technology/
challenges-for-cost-effective-microal-
gae-anaerobic-digestion.
Ueno, Y.; Kurano, N.; Miyachi, S., 1998:
Ethanol production by dark fermenta-
tion in the marine green alga; Chlorococ-
cum littorale. Journal of Fermentation and
Bioengineering 86, 38–43.
Ufnar, J. A.; Wang, S. Y.; Ufnar, D. F.;
Ellender, R. D., 2007: Methanobrevibacter
ruminantium as an indicator of domesti-
cated-ruminant fecal pollution in sur-
face waters. Applied and Environmental
Microbiology 73, 7118–7121.
Van Soest, P. J.; Robertson, J. B.; Lewis, B.
A., 1991: Methods for dietary fibre, neu-
tral detergent fibre and non starch
polysaccharide in relation to animal
nutrition. Journal of Dairy Science 74,
3583–3597.
Yaakob, Z.; Ali, E.; Zainal, A.; Mohamad,
M.; Takriff, M. S., 2014: An overview:
biomolecules from microalgae for ani-
mal feed and aquaculture. Journal of Bio-
logical Research 21, 6.
Zhou, Y. W.; Hernandez-Sanabria, E.;
Guan, L. L., 2009: Assessment of the
microbial ecology of ruminal methano-
gens in cattle with different feed effi-
ciencies. Applied and Environmental
Microbiology 75, 6524–6533.
Zhou, Y. W.; McSweeney, C. S.; Wang,
J. K.; Liu, J. X., 2012: Effects of dis-
odium fumarate on ruminal fermenta-
tion and microbial communities in
sheep fed on high-forage diets. Animal
5, 815–823.
9